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Leveraging covalent modification for diverse applications in antiviral discovery and kinase mechanism deconvolution

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Leveraging covalent modification for diverse applications in antiviral discovery and kinase mechanism deconvolution

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LEVERAGING COV ALENT MODIFICATION FOR DIVERSE APPLICATIONS IN
ANTIVIRAL DISCOVERY AND KINASE MECHANISM DECONVOLUTION

by

JOSHUA J. FENG

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

DECEMBER 2022

Copyright 2022 Joshua J. Feng
ii

Dedication
To xx, because you’re not here and you wouldn’t care:
How can you make a mirror by polishing a brick?

iii

Acknowledgements
Though this is surely backwards, I am finding that properly acknowledging all those due
mentioning is a more daunting task than drafting and completing the contents of this dissertation
proper. Along the course of my graduate studies, I have been privileged to have crossed paths with
so many inspiring, talented, and good people, to the extent that it feels slightly criminal to merely
send a nod of recognition in the form of a single – and in some cases, half – sentence of thanks.
Nevertheless, convention prevents me from submitting a novella qua acknowledgements section
(who even reads these, anyways?), so I pray that the appropriate spirit transmits, even if all that
weight falls on the simple vessel of a fumbled clause.
Firstly, I would like to thank my primary research advisor, Dr. Chao Zhang, who has – as I
hoped – been every bit of the mentor that I needed. I have tried my best, as I think we all do, to
put on the best fake-it-til-you-make-it performance throughout my time at USC. Though I suspect
that he is wise to some of my more “faked” moments, he has provided me with just the right
amount of independence and trust to reach a more-than-I-probably-deserve amount of “maked” in
my intellectual, scientific, and career development. I will be forever motivated to learn how to
learn because of the continual demonstrations of insight and wit that I witnessed from him under
his academic tutelage. I would also like to thank all my committee members (inclusively,
throughout the various stages along our Department of Chemistry conveyer belt): Drs. Lin Chen,
Mike Inkpen, Nicos Petasis, Peter Qin, and Kate White.
Perhaps no other paragraph deserves as much love as this one, dedicated to the members of
iv

the Zhang group, both past and present. Of those earlier days, I am supremely grateful to have
stumbled upon such rich friendships, which surely is what propelled me to somehow make it
through the later years of this program. To Arunika Ekanayake, Amir Assadieskandar, Cay Yu,
Renata Miranda, and Bob Yuan (to whom I must dedicate an additional – though empty –
parenthetical): thank you for bringing me into the group and nurturing my bright-eyed hopes. More
recently, in these darker times (said in lighthearted jest… something, something, grain of truth in
every joke), I owe so much gratitude to the newer generation of Zhang lab members. In particular,
I must point out: Biancha Espinosa (I guess we weren’t on the same page, but we still got each
other to the end of the book, I think), Chau Ngo (I really wouldn’t have been able to stay sane
digging around in my backyard, naked, in the dark – metaphorically – without your support and
companionship), Tai Nguyen (for silently – and loudly – reminding me to take on all things
responsibly and virtuously), Luna Kim (your presence alone was often enough of a platform to
prevent me from teetering towards total instability; to better times ahead, to BOTW2), Jiaqi Tang
( 对不起, 最后我还是一个垃圾中文学生), and Yida Li (for reminding me of all that I still need
to learn). Lastly, to Anusha Mubin, thanks for being such a patient member of our group and for
navigating through a (cat)-sea of riff-RAFf with me.
Most importantly, I must acknowledge my friends and family. In truly ironic(?) fashion, I
could never verbally spell these particular thanks out; my excuse is that I wouldn’t dare deface or
diminish my gratitude with such ugly and unreliable things that words can sometimes be. But: you
know who you are and, for better or for worse, know that you are in my heart.

v

Table of Contents

Dedication ....................................................................................................................................... ii

Acknowledgements ........................................................................................................................ iii

List of Figures ............................................................................................................................... vii

Abstract ........................................................................................................................................... x

Chapter 1: Covalent ligands: indispensable instruments in drug discovery and chemical biology 1
1.1 A resurgence of interest in drugging via covalent inhibition ............................................. 1
1.2 Probing cellular events and processes with biological and chemical techniques .............. 8
1.3 Conclusions and outlook .................................................................................................. 15
1.4 References ........................................................................................................................ 16

Chapter 2: Rational discovery of covalent inhibitors targeting viral-cycle-critical
SARS-CoV-2 non-structural proteins ........................................................................................... 22
2.1 Introduction ...................................................................................................................... 22
2.1.1 Emergence of the COVID-19 global pandemic ..................................................... 22
2.1.3 Overview of current antivirals, therapeutics, and prophylactics used against
SARS-CoV-2 ................................................................................................................... 30
2.1.4 Perspectives and roadmap of development ............................................................ 32
2.2 SARS-CoV-2 Nsp12: RNA-dependent RNA polymerase ............................................... 34
2.2.1 Introduction ............................................................................................................ 34
2.2.2 Results and discussion ........................................................................................... 38
2.2.3 Summary ................................................................................................................ 72
2.3 SARS-CoV-2 Nsp5: 3C-like/main protease ..................................................................... 73
2.3.1 Introduction ............................................................................................................ 73
2.3.2 Results and discussion ........................................................................................... 78
2.3.3 Summary .............................................................................................................. 109
2.4 Experimental details....................................................................................................... 109
2.5 References .......................................................................................................................116

Chapter 3: A methodological platform for chemical-genetic interrogation of RAF inhibitor
polypharmacology and biology................................................................................................... 134
3.1 Introduction .................................................................................................................... 134
3.1.1 Protein kinases in signal transduction .................................................................. 134
3.1.2 Mitogen-activated protein kinase signaling pathway .......................................... 137
3.1.3 Regulation of RAF activity and dimerization ...................................................... 138
3.1.4 Dysregulation of RAF in disease and therapeutic strategies ............................... 141
vi

3.1.5 Paradoxical activation: a transactivation model for inhibitor-induced RAF
activity........................................................................................................................... 143
3.1.6 Perspectives and motivations ............................................................................... 146
3.2 Engineering covalent complementation to probe RAF kinase activity and biology ..... 149
3.2.1 Design and development of isoform-selective RAF inhibitors ............................ 149
3.2.2 Functional assessment of Ele-Cys platform ......................................................... 151
3.2.3 Inhibitor-derived chemical probes to monitor RAF occupancy........................... 156
3.2.4 Visions for this methodology ............................................................................... 158
3.3 Results and discussion ................................................................................................... 158
3.3.1 In-depth characterization of covalent RAF inhibitors in Ele-Cys chemical-
genetic platform ............................................................................................................ 158
3.3.2 Broadly applying the Ele-Cys method to probing under-investigated
problems in RAF biology.............................................................................................. 163
3.3.3 Combining Ele-Cys with novel assay formats to probe dimer-dependent
paradoxical activation ................................................................................................... 174
3.3.4 Investigating RAF modulation for regulating embryonic stem cell self-
renewal .......................................................................................................................... 188
3.4 Summary ........................................................................................................................ 194
3.5 Experimental details....................................................................................................... 195
3.6 References ...................................................................................................................... 201

Bibliography ............................................................................................................................... 215

Appendix: Chapter 3 SpyCatcher/SpyTag information .............................................................. 252

vii

List of Figures

Figure 1.1 General mechanistic scheme of covalent inhibition ......................................................3
Figure 1.2 Representative panel of residue-selective covalent warheads .......................................6
Figure 1.3 Comparison of classical and chemical genetic philosophies .........................................9
Figure 1.4 Relationship between specificity and timescale of assorted methods for
perturbing protein function ............................................................................................................ 11
Figure 1.5 Allele-specific chemical genetic strategies for engineering specificity ......................13
Figure 2.1 Schematic diagram of the SARS-CoV-2 viral particle and genome ............................25
Figure 2.2 Coronavirus virion and life cycle ................................................................................28
Figure 2.3 Structures of the SARS-CoV-2 core polymerase complex and catalytic center ..........36
Figure 2.4 Structural outline of quinazoline-based inhibitors/probes ...........................................39
Figure 2.5 Schematic of CuAAC-mediated in-gel fluorescence analysis for covalently
labeled proteins ..............................................................................................................................41
Figure 2.6 Identification of Nsp12-interacting covalent inhibitors ...............................................42
Figure 2.7 Streptavidin enrichment of Nsp proteins and influence of CuAAC ............................44
Figure 2.8 Systematic evaluation of CuAAC reagents on Strep-Tactin® enrichment ..................45
Figure 2.9 Post-enrichment CuAAC protocol for visualizing in situ labeling by fluorescence ...46
Figure 2.10 Characterization of quinazoline inhibitor labeling of Nsp12 by on-resin CuAAC ...48
Figure 2.11 MS/MS determination of reactive labeled residue in Nsp12 .....................................49
Figure 2.12 Design of “open-chain” quinazoline analogs ............................................................50
Figure 2.13 Open-ring inhibitors promote an innate immune response upon viral infection .......52
Figure 2.14 First-generation of potential antiviral molecules derived from open-ring
quinazoline inhibitors.....................................................................................................................53
Figure 2.15 Effect of first-generation antivirals in SARS-CoV-2-infected Caco2 cells ...............55
Figure 2.16 Second-generation panel of putative antivirals .........................................................56
Figure 2.17 Third-generation panel of putative antivirals ............................................................59
Figure 2.18 Target engagement confirmation of CTPS1 ..............................................................62
Figure 2.19 Validating CTPS1 as mediator of antiviral effects ....................................................64
Figure 2.20 Profiles of SARS-CoV-2 viral engagement with activity-based probes derived
from putative antivirals ..................................................................................................................65
Figure 2.21 Antiviral competitor validation against viral proteins labeled by activity-based
probes .............................................................................................................................................67
Figure 2.22 Dose-dependent competitor titrations of best antiviral compounds against Nsp12 ..67
Figure 2.23 MS/MS identification of reactive binding residues in Nsp12 ...................................69
Figure 2.24 Confirmation of MS/MS site ID data through in situ labeling of Nsp12 alanine
mutants ...........................................................................................................................................70
Figure 2.25 Dose titration of 2.9 in Nsp12 mutants at lower concentration ranges ......................71
Figure 2.26 Dose titration of 2.34 in Nsp12 mutants at lower concentration ranges ....................71
Figure 2.27 Structure of the SARS-CoV-2 main protease homodimer .........................................75
Figure 2.28 Design rationale of inhibitors targeting the catalytic residues of
viii

SARS-CoV-2 Nsp5 ........................................................................................................................77
Figure 2.29 Attempts to identify Nsp5 binders from quinazoline-based inhibitors ......................79
Figure 2.30 Bacterial expression of Nsp5-6xHis in BL21 ............................................................81
Figure 2.31 Mammalian expression of Nsp5-TwinStrep in HEK293T ........................................83
Figure 2.32 First-generation Nsp5 inhibitors and kinetic characterization ...................................85
Figure 2.33 Activity-based probes derived from first-generation Nsp5 inhibitors
and initial attempts at in situ labeling ............................................................................................88
Figure 2.34 Detection of Nsp5 engagement on-resin, in vitro by vinyl sulfone ABPs .................90
Figure 2.35 Figure 2.35 First-generation ABPs fail to label Nsp5 in situ .....................................91
Figure 2.36 Second-generation Nsp5 inhibitors and kinetic characterization ..............................92
Figure 2.37 Irreversibility versus reversibility reaction kinetics with second-generation
inhibitors containing alkyne and nitrile warheads .........................................................................94
Figure 2.38 Third-generation Nsp5 inhibitors and kinetic characterization .................................96
Figure 2.39 Irreversibility versus reversibility kinetics of third-generation Nsp5 inhibitors .......97
Figure 2.40 Characterization of ABPs derived from third generation inhibitors in vitro .............99
Figure 2.41 In situ characterization of Nsp5 labeling with third-generation ABPs ....................101
Figure 2.42 Off-target labeling profile of third-generation ABPs in HEK293T and HeLa ........102
Figure 2.43 ABPs enable competitive titration of third-generation inhibitors in vitro
and in situ .....................................................................................................................................103
Figure 2.44 Measuring Nirmatrelvir residence time in live cells................................................104
Figure 2.45 Tuning reactivity of latent alkyne warhead by trifluoromethyl appendage .............106
Figure 2.46 Cytotoxicity of Nsp5 inhibitors in Vero-E6 cells ....................................................108
Figure 2.47 Antiviral analysis of Nsp5 inhibitors in SARS-CoV-2-infected
HeLA-ACE2 cells ........................................................................................................................108
Figure 3.1 Protein phosphorylation by kinases ...........................................................................135
Figure 3.2 RAS/MAPK signaling ...............................................................................................138
Figure 3.3 Conformational features of RAF activation ..............................................................140
Figure 3.4 Types of kinase inhibitors ..........................................................................................144
Figure 3.5 Transactivation model of type I
1/2
inhibitor-induced RAF dimerization ...................145
Figure 3.6 Allele-specific chemical genetic engineering of isoform-selective RAF inhibitors ..149
Figure 3.7 RAF inhibitors and quinazoline-based derivatives ....................................................151
Figure 3.8 Potent and selective inhibition of engineered BRAF
TM
by quinazoline inhibitors ...152
Figure 3.9 Effects of isoform-selective RAF inhibitors on MAPK signaling .............................154
Figure 3.10 Dimer formation effects in response to varied RAF inhibitors in multiple
contexts ........................................................................................................................................156
Figure 3.11 RAF quinazoline-based probe directly labels BRAF ..............................................157
Figure 3.12 Time-course engagement of 3.4 to BRAF
DM
...........................................................160
Figure 3.13 BRAF DNA titration and influence of protein abundance on inhibition .................162
Figure 3.14 Inhibitor titration against p61-BRAF
DM
and analysis of downstream signaling .....166
Figure 3.15 Ele-Cys platform and its application in non-canonical BRAF mutants ..................168
Figure 3.16 Ele-Cys platform and inhibitor-sensitized ARAF ....................................................172
ix

Figure 3.17 In vitro labeling of recombinant BRAF with a desthiobiotin-ATP probe ................176
Figure 3.18 Competitive labeling of transiently expressed RAF by desthiobiotin-ATP probe...178
Figure 3.19 Monitoring in situ dimer events and their effects on enhancing ATP-binding
affinity ..........................................................................................................................................180
Figure 3.20 DNA dose titration of SpyCatcher-Tag-induced RAF dimerization in situ .............183
Figure 3.21 Tuning in situ SpyCatcher-SpyTag engagement through dimer-disrupting
mutations ......................................................................................................................................185
Figure 3.22 Tuning in situ SpyCatcher-SpyTag engagement through exploration of
less-optimized peptide tags ..........................................................................................................186
Figure 3.23 Introduction of dimer-disrupting mutations into early-generation
SpyCatcher-Tag-RAF dimers .......................................................................................................188
Figure 3.24 Characterization of RAF knockout in mouse embryonic stem cells .......................191
Figure 3.25 Sanger chromatograms of CRISPR/Cas9 knock-in mice ........................................192
Figure 3.26 Monitoring mouse embryonic stem cell self-renewal after small-molecule
inhibition of RAF .........................................................................................................................194

x

Abstract
Covalent target modification has re-emerged in recent years as a promising modality for
therapeutic development and mechanistic biochemical analysis. Although slightly colored by some
decades’ worth of residual skepticism, current trends suggest a paradigmatic shift towards near-
centerpiece status for covalent drugs and probes within medicinal chemistry and chemical biology.
Recognizing the benefits that covalent modulators confer, we sought to address two pressing
clinical needs through methodologies reliant on small molecules acting through covalent
mechanisms. This dissertation details our efforts in the discovery of antiviral compounds for
combating the SARS-CoV-2 global pandemic and the demystification of RAF kinase physiology.
Chapter one contains an expository account of the history of covalent drugs and discusses
some of the advantages and disadvantages for their use. We describe some of the current
innovations and perspectives that possibly influence the trajectory of covalent drug development
in years to come. The chapter concludes with a discussion of alternative utilities for targeted
covalent protein modification by small molecules in the domain of chemical genetics. We describe
the scientific necessity of a chemical genetic approach to biology and biochemistry as a response
to more classic genetic methods.
In chapter two, we discuss two inverse, yet complementary, approaches to designing covalent
small-molecule inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (Nsp12) and
main protease (Nsp5). Of the former, we performed a forward pharmacological, phenotypic screen
of inhibitors and selected for those successful in preventing/mitigating SARS-CoV-2 viral
xi

infection. From our most potent lead compounds, we designed activity-based probes and identified
viral factor Nsp12 as the bona fide pharmacological target presumably responsible for mediating
antiviral effects upon covalent inhibition. These drug candidates have verified in vivo efficacy and
provide a launchpad for the design of future potent and specific polymerase inhibitors. Of the latter,
we executed a reverse pharmacological, target-based optimization of peptidomimetic scaffolds
shown to exhibit efficacy in inhibiting Nsp5. Specifically, we pursued a systematic evaluation of
covalent warheads and assessed their in vitro and in situ labeling properties across three
generations of ligand structures. The culmination of this work has been the characterization of an
irreversible, latent alkyne warhead that can be sterically and electronically tuned to adjust
reactivity and off-rate kinetics, providing groundwork for the synthesis of appropriately
derivatized activity-based probes for in situ and in vivo applications. Additionally, inhibitors
bearing the parental, electrophilic alkyne show marked improvement over current FDA-approved
drugs targeting Nsp5 and thus represent a class of warheads with verifiable clinical potential.
Finally, chapter three documents our efforts to thoroughly characterize and broadly apply a
chemical genetic platform based on covalent complementation for the illumination of various
RAF-centered nuances within the MAPK signaling pathway. This so-called Ele-Cys methodology
utilizes isoform-specific covalent inhibitors that engage with a cognate, sensitized RAF protein,
enabling users to probe into the mechanistic subtleties of RAF kinase biochemistry. While we
demonstrate that several under-explored niches in RAF research are amenable to our approach, we
ultimately envision our system providing an avenue into unraveling some of the mysteries of
inhibitor-induced RAF dimerization and subsequent drug resistance observed in RAF-implicated
xii

lesions. Carefully deconstructing the “transactivation” model that has been proposed to account
for this phenomenon requires exquisite control over inhibitor occupancy and dimer permutation –
both of which we attempt to navigate with Ele-Cys supplemented by the appropriate biochemical
technologies.

1

Chapter 1: Covalent ligands: indispensable instruments in drug discovery and chemical
biology

1.1 A resurgence of interest in drugging via covalent inhibition
Qualms about covalency in the face of industrial prevalence
Traditional drug discovery programs have been historically marred by an overall reluctance
to pursue the intentional development of small-molecule therapeutics operating by way of covalent
modification of their targets. Indeed, it seems that the general disposition of medicinal chemists
and pharmacologists is biased against the permanence of such an irreversible interaction.
1,2

Perhaps the origin of such fears can be traced back to some pioneering toxicological studies in the
early 1970s, in which hepatotoxic properties of some common drugs – acetaminophen,
bromobenzene, etc. – were linked to the covalent modification of liver proteins by highly reactive
drug metabolites.
3,4
Since then, there has been a pervasive attitude within the drug development
community: drugs that reversibly engage with proteins likely represent a superior drug class, as
they are unlikely to present similar risk factors as their covalent counterparts.
5
Interestingly enough,
this attitude amongst the community does not necessarily reflect upon the actual representation of
drugs acting through covalent mechanisms in the industry. On the contrary, covalent drugs have
enjoyed quite a profound presence in the clinic, as measured through both economic and
pharmacological standards.
6
Of the former, covalent drugs (e.g., esomeprazole, lansoprazole, and
clopidogrel) consistently feature in top-selling drug lists in the United States and have collectively
2

been valuated as netting multiple billions of dollars worldwide annually.
7
Regarding the latter, an
extremely wide variety of therapeutic indications are treated by covalent inhibitors, ranging from
cancer to gastrointestinal disorders, infectious diseases, nervous system disorders, and more.
8,9

Almost all types of proteins can be found as targets in these applications, including a plethora of
enzymes and membrane proteins such as proton pumps and even G-protein coupled receptors
(GPCRs).
10,11
In fact, it is estimated that more than one-third of all enzyme targets of FDA-
approved drugs are treatable by at least one covalent inhibitor.
12

The considerations mentioned above do not reveal hypocrisy from within our institutions.
Rather, our earliest covalent drugs were discovered serendipitously for their abilities to induce a
phenotype; elucidation of covalent mechanisms of action have generally occurred post hoc.
Fortunately, these considerations are also not implicative of obstinance; developments in recent
decades have driven a shift in the zeitgeist, such that covalent drugs are not only merely accepted,
but are the primary motivations behind many development projects.

Mechanistic/pharmacological features and consequent advantages of irreversible, covalent
inhibitors as drug candidates
Present-day acceptance of covalent drugging is best appreciated in light of the inherent
advantages that they offer, which ultimately stem from some unique pharmacological properties.
Mechanistically, the covalent inhibition event can be described by two sequential steps, wherein
an initial non-covalent binding event is followed by irreversible bond formation (Figure 1.1). The
first step is fundamentally identical to how traditional drugs engage their targets. The putative
3

inhibitor must first interact with the target protein (in this case, an enzyme) to form the enzyme-
inhibitor complex, represented by E•I. Inhibitor binding is described the inhibitor constant, Ki,
providing a measurement of binding affinity dictated by non-covalent interactions such as
hydrogen-bonding, ionic interactions, and Van der Waals forces. Formation of this complex usually
facilitates the placement of the inhibitor’s reactive moiety in proximity to a correspondingly
reactive residue of the enzyme. This promotes proximity-driven reactivity between these two
components and produces the final inhibited complex, E-I. Rate of terminal bond formation is
described by the kinact or k2, and is primarily governed by the intrinsic reactivity between
nucleophile (generally residues on the enzyme, such as cysteine, serine, and lysine) with
electrophile (of the inhibitor). If given enough time to react, irreversible covalent inhibitors will
yield stable covalent complexes, i.e., the kinact is significantly enhanced over the rate of the reverse
reaction, described by k-2. Conversely, reversible covalent inhibitors feature a k2 : k-2 ratio that
approaches unity, so that covalent complexes are transient and disturbed upon covalent bond
cleavage.

Figure 1.1 General mechanistic scheme of covalent inhibition.

The goals of any drug discovery program are hallmarked by two objectives: potency and
specificity. These two facets of drug optimization are discussed below.
4

Because covalent inhibitors proceed by a two-step mechanism, they provide two separate
avenues for honing drug potency. A major limitation of reversible inhibitors is that their binding
affinities (read, potencies) are determined entirely by the extent of their non-covalent interactions
with target protein.
13
Because these interactions are a function of the size of the molecule, they are
constrained by the upper bounds of ligand efficiency, assuming the molecule exhibits Lipinski-like
qualities.
14
Since drug-like molecules – at least, conventionally – do not exceed a molecular weight
of 500 Da (else they face complications of poor bioavailability, pharmacokinetics, bio-absorption,
etc.), reversible drug developers must maximize the value of each single heavy atom incorporated
in the drug structure.
15
This essentially forces an upper limit on drug potency, as even under perfect
conditions, only roughly 0.3 kcal of free binding energy can be achieved per mol of drug per heavy
atom.
16
Herein lies the first advantage of covalent inhibitors: not only can non-covalent
interactions be tuned to refine Ki, but the covalent reactive handle can also be adjusted to refine
kinact and lead to net improvements of overall inhibitor potency. Previous computational efforts
have reported that drug-enzyme complexes reliant on a covalent bond formation event routinely
exceed ligand efficiency limits, providing evidence that targeted covalent inhibition can improve
upon reversible, transient interactions in previously inaccessible ways.
17,18
An additional
advantage is that some molecular real estate of the drug can therefore be sacrificed since warhead
(i.e., electrophile) potency can potentially compensate for fewer non-covalent interactions. This
thus lends the opportunity for reaping the pharmacokinetic benefits associated with smaller drugs.
Finally, because irreversible covalent inhibitors have negligible off-rates, they tend to have
particularly high target residence times.
19
The upshot of which, is that prolonged and sustained
5

inhibition is possible, as catalytic output can only be restored upon protein turnover and not by
inhibitor disengagement of the active site. This has important implications for dosage regimens;
drugs with covalent mechanisms of inactivation may not require as frequent administration as
those acting non-covalently.
Selectivity of a covalent inhibitor is primarily dictated by structural interactions in the initial
binding step. Assuming high-enough degrees of complementarity between the drug and enzyme,
the lifetime of the E•I can be sufficiently large to ensure that the bond-forming step happens. Thus,
selectivity is optimized through a balancing act between Ki and kinact: relative to Ki, the warhead
must be sufficiently reactive so that the reaction proceeds within the window of E•I formation and,
simultaneously, sufficiently non-reactive to prevent indiscriminate reactions with intrinsically
nucleophilic residues across all biological -omes. Rational discovery programs, such as those
under the targeted covalent inhibition domain, navigate this issue by targeting specific nucleophilic
residues within target protein active sites that are poorly conserved in neighboring homologs and
paralogs.
20,21
By labeling rare, or even entirely unique, nucleophiles, inhibitors can display
exquisite selectivity over off-targets, even from those within the same protein family.
22
In the
undercurrent of these issues is a tacit expression of caution for warhead selection. The naïve
approach is to assume that the best warheads are the most reactive, but clearly this is not the case.
In fact, extreme judgment must be exercised to utilize warheads that are appropriately matched
with the goals of the development program (i.e., does it adequately fit the demands for target
engagement, does it lead to an acceptable off-target profile, among other considerations).
Accordingly, warhead development is a nuanced and rich field, which is constantly advancing in
6

the discovery of residue-specific/selective (including cysteine, lysine, methionine, and more)
electrophilic handles (Figure 1.2).
23–26

Figure 1.2 Representative panel of residue-selective covalent warheads. Semi-selective
covalent warheads and their targeted amino acids coded by color. Figure adapted from Dalton, S.
E. et al., ChemBioChem. 2019.
26

Prospective and outlook for the covalent drug landscape
Clearly, properly harnessing covalent inhibition is an excellent strategy for innovating drug
discovery. Despite an early vague hesitancy in adopting covalent drugs outright, the recent
trajectory for incorporating irreversible inhibition as a small-molecule modality is moving sharply
in the forwards direction.
27
More and more covalent drugs are being granted FDA approval, most
of which are not covalent by happenstance, but after rational design with the intention of effecting
7

an irreversible protein inactivation event, often leading to significant improvements in inhibitor
potency and efficacy. Several examples of this now abound, but perhaps the most landmark case
is the discovery of covalent KRAS
G12C
inhibitors, a target which was previously thought to be
“undruggable.”
28
In contrast to a more traditional approach for covalent drug development – one
which involves retrofitting reversible inhibitor scaffolds with covalent warheads – the KRAS
program was initiated from a screen of small reactive fragments. The final inhibitor structure was
built around leads from the screen, eventually culminating in a molecule that is remarkably
effective against a protein which does not contain any pronounced binding pockets.
29,30

Perhaps one of the most important indications in support of covalent inhibition as a new
paradigm in drug discovery is the advent of proteomics as a complementary discipline. As
mentioned above, covalent drugs are primarily plagued by worries of off-target reactivity. However,
due to chemoproteomic screening methods, most of those worries have become invalidated. The
method essentially enables the complete proteomic profiling of reactive cysteines (or other
residues) which enables the direct identification of specific sites modified by a candidate drug
molecule.
31–33
In the words of Ben Cravatt, perhaps the major pioneering figure, this allows for a
“demystification” of the covalent drug discovery process that enables very rational decision-
making with respect to inhibitor design. Strikingly, this methodology arguably elevates covalent
drugs over reversible ones, since direct detection of reversible, non-covalent interactions is not
possible in a likewise manner. The steady rise of covalent inhibitors in the past decade, coupled
with the emergence of novel technologies reinforcing their utility potential, suggests a deep-set
position for their future in pharmacology.
34

8

1.2 Probing cellular events and processes with biological and chemical techniques
Classical forward and reverse genetic screens
Cells are host to a wide range of highly dynamic molecular events including gene expression,
intracellular transport, cell division, organelle biogenesis, among an impossibly long list of other
processes.
35,36
The manifestation of these events can depend on cues that are both internal and
external to the cell, ultimately altering the coordinated activities of an ensemble of
biomacromolecules. At the heart of a developed organism’s ability to perform complex tasks is a
correspondingly complex network of biochemical events arising from a vast diversity of DNA,
RNA, and proteins. For all the functional diversity present within human cells, there are only about
30,000 protein-coding genes in the human genome, a mere five-fold increase compared to the yeast
genome.
37
Two primary mechanisms account for this rich array of cellular activities: RNA splicing
and post-translational modifications (PTMs) of proteins, both of which constitute a diversity-based
mechanism of genomic evolution, by which relatively few genes can yield rich legions of gene
products with manifold structural and catalytic responsibilities. While this is advantageous from
an evolutionary perspective, it presents a scientific challenge for molecular biologists. That is, the
inherent familial similarities widespread in the genome, transcriptome, and proteome can
convolute the physiological roles of specific macromolecules such that the precise function of any
given biological entity can be difficult to relate to a cellular phenotype.
Traditional approaches in classical molecular genetics have been instrumental in discovering
relationships between genotype and phenotype. Classical genetic screening methods are divided
into two categories: forward and reverse (Figure 1.3).
38
Generally speaking, both methods involve
9

the detection of phenotypic variations and subsequent correlation of those qualities to either
induced/site-directed or random changes in nucleic acid sequences. However, the two methods
diverge at the level of systems perturbation and direction of investigation.

Figure 1.3 Comparison of classical and chemical genetic philosophies. Figure adapted from
Kawasumi, M. et al., J. Invest. Dermatol. 2007.
38

At its most bare, forward genetics screens begin with the identification of a phenotype-of-
interest, often arising from random mutation after exposure of a model organism to mutagens (e.g.,
ethylmethanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), radiation, and others).
39
The
affected gene sequence responsible for either loss-of-function or gain-of-function phenotypes can
then be identified using traditional gene mapping techniques, through either genome-wide
association studies (GWAS) or newer screening methods reliant on DNA barcoding.
40
Validation
experiments can then be performed – typically carried out by various complementation tests – to
ensure that the observed phenotype is indeed determined by the putative genotype.
41
By contrast,
reverse genetics screens – as is implied by its name – operate in the opposite direction and begin
10

with intervention at the genomic level.
42
Due to recent advances in molecular genetics
methodologies, robust and specific genetic manipulations are possible, such that researchers may
execute exact edits of a gene-of-interest and relate consequent phenotypes to the modification
applied. Some technologies include site-directed mutagenesis for targeted deletions and point
mutations, RNAi-induced gene silencing, and CRISPR-Cas9-induced gene knockdowns and
knock-ins.
43,44

Certainly, the major strength of the classical genetic methods described above is the robust
nature of the screens. Specific changes of a single or small clusters of nucleotides can be related
precisely to apparent dysregulation at the protein level. However, some major shortcomings of
these screening strategies present problematic obstacles for garnering a complete picture of cell
physiology (Figure 1.4). First, traditional RNA silencing and knockout methods are slow
perturbations, sometimes requiring a timescale of hours or more to achieve the genetic
alternation.
45
This is not suitable for studying fast, dynamic processes; intervention methods
operating across longer stretches of time are “mis-matched” with most biological processes of
interest, such as PTMs and signal transduction events, which often occur within milliseconds to
minutes.
46
Additionally, classical genetic methods which entail wholesale loss of a protein by
knockdown or knockout are not able to adequately uncouple the enzymatic and structural functions
of a protein.
47
In other words, it can be difficult to conclude from the absence of a protein whether
an emerging phenotype is a result of an absence of catalysis (assuming the protein-of-interest is
enzymatic) or an absence of a critical protein-protein interaction (PPI).

11

Figure 1.4 Relationship between specificity and timescale of assorted methods for perturbing
protein function.

Emergence of chemical-genetic interventions by small-molecule modulation
An alternative approach to perturbing biological systems has become more feasible in recent
decades due to advances in chemical biology.
48
The fundamental chemical biological philosophy
is to use small molecules as tools to interrogatively probe a system under study.
49
Because proteins
have evolved in a manner to bind small-molecule partners (i.e., cofactors and ligands) to execute
their functions, they are amenable to binding events between themselves and rationally designed
cofactor/ligand mimetics. This is the guiding principle behind chemical genetics: as an alternative
to classical genetic screening methods, chemical genetics harnesses the dependence of proteins on
small chemical entities as a perturbation strategy rather than by direct genetic manipulation.
50–52

Small molecules constitute an immediate solution to the previously mentioned limitations of
classical genetics. For one, small-molecule modulation of protein targets takes place on a kinetic
scale that is better aligned with the physiological events in a cell.
53
Instead of being constrained to
12

a lengthy knock-out/knockdown procedure, protein function can be modulated rapidly by small
molecule probes, as they are bound only by membrane permeability properties and diffusion
limits.
54
The second point is that phenotypic variation resulting from small-molecule modulation
can be readily attributed to either catalysis or structure unambiguously. Because probes tend to be
tailor-made towards a specific type of perturbation (i.e., inhibiting catalytic activity vs. disrupting
a PPI), resultant effects in the organism/cell can be traced back to the properties of the small
molecule, so long as they are properly characterized. A classic example demonstrating this
advantage involves a study performed on Aurora kinases, which are involved in cell cycle
regulation by acting within spindle assembly checkpoint pathways.
55
In this work, small-molecule
inhibition specifically inhibited kinase catalytic activity of Aurora kinases while leaving its
scaffolding functions intact, thereby uncoupling activity from function.
56

Although chemical genetics certainly offers numerous advantages over classical genetics, the
description laid out above is slightly rose-tinted. Because of the diversity-based mechanisms
driving protein evolution described previously, members within a protein family often share the
same or similar co-factors and may be highly homologous in their ligand binding sites. In light of
this, the polypharmacological properties of small-molecule modulators present a significant
obstacle to successful application. In order for a chemical genetics approach to truly offer utility,
a researcher’s toolbox must be outfitted with inhibitors exhibiting exquisite isoform/paralog
specificity.

13

Allele-specific chemical genetics
One simple strategy for achieving a high degree of isoform selectivity is to iteratively optimize
ligand/inhibitor chemical structure to better establish complementarity with the protein target.
Despite marked improvements in the field of organic synthesis over the last century, this is still
not (and may never be) a viable approach, in isolation. Instead, a preferable solution is the
simultaneous optimization of the both sides of the protein-ligand interface. This strategy entails
engineering a protein by mutagenesis to create artificially high, mutual affinity with a matching
synthetic cofactor/ligand analog. The alterations are applied in a way such that the engineered pair
remains biochemically competent but is orthogonal to the native system. Mechanistically, the
design principles conferring allele specificity ultimately rely on introducing new interactions
(covalent and non-covalent) that are absent in wild-type proteins. Accordingly, this methodological
blueprint is referred to as allele-specific chemical genetics (ASCG).
57,58

Figure 1.5 Allele-specific chemical genetic strategies for engineering specificity. Inhibitor
selectivity is poor in A) native, wild-type systems, but can be engineered through the introduction
of orthogonal B) steric or C) covalent complementation.

14

Many flavors for introducing complementarity abound, though by far the two most common
design principles are centered on either an engineered steric or covalent interaction (Figure 1.5).
The steric approach, known colloquially as the bump-hole method, was first developed by
Schreiber and colleagues for studying the cyclosporine-cyclophilin interface and later pioneered
by the Shokat group in their applications on protein kinases.
59,60
Briefly, this method relies on
manipulating hydrophobic interactions between a protein and its ligand by the replacement of
bulky hydrophobic residues within a ligand-binding pocket with amino acids possessing smaller
side chains. The alteration results in a void space or “hole” that can be complemented by a ligand
appended with a correspondingly large hydrophobic substituent or “bump.” The modified ligand
thus restores some of the interactions lost by the native cofactor, increasing its relative affinity to
the engineered protein. On the other hand, covalent complementation can be achieved by the
exogenous introduction of a reactive cysteine (or any nucleophilic/reactive side chain), which
sensitizes the engineered protein to a ligand possessing a corresponding electrophilic warhead.
61–
63
Reaction between the engineered pair produces a covalent adduct that is otherwise impossible
with the analogous endogenous components, as the wild-type protein lacks a cysteine residue in
the equivalent position. This method has enjoyed widespread success in creating kinase-ligand
complementary pairs, wherein exchange of residues on the hinge region for cysteines renders the
kinases reactive towards electrophile-containing inhibitors.
64
Strategies relying on covalent
complementation between an electrophilic ligand and a reactive cysteine are appropriately named
“Ele-Cys” approaches.
65

15

1.3 Conclusions and outlook
The concepts introduced in this chapter serve as a focal point for our research. Recent trends
suggest permanence and an upwards trajectory for covalent inhibition in drug discovery. We sought
to capitalize on this and perform a forward pharmacological screen of reactive, covalent molecules
against important novel coronavirus proteins. This led us to the discovery of an irreversible binding
interaction between a viral polymerase (Nsp12) and several nucleotide analogues bearing covalent
warheads. These molecules were culled for their ability to induce antiviral effects and thus
represent potential therapeutic candidates for treating viral infections. Simultaneously, we
performed traditional drug optimization of peptide scaffolds reported to be potent inhibitors of a
separate viral target (Nsp5). We explore the impact of appending substrate mimics with latent,
irreversible warheads and ultimately produce enhanced covalent inhibitors and activity-based
probes for this viral protease. The latter body of our work features an allele-specific chemical
genetics study, made possible by engineering kinases to be sensitized against covalent inhibitors
serving as functional proxies for various clinical drugs. We apply this technology for the
deconvolution of drug-resistance mechanisms and demonstrate its utility in the functional
dissection of a wide range of related unsolved biological problems.

16

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22

Chapter 2: Rational discovery of covalent inhibitors targeting viral-cycle-critical SARS-
CoV-2 non-structural proteins

2.1 Introduction
2.1.1 Emergence of the COVID-19 global pandemic
Late in 2019, several health facilities in Wuhan, China reported the first appearance of unusual
pneumonia-like cases that were vaguely reminiscent of patients diagnosed with severe acute
respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).
1
These two diseases
were caused by highly pathogenic coronaviruses (SARS-CoV/MERS-CoV) that ultimately spread
to reach epidemic status in 2002 and 2012, respectively.
2,3
Patients in Wuhan displayed symptoms
typical of respiratory viral illnesses, including fever, dry cough, dyspnea, and chest pain.
4
On New
Year’s Eve of 2019, the Wuhan Municipal Health Commission publicly declared a pneumonia
outbreak – which, at the time, had an unknown cause – and alerted the World Health Organization
(WHO) of a potential epidemic/pandemic threat.
5
Isolation of the virus from bronchoalveolar fluid
samples of patients with pneumonia-like symptoms enabled metagenomic RNA sequencing, which
allowed independent groups of researchers to eventually determine that the causative agent was an
unprecedented, unknown betacoronavirus.
6,7
Complete genome sequences of this betacoronavirus
were determined as early as January 12 and were published on the Virological website and
documented in the GISAID database.
8

Early attempts to track the origin of the betacoronavirus led to the discovery that 27 of the
23

earliest hospitalized patients were linked to the Huanan Seafood Wholesale Market, known to sell
seafood, live animals, and other wildlife.
9
Despite this, the true genesis story for the virus has been
obscured by several competing datapoints, including an early report from France detailing positive
identification of the virus by PCR from a patient’s saliva sample in late 2019.
10
Regardless, less
than one month after the initial Wuhan cases, infections presumably caused by the novel
coronavirus had appeared in as many as 34 provinces in China. Evidently, the virus was capable
of rapid human-to-human transmission, likely exacerbated by the increase in city-to-city travel in
anticipation of lunar New Year festivities.
11
This prompted the WHO to declare the outbreak a
public health emergency of international concern on January 30.
12
By February 11, the infectious
disease was named ‘COVID-19’ and was established as being caused by the ‘SARS-CoV-2’ novel
coronavirus.
13
Thanks to very severe early containment measures in China – which included travel
and gathering restrictions – daily number of new cases were on the decline by late February and
essentially controlled in March 2020.
14
However, elsewhere in the world, the abundance of
international travel had spurred unchecked propagation of the virus. By August 2020, 216 countries
from all 6 continents globally had reported more than 20 million cases of COVID-19, with over
700,000 documented deaths.
15
As of July 29, 2022 – over 2 years after the first reports of the
disease – there have been over 550 million documented COVID-19 cases globally and nearly 6.5
million deaths and is still considered an ongoing pandemic.
16

2.1.2 Coronaviruses and SARS-CoV-2 pathogenesis
Generally, coronaviruses of the family Coronaviridae have been associated with respiratory
24

or intestinal infections in humans and other animals, though with varying severities amongst
different strains.
17,18
They have been so named due primarily to a historical quirk, as electron
microscopy was the primary means of characterization in early-day virology. Indeed, viruses in
this family contain a circlet of protein protrusions and resemble a crown-like structure.
19
Originally,
coronaviruses were categorized into groups based on their antigenic relationships. However,
contemporary classification includes four genera of the alpha-, beta-, gamma-, and delta-
coronaviruses.
20
Across the four genera, coronaviruses are, by majority, common pathogens that
are endemic among humans. The most prevalent strains of human coronavirus (NL63, 229E, OC43,
and HKU1) infect cells found within the upper respiratory tract and cause common cold
symptoms.
21
The three coronaviruses responsible for the outbreaks described above originate from
zoonotic transfer events occurring due to spillover from animal reservoirs.
22
In contrast to the more
benign coronaviruses, these three strains can replicate within the lower respiratory tract and lead
to more severe respiratory infectious diseases such as potentially fatal acute respiratory distress
syndrome (ARDS).
23

Strikingly, among all known RNA viruses, coronaviruses possess the largest genomes, ranging
from ~26 kbp (HKU12) to ~32 kbp (SW1).
24
SARS-CoV-2 is a novel and distinguished
betacoronavirus with a positive sense, single-stranded 29 kbp genome that shares 79% genome
sequence identity with SARS-CoV and 50% with MERS-CoV .
25
The viruses found within this
genus share similar genomic organization characterized by six functional open reading frames
(ORFs) encoding for the following elements: replicase (ORF1a/ORF1b), nucleocapsid (N), spike
(S), envelope (E), and membrane (M). Additional accessory proteins are also interspersed between
25

the S, E, M, and N structural genes (Figure 2.1B).
26

Figure 2.1 Schematic diagram of the SARS-CoV-2 viral particle and genome. A) Four
structural proteins, N, S, M, and E, provide structural stability to the virus and create a particle that
resembles a crown. B) SARS-CoV-2 genome illustrating the non-structural proteins encoded by
ORF1A and ORF1B. Cleavage sites of S protein are detailed. Figure adapted from Zhang, Q. et.
al., Signal Transduct. Target. Ther. 2021.
27

These structural elements give rise to a large, spherical viral particle that contains obvious
surface projections (Figure 2.1A). The SARS-CoV-2 viral particle is very typically sized –
measured at about 100 nm – rendering it susceptible to N95 filtration.
28
Within the interior of the
virus is the long ssRNA genome packaged by N proteins. Of the SARS-CoV-2 structural proteins,
very little is known about the N protein, likely due to its highly disordered nature, which presents
a major barrier to conventional biophysical characterization. Recently, the N proteins have been
shown to undergo phase separation with RNA, which may underlie its ability to organize and
compact the rather long genomic molecule.
29
These internal components are enveloped by a lipid
26

bilayer, which is derived from the endoplasmic reticulum (ER) of host cells.
30
Embedded within
the membrane are the better-studied M and E proteins, responsible for the maintenance of overall
particle size and preservation of structural integrity. Between the two, M proteins are considered
the main structural element and are type III membrane proteins, with a prominent triple-spanning
transmembrane domain that orients a large C-terminal matrix-like lattice towards the cytosolic
interior of the protein.
31
Interestingly, M proteins have been noted to be homologous to the
prokaryotic sugar transport protein, SemiSWEET, and was suggested to play a part in regulating
S protein glycosylation, in addition to its more traditional structural roles during viral assembly
and budding.
32
E proteins, on the other hand, are thought to provide only minor contributions to
the virus exterior. They contain an extracellular C-terminal domain and are most known for their
high α-helical content, which constitute a pentameric transmembrane domain that resembles an
ion channel.
33
There is some functional redundancy between M and E proteins, though the latter
has been shown to act as a viroporin, disrupting ion homeostasis in host cells and facilitating viral
release.
34

Perhaps the best-studied structural SARS-CoV-2 element – certainly, the component that has
received the most attention in popular science/medicine contexts – is the S protein and represents
the now-distinguishing visual signature of the virus. The S proteins are homo-trimeric class I
fusion glycoproteins that can be divided into two functional domains, named the S1 and S2
subunits.
35
Normally, S proteins exist in a dormant, metastable prefusion conformation that
undergoes extensive rearrangements during host cell fusion. The initial host interaction is mediated
through the extracellular N-terminus found within the S1 domain, containing the receptor-binding
27

domain (RBD) which features a signal peptide that specifically engages host cell receptors.
36
The
transmembrane S2 domains contain heptad repeat regions and a fusion peptide that enables
progression of endocytosis upon radical reorganization of S protein tertiary structure. Due to its
ability to engage with host cell receptors, S proteins are the major determinant of cellular tropism
and initiate the infection cycle.
37

SARS-CoV-2 targets the same host cell receptors as SARS-CoV , namely the angiotensin-
converting enzyme 2 (ACE2) receptor found attached to the cell membrane of enterocytes located
in the small intestine, duodenum, liver, testis, and kidney.
38
Surprisingly, though ACE2 receptors
are found within the respiratory system in upper bronchial and nasal epithelia, their expression is
relatively limited compared to alternate locations in the human body.
39
Despite targeting identical
entry points, SARS-CoV-2 S has been shown to bind human ACE2 with much greater affinity than
SARS-CoV S, exhibiting about a 20-fold improvement in its dissociation constant. Once receptor
engagement has occurred, viral infection requires the action of host cell proteases to progress viral
fusion. Specifically, through the coordinated enzymatic activities of transmembrane protease
serine protease 2 (TMPRSS2), cathepsin L (catL), and furin, host-bound S proteins become
cleaved, separating the S1 and S2 subunits and enabling uptake at the endosomal membrane.
40

Following entry, genomic SARS-CoV-2 RNA is released and uncoated, which allow
immediate translation of ORF1a and ORF1b by host cell machinery to produce the pp1a and pp1ab
polyproteins, respectively (Figure 2.2). These contain 16 total non-structural proteins (Nsps), for
which Nsps1-11 are translated from the pp1a cassette.
41
The latter 5 Nsps are encoded by
polyprotein pp1ab, which results from a programmed -1 ribosomal frameshift. Interestingly,
28

ribosome profiling has revealed a frameshift rate between 45-70% in SARS-CoV-2, which
determines a polyprotein stoichiometry such that there is a rough two-fold excess of pp1a.
42
Co-
translational and post-translational processing of the entire Nsp1-16 cassette produces the
individual proteins that constitute the viral replication and transcription complex (RTC).
43
This is
critically executed through the actions of two viral cysteine proteases: the main protease (M
pro
,
also known as Nsp5) and the papain-like protease (PL
pro
, which is the protease domain of the multi-
functional Nsp3).
44,45
Through independent amide-bond hydrolysis responsibilities at defined
cleavage motifs in both polyprotein substrates, the two proteases bring about the maturation of the
functional replicase.

Figure 2.2 Coronavirus virion and life cycle. Following ACE2-receptor-mediated entry (1) into
cells, the viral particle uncoats and releases its genome (2). ORF1A/B translation (3) occurs
immediately to produce the non-structural proteins of the replisome, upon cleavage by the main
protease. Further genomic replication then proceeds (4a-5), which results in the translation of viral
29

structural proteins that translocate to the ER (6). Associations between the structural proteins and
nucleocapsid-complexed genomic material (7) leads to the formation of a mature virion (8), which
is released by the host cell via exocytosis (9). Figure adapted from Burmer, G. LifeSpan
Biosciences, Inc.
46

Polyprotein processing begins with a rapid release of Nsp1, which enables it to perform its
roles in targeting host-cell translation machinery and suppressing the innate immune response.
47

This is followed by the core components of the RTC, Nsps2-16, which collectively play parts in
RNA synthesis, proofreading, and modification. Replication of the viral genome is carried out by
the RNA-dependent RNA polymerase (RdRp, Nsp12), which require cofactors with primase and
3’-terminal adenylyltransferase activity (Nsp7 and Nsp8).
48
Proofreading functions are mediated
by a 3’-5’ exonuclease (Nsp14).
49
Finally, the lesser understood RNA modifications are performed
through the combined actions of the capping machinery, while include a cofactor (Nsp10), 5’-
triphosphatase (Nsp13), N7-methyltransferase (Nsp14), and 2’-O-methyltransferase (Nsp16).
50

The combined efforts of the RTC will culminate in the synthesis of newly formed RNA genomic
material that, upon encapsidation with translated N proteins, migrates to the ER-to-Golgi
intermediate compartment (ERGIC), which is formed from the combination of translated structural
proteins and host lipid/endosome material. The coalescence of these ingredients triggers budding
events in the lumen of secretory vesicular compartments that will ultimately be released from the
host cell via exocytosis.

30

2.1.3 Overview of current antivirals, therapeutics, and prophylactics used against SARS-
CoV-2
Discrete steps in the SARS-CoV-2 replication cycle represent promising inlets as targets for
antiviral development. Some of the most critical biochemical events that are conducive to viral
proliferation include the entry/fusion phase, polyprotein processing, genomic replication, and
virion maturation/budding.
51
As such, most antiviral drugs are designed with the intention of
interfering or disrupting these viral life cycle components.
It is worth briefly mentioning the parallel evolution - at least in terms of timeline, though also
by rate of development progress – of COVID-19 vaccinations. There was an immediate surge of
urgency for producing effective antivirals, primarily for the purpose of addressing patient care for
those who have already been infected. Such was the impetus for antiviral development, as small-
molecule therapeutics were required to lessen the load faced by hospitals and medical care
providers. However, the endgame for addressing a viral pandemic had always hinged on herd
immunity and/or vaccination. The earliest vaccines developed came from Pfizer-BioNTech and
Moderna, both of which utilized mRNA encoding the S protein, and are both widely in use today.
52–
54
Meanwhile, Johnson&Johnson had also developed an adenovirus vector-based vaccine which
had slightly lower reported efficiency in protecting against SARS-CoV-2 infection.
55
Most recently,
Novavax has offered their own contribution to the arsenal of available vaccines, providing a novel
nanoparticle formulation of the S protein itself.
56,57

The rationale behind preventing entry of viral particles is intuitive enough: if the virus is
unable to access the cell, it will therefore be unable to replicate its genome and propagate. Entry
31

inhibition features mechanistic principles which are bipartite. First, therapeutics which can attach
to the viral particle may neutralize or preclude productive interactions between the S protein and
host cell receptors (e.g., ACE2). Second, enzymes which are critical for the progression of viral
fusion and uptake may lead to stalled complexes between virus and host cell upon inhibition.
Attractive targets of this second class include host-cell proteases, which are responsible for
cleaving S proteins and enabling conformation rearrangements necessary for endocytic
competence. Of the former, various classes of molecules have been developed and are at various
stages of approval for clinical use. These include immunologic-based therapies (monoclonal
antibodies, single-domain antibodies, and polyclonal antibody preparations) and soluble ACE2
“decoys.”
58,59
Antibody-based therapies can not only neutralize invading viral particles but can
also recruit host effector pathways to destroy virus-infected cells. Some of the more successful
examples include casirivimab, bamlanivimab, and cilgavimab.
60–62
ACE2 decoy strategies employ
mimics of the ACE2 ectodomain and have been shown to bind to SARS-CoV-2 particles via S as
tightly as monoclonal antibodies.
63
Some recent avenues of optimization for ACE2 decoys involve
the identification of minimal binding motifs, producing smaller and smaller regions of ACE2 that
can out-compete viral attachment to the full-length receptor.
64
The latter class of entry inhibitors
feature several small-molecule protease inhibitors originally used for other indications. These have
been adapted and repurposed to prevent S protein release and subsequent transitions to a fusion-
ready state. Noteworthy examples include camostat and nafamostat, which are serine protease
inhibitors that have been documented to inhibit TMPRS22.
65,66
Additional TMPRS22 inhibits in
earlier stages of approval are Alpha-1 antitrypsin (α1-AT), aprotinin, and bromhexine.
67,68

32

Early attempts at SARS-CoV-2 antiviral discovery were rather disjointed and hurried, largely
due to the urgency for finding viable therapeutics that could retard pandemic progression. Many
molecules purported to have antiviral effects emerged during these initial large-scale screening
efforts – some of which have been obscured by exaggerated reports of potency or are backed by
questionable evidence – which are best characterized as drug repurposing campaigns.
69–71
The idea
was that a pathway to clinical use after discovery could be expedited if positive leads had pre-
existing FDA-approval. While some of these putative leads have been debunked, the remaining
candidates often fall into a class of therapeutics for which their mechanisms of action are unclear
or unknown. Some interesting examples, for both scientific and historical reasons, include
chloroquine, niclosamide, clofazimine, emetine, and ivermectin.
72–75

By far, the two most popular classes of antivirals are those that act on the RdRp (Nsp12) or
the proteases (primarily Nsp5 though to some extent, Nsp3 as well). These small molecules
compromise the integrity of the viral replication machinery and prevent plain-sailing progression
of the viral infectious cycle, either through the prevention of genome replication or polyprotein
processing, respectively. These two viral targets will be the subjects of this chapter and are
discussed in more detail in the following sub-sections.

2.1.4 Perspectives and roadmap of development
The severity of the SARS-CoV-2 pandemic is somewhat unprecedented, at least in recent
memory, and is set to have lasting impacts on global society and culture. At the moment, the
prospective outlook on COVID-19 is uncertain; it is difficult to predict our ability to contain the
33

virus (against newly emerging variants, among other concerns) and the prospect of future
pandemics arising from similar (or worse yet, dissimilar) virus spillover is both frightening and
real.
76,77
As such, there is a pressing need to advance our arsenals in the evolutionary arms race of
host versus virus. For SARS-CoV-2 specifically, we, and others, have fixated on two viral targets
that invite inhibitor development due to their critical roles in mediating the viral life cycle. These
are the Nsp12 RdRp and the Nsp5 main protease proteins. The following sub-sections detail the
opposite, yet complementary, approaches that we have taken in the development of covalent,
irreversible inhibitors of either viral protein. Our work on Nsp12 features a forward
pharmacological screen of reactive small molecules possessing covalent, electrophilic warheads
that were observed to induce positive antiviral phenotypes. After confirmation of efficacy, we
ultimately identified the RdRp as a bona fide target. Conversely, we pursued a traditional SAR
optimization strategy against Nsp5, utilizing tightly binding inhibitor scaffolds and systematically
evaluating potency enhancements after varying reactive covalent warheads.

34

2.2 SARS-CoV-2 Nsp12: RNA-dependent RNA polymerase

2.2.1 Introduction
Functional role of Nsp12 in coronavirus infection and proliferation
Following polyprotein cleavage and processing, various Nsps of the viral replisome assemble
to produce the machinery necessary for transcription and replication of genomic RNA. Among
them, Nsp12 is the core, catalytic component that presents RNA polymerization activity.
78
Studies
have demonstrated the ability of SARS-CoV Nsp12 homologs to exhibit polymerase activity
independently of any additional cofactors, though with very low efficiency. Upon stimulation by
Nsp7 and Nsp8 association, the polymerase complex enjoys a significant enhancement in catalytic
output.
79
The three-part complex of Nsp12, Nsp7, and Nsp8 is considered the minimal motif
required for SARS-CoV (and by extension, SARS-CoV-2) RNA synthesis. Additional auxiliary
replicase components, including Nsp10, Nsp13, Nsp14, and Nsp16, assemble the complete
holoenzyme, through which non-essential RNA synthesis functions are conferred.
80

Structural features of the SARS-CoV-2 replicase (Nsp7+8+12)
Various structures of the SARS-CoV-2 core polymerase complex have been proposed through
X-ray crystallography and cryogenic electron microscopy (cryo-EM) experiments.
81,82
Similar to
structural analyses performed on SARS-CoV RdRp components, various residues near the N-
terminal regions of Nsp12 and Nsp8 are unresolved and thus produce structural information
covering approximately 80% of the minimal polymerase complex.
83

35

The polymerase complex consists of the core catalytic component Nsp12 bound to an Nsp7-
Nsp8 heterodimer, with an additional binding event to a single Nsp8 subunit at a distinct site
(Figure 2.3A).
84
The C-terminal polymerase domain found within Nsp12 assumes traditional
polymerase structure, adopting a cupped “right-handed” configuration that is comprised of finger,
palm, and thumb domains (Figure 2.3B). Additionally, there is a nidovirus RdRp-associated
nucleotidyltransferase (NiRAN) domain within the Nsp12 N-terminal region that is shared by all
polymerases found in the Nidovirales order. The NiRAN associates near the back side of the
polymerase domain and creates an interface that bridges a connection to the finger domain.
85
The
polymerase domain itself contains seven essential catalytic motifs (labeled A-G), for which the
first six are highly conserved amongst all viral polymerases (Figure 2.3C). Motif G is considered
unique to positive-sense RNA viruses, which require additional interactions with the RNA primer
to initiate RNA synthesis. Nsp12 polymerase domains feature a hallmark fingertip, made from
motif C, that protrudes into the catalytic cavity and interacts with finger extension loops and the
thumb domain (Figure 2.3D). Interactions between the fingertip and finger extension are stabilized
by the Nsp7-Nsp8 heterodimer, which secures these loops mainly through the action of Nsp7.
Structural studies have shown that, in the absence of the Nsp7-Nsp8 heterodimer, this
fingertip+extension loop is highly disordered and can prevent nucleotide engagement.
86
The lone
Nsp8 subunit primarily provides a clamp near the top of the finger domain and provides
stabilization of the interface between NiRAN and the polymerase domain. Finally, there are two
metal-binding sites identified in the Nsp12 protein, comprised of cysteine and histidine residues
thought to coordinate with zinc ions.
87
Both of these sites are distinct from the catalytic site and
36

are accordingly hypothesized to provide structural support during the proper folding of the
polymerase domain.

Figure 2.3 Structures of the SARS-CoV-2 core polymerase complex and catalytic center. A)
Schematic representation of Nsp12 domain architecture and Nsp7/Nsp8 subunit interactions.
Domains are uniquely color-coded. B) Density map (left) and atomic model (right) of intact
complex. Black arrow depicts the finger extension and red arrowhead marks the fingertip on the
atomic model. C) Domain architecture of catalytic polymerase region. Catalytic motifs are
uniquely color-coded within the context of the fingers, palm, and thumb domains. D) Atomic
model of the RdRp catalytic active site. Figure adapted from Peng, Q. et. al., Cell Rep. 2020.
84

In terms of its catalytic function, RNA synthesis proceeds upon the entrance of template strand
via a chamber shaped by the finger extension loops and fingertip. This structure provides a guide
for the 3’ end of the viral RNA. The incoming nucleotide triphosphate (NTP) enters the catalytic
chamber from a channel behind the palm subdomain. The duplex formed between the template
and product strands exit the polymerase active site from the front and are unwound by other Nsp
subunits to yield the functional single-stranded genomic RNA.
88

37

Antiviral strategies targeting the replicase complex
A handful of inhibitors targeting the Nsp12 polymerase subdomain have been proposed, to
varying degrees of success. The unifying theme amongst Nsp12 drugs involves a structure that
mimics nucleotides (often, purine analogs are used) which “tricks” Nsp12 into incorporating the
drug molecule and produces a replication-hampering effect, either directly/immediately or
indirectly. For better or worse, the most popular example might be the monophosphoramidate
prodrug, Remdesivir (brand name, Veklury), which was the first FDA-approved drug used for the
treatment of COVID-19.
89,90
Remdesivir is a nucleoside analog, from which the active NTP form
is produced after triphosphorylation of the unveiled metabolite upon hydrolytic activation.
91

Antiviral activity of Remdesivir was demonstrated in cell culture and animals, though current
consensus holds that the drug is largely ineffective and does not offer any therapeutic benefits.
92–
94
The mechanism of Remdesivir was originally proposed to proceed via stalling of the RdRp.
95

Remdesivir would be incorporated into the elongating RNA strand, which allows subsequent
polymerization of three additional nucleotides before an eventual steric clash prevents further
strand translocation. This barrier arises from the effect of the C1’-cyano group, which clashes with
a serine residue in Nsp12. Some additional examples of purine analogs that have shown some
promise as COVID-19 therapeutics are Favipiravir and Ribavirin, both of which mimic the
structure of guanine nucleobases.
A newly emerging drug candidate targeting Nsp12 is Molnupiravir, which, like Remdesivir, is
a nucleoside derivative prodrug.
96
It is distinguished by an N
4
-hydroxycytidine nucleobase, which
is a pyrimidine mimic of cytosine. Originally developed as an influenza therapeutic, it is now being
38

used for COVID-19 treatment. In December 2021, it received emergency use authorization (EUA)
for individuals unresponsive to other treatment options, but only by a narrow margin. Interestingly,
the reason behind hesitancy to adopt Molnupiravir as a first-line therapeutic is related to its
purported mechanism of action.
97
In contrast to Remdesivir, which directly interferes with the
immediate ability of Nsp12 to elongate RNA, Molnupiravir compromises viral reproduction by
promoting mutations in the RNA genome via induction of increased error-rate in Nsp12. The
nucleobase of Molnupiravir contains an oxime motif that exists between two tautomeric forms,
one that mimics cytosine and the other, uridine. Importantly, Molnupiravir evades proofreading
activity of the replicase complex so that it becomes permanently incorporated into the newly
synthesized RNA strand. This drug-containing strand can serve as a template for future RNA
synthesis, such that the Molnupiravir nucleobase, equally existing in both tautomeric forms,
induces equal misincorporation of guanine or adenine. There are persisting safety concerns about
such a mechanism of action; inducing mutation may be accelerating evolution of drug-resistant
viral strains. Thus, there is a need for the discovery of safe and effective Nsp12 inhibitors.
In the following section, we outline a series of discoveries that led to the ultimate identification
of small molecules that covalently bind to Nsp12.

2.2.2 Results and discussion
2.2.2.1 Early indications of Nsp12 druggability
Screen and identification of Nsp12-interacting quinazoline inhibitors
During the early stages of the SARS-CoV-2 outbreak, there was relatively little information
39

widely known about viable drug targets and the structures of potential antiviral inhibitors. We had
previously developed several quinazoline-based covalent inhibitors possessing electrophilic
warheads of varying reactivities. These inhibitors had been proven to be highly potent against
enzymes such as vacuolar H
+
ATPase (V-ATPase), guanosine monophosphate synthase (GMPS),
and various kinases.
98,99
These enzymes all possess binding sites that are amenable to interactions
with adenine, guanine, and/or structural mimics of these nucleobases. We therefore hypothesized
that a broad screen with a small panel of quinazoline (structurally and electronically analogous to
purines) inhibitors against important SARS-CoV-2 replicase complex proteins could lead to the
discovery of promising viral protein-inhibitor pairings (Figure 2.4A).

Figure 2.4 Structural outline of quinazoline-based inhibitors/probes. A) 4-aminoquinazoline
scaffold structure. CuAAC-reporter groups (R) and electrophilic warheads (E) are appended at
variable positions. Commonly employed warheads used in experiments: CA=chloroacetamide,
AA=acrylamide, ITC=isothiocyanate. B) Structures of CA-bearing probes.

Our first attempts at screening experiments involved transient co-transfections of HEK293T
40

cells with Nsp7, Nsp8, and Nsp12 genes (components of the fully intact RdRp) containing a C-
terminal Twin-Strep-tag®.
100
We treated expressing and non-expressing cells with growth medium
supplemented with a batch cocktail of quinazoline inhibitors with chloroacetamide (CA),
acrylamide (AA), or isothiocyanate (ITC) warheads. Whole cell lysate and Streptavidin-enriched
samples were resolved by SDS-PAGE and visualized by in-gel fluorescence after CuAAC of
propargyloxy reporter groups with TAMRA-N3 (Figure 2.5). The data indicate that there is clear
Nsp12 (~106.7 kDa) engagement by the CA and AA cocktails (Figure 2.6A). Separate treatment
of replicase-expressing cells with the individual inhibitor components of the AA (Figure 2.6C) and
CA (Figure 2.6B) mixtures revealed mixed results. Although none of the individual AA-inhibitors
produced an obvious Nsp12-labeling result, several CA-inhibitors were successful in doing so.
Specifically, inhibitors with the warhead installed at the meta-position of the aniline ring (2.1) and
the 6- and 7- positions of the quinazoline core (2.2, 2.3, respectively) had pronounced Nsp12
labeling (Figure 2.4B). Encouraged by these findings, we performed dose-course evaluations of
2.1, 2.2, and 2.3 from whole cell lysate samples (Figure 2.5D). From these preliminary results, 2.1
displayed the best Nsp12 engagement behavior.
41

Figure 2.5 Schematic of CuAAC-mediated in-gel fluorescence analysis for covalently labeled
proteins. Irreversible labeling of a target protein by a small-molecule probe bearing a reporter
alkyne affords a covalent adduct. Reaction between the adduct and a fluorophore bearing a
complementary CuAAC-reactive counterpart produces a fluorescent covalent complex which can
be resolved and visualized by SDS-PAGE.

42

Figure 2.6 Identification of Nsp12-interacting covalent inhibitors. In situ labeling of SARS-
CoV-2 replicase complex was monitored after TAMRA-N3 CuAAC with cell lysate. Red bracket
indicates anticipated position of Nsp12 (~106.7 kDa). Quinazoline probes displaying poor labeling
followed naming convention of Q-x-yy, where Q=quinazole, x=position of electrophile, y=identity
of electrophile. A) Batch screening of inhibitor cocktails. Individual inhibitor concentrations:
CA=3 µM, AA=5 µM, ITC=10 µM. Labeling by individual components from B) AA and C) CA
inhibitor cocktails. D) Dose-response titration of best-performing compounds.

Optimization of on-resin click chemistry
Although preliminary experiments with samples generated from whole cell lysate were
intriguing, it was difficult to definitively pinpoint Nsp12-based fluorescence amidst the
43

background of non-specific labeling by the 3 inhibitors tested. We thus found it necessary to
optimize a pulldown procedure for Twin-Strep-tag®-containing proteins that is compatible with
CuAAC-mediated installation of a fluorescent reporter. Our initial experiments featured an attempt
at a Streptavidin agarose enrichment, but the fluorescence results were indistinguishable from
whole cell lysate conditions; enrichment efficiency was very low/essentially zero.
In the absence of the CuAAC reaction, we were able to enrich Nsp12, Nsp5, and Nsp8 proteins
from HEK293T cell lysate with very high purity (Figure 2.7A). Although Nsp7 was included in
the replicase co-transfection condition, it was unable to be resolved by SDS-PAGE due to its low
molecular weight.
Our suspicion was that the CuAAC reaction was somehow influencing the enrichment
efficiency and precluding the pull-down of pure Twin-Strep-tag® proteins (note that the CuAAC
reaction was performed before enrichment with Strep-Tactin® resin). To test this, we incubated
replicase-expressing cells with AA-inhibitors and evaluated enrichment quality by in-gel
fluorescence and silver stain. Protein samples were eluted from Strep-Tactin® by heat and biotin
competition (Figure 2.7B). Under both sets of elution conditions in the absence of CuAAC, Nsp12
is successfully enriched and highly pure. However, attempts at enriching Nsp12 after CuAAC were
unsuccessful, as evinced by intense smearing in the fluorescent and silver stain readouts.

44

Figure 2.7 Streptavidin enrichment of Nsp proteins and influence of CuAAC. A) Enrichment
profiles for mock, Nsp5, and Nsp7+8+12 expression. Anticipated PAGE positions indicated for
Nsp12 (red bracket), Nsp5 (33.8 kDa, black arrow), Nsp8 (21.9 kDa, black arrowhead). Nsp7 (9.2
kDa) unable to be resolved. B) In-gel fluorescence and silver stain profiles obtained after
Streptavidin enrichment of CuAAC-treated cell lysate. Gel samples were retrieved after either
heating or biotin elution from resin.

Next, we set out to dissect the CuAAC reaction under the premise that either an individual or
multiple CuAAC reagents were responsible for the poor enrichment. The reaction mixture contains
four separate components: tris(2-carboxyethyl)phosphine (TCEP), Tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (TBTA), CuSO4, and TAMRA-azide (refer to experimental details for
specific reaction conditions). We reasoned that systematic removal of an individual component
would reveal the compromising culprit (Figure 2.8A). Indeed, while removal of an individual
CuAAC component could not salvage fluorescence profiles, CuAAC cocktails lacking CuSO4
produced relatively clean enrichment of Nsp12. Armed with the knowledge that Cu
2+
/Cu
+
is
responsible for the low enrichment quality, we wanted to tune the CuAAC conditions to be
productive towards pure pulldown and enable visualization of a signal by fluorescence.
45

Unfortunately, these efforts were stymied in light of trying several conditions including: spiking
in excess copper-chelating agents (Figure 2.8B), altering the reducing reagent and chelating ligand,
and tuning ligand:Cu
2+
ratios (Figure 2.8C,D).

Figure 2.8 Systematic evaluation of CuAAC reagents on Strep-Tactin® enrichment.
Comparison of SDS-PAGE profiles by in-gel fluorescence and silver stain before and after
enrichment. Specific CuAAC conditions can be found in experimental details; modifications
included: A) Systematic removal of individual CuAAC reaction components. Agarose resin was
compared against Sepharose. B) Inclusion of Cu
2+
-chelating reagent for 15 minutes, post CuAAC,
C,D) Substitution of corresponding CuAAC component for indicated reagent. THPTA=(tris-
hydroxypropyltriazolylmethylamine).

Because of repeated failures at identifying CuAAC conditions that could be compatible with
Strep-Tactin® pulldown, we turned our attention to modifying the enrichment workflow to prevent
conflicting procedural conditions. We reasoned that we could circumvent problems caused by the
exposure of Cu
2+
/Cu
+
to the Sepharose resin by simply performing CuAAC after whole cell lysate
46

has been enriched. Reorganization of the protocol thus allowed for CuAAC to be performed on
enriched proteins only, which, based on previous enrichment data, would be exclusively proteins
containing a Twin-Strep-tag®. Surely enough, by performing an on-resin (post-enrichment)
CuAAC reaction, the enrichment efficiency was extremely high and produced only a single signal
corresponding to Nsp12 by fluorescence readout (Figure 2.9).

Figure 2.9 Post-enrichment CuAAC protocol for visualizing in situ labeling by fluorescence.
Three alternative CuAAC protocol workflows were evaluated for enrichment efficiency and in-gel
fluorescence readout. A = original CuAAC procedure: CuAAC of whole cell lysate, enrichment,
and heat/biotin elution. B = Enrichment of whole cell lysate after in situ inhibitor treatment,
CuAAC of enriched proteins on-resin, heat elution. C = Enrichment (identical to “B”), biotin
elution, and CuAAC of eluent proteins.

Characterization of 2.1 and 2.3 as Nsp12-binding small molecules
Having determined an optimized workflow that enables target engagement analysis of Twin-
Strep-tag®-bearing Nsp12 by covalent inhibitors, we set out to further characterize in situ binding
properties after protein enrichment. Using the revamped method, replicase-expressing cells were
subjected to dose-course treatment of either 2.1 or 2.3 and analyzed for target engagement by
47

fluorescence after CuAAC (Figure 2.10A). Both inhibitors appeared to label Nsp12 robustly and
dose-dependently, with 2.3 exhibiting labeling potency roughly 3-fold greater than 2.1. In an effort
to validate these results, we attempted a dose-dependent competition experiment, where replicase-
expressing cells are treated with a pulse of inhibitors 2.4, 2.5, or 2.6 lacking an alkyne reporter
group (Figure 2.10C), then subsequently chased by reporter-containing 2.1 or 2.3 after a PBS
washout (Figure 2.10B). The pulse-chase combination was selected based on complementary
warhead positions after applying a structural overlay. Although the conditions did not produce an
adequate fluorescent signal from probe 2.1, near-complete competition against 2.3 by 2.5 was
achieved at the highest competitor concentration used. The same result was observed with
competitor 2.6, though to a lesser extent because of the low resolution.

48

Figure 2.10 Characterization of quinazoline inhibitor labeling of Nsp12 by on-resin CuAAC.
Workflow B CuAAC (see Figure 2.8) adapted for producing labeling profiles. A) Dose-response
labeling of Nsp12 in situ by either 2.1 or 2.3. B) Target validation by competitive pre-treatment of
inhibitor analogs lacking CuAAC reporter groups. Treatment conditions in situ include dose-
course incubation of competitor, followed by PBS washout, and final incubation with 2.1 or 2.3.
C) Structure of structural analogs used in competition experiments.

Based on these results, we were confident that Nsp12 was being covalently labeled by probes
2.1 and 2.3 at a nucleophilic residue. We were interested in determining the specific reactive site
and proceeded to determine this by MS/MS. Briefly, replicase-expressing cells treated with either
probe were enriched, resolved by SDS-PAGE, and the gel slice corresponding to Nsp12 was sent
for MS/MS fragmentation analysis after tryptic digest (Figure 2.11A). Peptide fragments
containing a mass corresponding to a probe-adduct can reveal the site of modification (Figure
2.11B,C,D). Although initial attempts suffered from low or poor protein coverage, we were
49

eventually able to generate enough Nsp12-probe sample to identify C564 as a high-confidence
reactive site. Thus far, the results indicate that from a small panel of covalent purine analog
inhibitors, we were able to identify a pair of moderately potent Nsp12 binders and traced the
reactive residue to a cysteine located along the brim of the active site.

Figure 2.11 MS/MS determination of reactive labeled residue in Nsp12. A) Enriched Nsp12
after in situ treatment with probe was resolved by SDS-PAGE and processed for MS/MS. B)
Identification of peptide fragment containing probe-protein adduct revealed C564 as site of
modification. Fragment spectra for C) 2.1 and D) 2.3 adducts.

2.2.2.2 Forward pharmacological screens for SARS-CoV-2 antiviral compounds
Enhanced IFN activation in viral-infected cells treated with open-ring quinoline/isoquinoline-
derived inhibitors
Encouraged by the ability of quinazoline-based inhibitors to interact with Nsp12, we decided
to undertake a more serious investigation of their potential as antivirals for the treatment of SARS-
50

CoV-2. While simultaneously exploring binding characteristics of quinazoline inhibitors with
various electrophilic warheads, we were also attempting to remodel probe 2.2 based on some
intriguing labeling results with its protein target, GMPS. Specifically, from two analogs of 2.2 –
one with a quinoline core (2.7) and another with isoquinoline (2.8) – we determined that only 2.8
was capable of GMPS labeling, indicating that only a single nitrogen atom within the quinazoline
scaffold was necessary for binding (Figure 2.12A). This prompted us to dissolve the rigidity
present on the ring and synthesize an “open,” flexible analog 2.9, which contains a propargyloxy
reporter group for CuAAC downstream applications and 2.10, a non-reporter methoxy analog. For
comparison’s sake, an open-ring scaffold variant (in which the amide functionality is reversed) of
2.9 was also synthesized, named 2.11, and included in our miniature panel for future experiments
(Figure 2.12B).

Figure 2.12 Design of “open-chain” quinazoline analogs. A) Removal of nitrogen atoms within
quinazoline heterocycle affords quinoline 2.7 (red path) or isoquinoline 2.8 (blue path). De-
rigidification of isoquinoline analog produces an open-ring amide analog that preserves hydrogen-
bond donor/acceptor properties. B) Structures of non-reporter methoxy analog 2.10 and “reverse-
amide” analog 2.11 derived from 2.9.

51

Upon viral infection, it is understood that the transcriptional and replicative machineries of
host cells and viruses are rapidly churning in order to meet the high DNA and RNA synthetic
demands of viral propagation. Because the primary objective in the viral life cycle is to
spread/reproduce, it is natural that there is an up-tick in the activity of viral factors and hijacked
host cell factors that play a role in nucleotide turnover, both in its de novo biosynthesis and as a
substrate for oligonucleotide extension.
101
Accordingly, we hypothesized that small molecule
mimetics of purines and pyrimidines may serve as competitive inhibitors against enzymes that may
be critical for maintaining viral replication. In collaboration with the Feng lab (USC School of
Dentistry), we performed a preliminary screen of quinazoline-derived inhibitors to assess their
potential as antiviral agents. Previous results from the Feng lab had revealed that the expression
of various SARS-CoV-2 polypeptides blunted the induction of pro-inflammatory cytokines – such
as interferon-β (IFN-B) – and were thus able to suppress and evade host cell innate immune
responses.
102
We reasoned that one marker for an effective antiviral would consist in its ability to
stimulate and/or rescue IFN-B production (or any other related cytokine) upon viral infection. Our
initial hypothesis was that a candidate antiviral would inhibit the activity of SARS-CoV-2
polypeptides exerting an inhibitory effect against cytokines or would be able to directly stimulate
cytokine production. A549 lung cancer cells were infected by Sendai virus (SeV), a prototypical
RNA virus, which was utilized as a proxy for SARS-CoV-2 infection and the relative levels of
IFN-B mRNA transcription were measured. After dose-course treatment with 2.9, 2.10, and 2.11,
the results somewhat surprisingly indicated that all three molecules exhibited a dose-dependent
increase in IFN-B transcription, compared to baseline SeV infection alone (Figure 2.13). This
52

result was especially pronounced in the highest dose of 2.10. Of note, only the three open-ring
quinoline and isoquinoline analogs produced this effect; quinazoline inhibitor 2.2 was not able to
stimulate cytokine production to any extent.

Figure 2.13 Open-ring inhibitors promote an innate immune response upon viral infection.
A549 cells infected by Sendai Virus (SeV) were measured for innate immune activity by IFN
reporter assay. DON = 6-diazo-5-oxo-L-norleucine, glutamine antagonist, previously shown to act
on the RIG-I pathway, serves as a positive control.

Design, synthesis, and characterization of first-generation putative antivirals
Following the success of our first immunology-based antiviral screens, we decided to launch
an SAR optimization of molecules inspired by 2.9 and 2.10, in the hopes of augmenting their
immune-boosting effects (Figure 2.14A). Our initial strategy was to simply modify the substitution
patterns present on the aniline ring, yielding para-methoxy (2.14) and ortho-methoxy (2.15)
analogs. In order to investigate the effect of heterocycle identity at this position, we included an
analog (2.16) which has an imidazole ring in place of the aniline. We also synthesized 2.12 which
has the aromaticity of the aniline removed and 2.19, which removes the cyclic character of the
53

aniline altogether. We also prepared 2.13, featuring N-methylation at the linking amide moiety,
which removes hydrogen-bond donor functionality at that position. Finally, we included 2.17 and
2.18, which contains a thiophene bridge rather than the original amide.

Figure 2.14 First-generation of potential antiviral molecules derived from open-ring
quinazoline inhibitors. A) Structures of molecules contained within this panel. SAR
optimizations from 2.9/2.10 highlighted in red. B) Determination of dose-response effects of first-
generation inhibitors measured by IFN reporter assay, as done previously. C) Selection of the best-
performing inhibitors were repeated at a lower dose range. Molecule exhibiting the best dose-
response curve is marked by the red box.

With this new panel of inhibitors in hand, we repeated the IFN-B reporter experiments and
evaluated whether any of the first-generation inhibitors had improved effects compared to 2.10.
54

Of the compounds tested, 2.14, 2.15, 2.17, 2.18, and 2.19 contained dose treatments that led to
strong responses of IFN-B (Figure 2.14B). We were optimistic about these findings, as these
inhibitors more-or-less improved upon the efficacy of parental molecule 2.10. Within the
concentration ranges applied, none of the inhibitors exhibited a standard dose-response
relationship, likely due to cell death or some other counterproductive effect at very high levels of
the inhibitor. Because of this, we repeated the experiment for our top-performing molecules at a
lower concentration range (Figure 2.14C). From this experiment, we judged that 2.19 displayed
the most robust dose response and induced the promising IFN-B response. Structurally, this analog
has an n-pentyl chain instead of the parental aniline, which suggests that highly flexible elements
with relatively greater degrees of conformational freedom would be preferable at this position. We
noted that 2.17 and 2.18 were also among the better-performing candidates, which was interesting
to us because they contain the structurally unique (amongst this panel of 8) thiophene linkers.
We turned our attention to evaluating the top 5 compounds in a SARS-CoV-2 infected
experimental model utilizing Caco2 cells. Pre-treatment of the drugs were performed for 2 hours
before viral infection. Post-infection, drug-supplemented medium was replaced every 24 hours
with fresh drug for 3 days. The ability of the compounds to act as antivirals were assessed by
measuring viral titer and relative expression of select SARS-CoV-2 genes measured by quantitative
PCR (qPCR). Based on the viral titers, 2.15, 2.17, and 2.19 were the most effective in diminishing
the propagation of the virus, with 2.19 being the standout performer (Figure 2.15A). These results
were essentially mirrored in the relative abundance of SARS-CoV-2 transcripts, Nsp1, N, and E
and were strongly suggestive of opportunities for further enhancement (Figure 2.15B).
55

Figure 2.15 Effect of first-generation antivirals in SARS-CoV-2-infected Caco2 cells. A) Cells
were treated with 2 uM of each compound before SARS-CoV-2 infection. Cells were passaged for
72 hours with daily replenishment of drug-supplemented growth medium. Viral titer was measured
by plaque assay. Statistically best-performing drugs are marked by an asterisk. B) Relative
expression of viral Nsp1, N, and E genes were monitored by qPCR in the presence of the indicated
drug or DMSO.

Design, synthesis, and characterization of second-generation putative antivirals
Guided by structural elements from the 3 most promising antiviral candidates, we continued
our SAR optimization by tuning the structures of 2.15, 2.17, and 2.19 (Figure 2.16A). Within the
2.15 “lineage,” we introduced alternative substituents while maintaining the ortho-substitution on
the aniline ring. This afforded new inhibitors with ortho-methyl (2.20), ortho-chloro (2.21), and
ortho-ethyl (2.22) groups. We were also slightly inspired to delve more deeply into the influence
of the thiophene linker and decided to graft structural moieties from previously well-performing
inhibitors onto the thiophene template scaffold. We were thus able to generate molecules derived
56

from 2.17 with the following additions to the aniline ring: ortho-methoxy (2.23), meta-methoxy
(2.24), and n-pentyl (2.25). Finally, our best antiviral candidate 2.19 was modified in a manner to
probe the role of the flexible alkyl chain. We incorporated an oxygen atom within the pentyl chain
(2.26) to see how increased polarity influenced its effects and closed the alkyl chain to form a
cyclopentyl ring moiety (2.27).

Figure 2.16 Second-generation panel of putative antivirals. A) Structures of second-generation
molecules. Parental structure providing basis of SAR analysis is shown to indicate lineage of
development. SAR motifs are highlighted in red. Characterization of antiviral efficacy was
performed under identical conditions as the prior generation and was monitored by qPCR
determination of B) N or C) E expression. Significant performers are indicated by a blue asterisk.
D) SARS-CoV-2 viral titer as determined by plaque assay revealed best antiviral compounds,
denoted with a red asterisk. Compounds 2.23 and 2.24 were toxic to cells.

57

Characterization of the second-generation panel of molecules was executed in an identical
manner as described above. Initial analysis of the results indicated that 2.25, 2.23, 2.20, and 2.24
were most effective in decreasing the expression of SARS-CoV-2 nucleocapsid (Figure 2.16B) and
envelope proteins (Figure 2.16C). However, during our measurements of viral titer in the cell
growth medium supernatant, we determined that 2.23 and 2.24 efficacy was a false positive
appearing as an artifact due to cell toxicity (Figure 2.16D). The two remaining compounds, 2.25
and 2.20, were consistently effective between the two sets of experiments and superior to the first-
generation parent 2.19. In terms of structural features, 2.20 stood out as a noteworthy molecule
because it contains combined elements from the best-performing inhibitors of previous generations:
it unites the thiophene ring and the n-pentyl flexible alkyl chain, which we hypothesized were
responsible for antiviral efficacy in past screens. Further bolstering this observation was the fact
that, despite no longer being the most potent molecule, 2.19 still one of the better performing
inhibitors. We reasoned this may be due the n-pentyl group and emphasizes a potential privileged
role of this flexible chain in its inhibitory mechanism.

Design, synthesis, and characterization of third-generation putative antivirals
For our final panel of inhibitors included in these broad screens, we decided to press further
in determining the importance of a few motifs previously shown to be critical or, at least,
productive towards antiviral potency (Figure 2.17A). All 6 molecules synthesized in this set
contain cyclic substituents extending from the amide nitrogen. This would allow us to decipher the
extent of contribution from the flexible, n-pentyl chain. Compounds 2.28, 2.29, and 2.31 were
58

designed with thiophene linkers but with closed ring substituents of cyclopentyl, cyclohexyl, and
2-methylcyclopentyl, respectively. The last analogue was included to determine the role of the
small, hydrophobic methyl in a position that is analogous to an ortho-substituted phenyl ring. This
design principle was totally exhausted by the inclusion of 2.30, 2.32, and 2.33, which have 2-
cyclopentyl, 2,6-dimethylphenyl, and 2-methylcyclohexyl groups, respectively, attached to the
amide nitrogen and bridged by the original phenyl ring. To reiterate, these molecules were designed
primarily to assess the essentiality of a closed ring versus an open chain substituent attached to the
amide nitrogen.

59

Figure 2.17 Third-generation panel of putative antivirals. A) Structures of third-generation
inhibitors. Expression of B) N and C) E SARS-CoV-2 proteins were determined by qPCR. Dose-
course evaluation of top-performing inhibitors 2.19, 2.25, and 2.20 against D) N and E) E
expression. Red box indicates best antiviral across the three generations.

Third-generation inhibitors were evaluated for their influence on SARS-CoV-2 nucleocapsid
and envelope protein expression, as described above. Unfortunately, none of the molecules in this
newest panel were competent antiviral compounds. Inclusion of 2.19 as a reference standard
60

showed that all the new molecules fared significantly worse; though there was modest, but
essentially negligible, decrease in N and E transcription after treatment with some of the drugs,
none of them contained structural modifications that improved antiviral activity (Figure 2.17B,C).
These results seem to indicate that having a medium-sized, flexible alkyl chain extending off of
the amide nitrogen is absolutely critical for having anti-SARS-CoV-2 activity.
Across all three panels of inhibitors, 2.19, 2.25, and 2.20 appeared to be the most promising
lead molecules in preventing SARS-CoV-2 infection. We decided to repeat qPCR measurements
of nucleocapsid and envelope protein expression during a dose-course application of these three
molecules. Although all three molecules resulted in decreased detection of the viral structural
proteins, only 2.19 displayed a robust dose-response relationship. Moreover, 2.19 was clearly the
most potent, as nucleocapsid levels were reduced by nearly 3-fold compared to 2.25 and 2.20
(Figure 2.17D,E).
The data compiled thus far points to 2.19 as a lead molecule worth exploring further due to
initial results suggestive of its ability to prevent SARS-CoV-2 infection. We were interested in
pursuing a more detailed analysis of its antiviral mechanism and proceeded to synthesize a probe
version of the inhibitor containing a reporter alkyne at the terminal end of the n-pentyl chain. This
yielded compound 2.34, which we envision could be used in combination with our on-resin
CuAAC experiments to determine host/viral factor targets and validate their in situ interactions
(Figure 2.20A).

61

Validating CTPS1 as a bona fide mediator of inhibitor-induced antiviral effects
Mechanistic investigations carried out by the Feng lab had established a potential role for
cytidine triphosphate synthetase 1 (CTPS1) in mediating SARS-CoV-2 evasion of host cellular
innate immune responses.
102
CTPS1 is a host factor enzyme primarily functioning within the de
novo biosynthetic pathways of pyrimidines.
103
It possesses a glutamine amidotransferase (GAT)
domain that catalyzes the reversible transformation of uridine bases to cytidine by interconversion
of the 4-oxo and 4-amino groups.
104
The Feng lab discovered that the amidation status of interferon
regulatory factor 3 (IRF3) was dependent upon the expression and catalytic competence of CTPS1.
Specifically, CTPS1 was shown to deamidate IRF3 (producing an N85D mutant), altering a critical
transcription factor-DNA phosphate backbone interaction necessary for transcription of pro-
inflammatory genes. The group hypothesized that certain SARS-CoV-2 viral factors (in particular,
ORF8 appeared to be a prominent mediator) promoted CTPS1 activity to indirectly mute
transcription of immune response elements. We therefore considered the possibility that our
previous panels of antivirals were able to diminish viral proliferation through CTPS1 inhibition.
Given that these inhibitors were derived from a highly potent and specific GMPS binder,
which also contains a GAT domain featuring a conserved and CTPS1-homologous catalytic triad,
we had strong reason to believe that our electrophilic drugs were targeting the same reactive
cysteine. We performed some preliminary analyses of CTPS1 target engagement by the previously
described GMPS inhibitor analog 2.9, which provided the basis for design of our antiviral panels.
This compound displayed decent antiviral effects, as determined by its ability to promote IRF-B
transcription, and importantly, possesses a terminal alkyne which we could use as a handle to
62

install a fluorogenic reporter. A construct of CTPS1-FLAG in pcDNA3.1 was transiently expressed
in HEK293T cells treated with a broad concentration range of 2.9, from which CTPS1 was
immunopurified, subjected to CuAAC with TAMRA-N3 after competitive 3xFLAG-peptide
elution, and analyzed for inhibitor binding by in-gel fluorescence. The results clearly indicate that
a CTPS1-inhibitor adduct was formed and, moreover, produced a reasonable dose-response curve
(Figure 2.18A). However, the binding interaction was only moderate, requiring up to 5 µM for
robust fluorescence, at which signal saturation was still not achieved.

Figure 2.18 Target engagement confirmation of CTPS1. A) Cells transiently expressing
CTPS1-FLAG were treated with a range of concentrations of 2.9. Cell lysate was enriched by
immunopurification and the eluent was subject to TAMRA-N3 CuAAC for eventual readout by in-
gel fluorescence. B) Cells transiently expressing CTPS1-TwinStrep were harvested and enriched
by StrepTactin pull-down following dose-course treatment with either 2.9 or 2.11 in situ.

We attributed signal weakness to potentially low abundance of protein after the immuno-
purification procedure. We decided to sub-clone the CTPS1 construct into a pLVX-Twin-Strep-
tag® vector and repeat the labeling experiments using the Strep-Tactin® Sepharose resin, from
which we had previously demonstrated high-quality in-gel fluorescence results after mammalian
63

cell expression and purification. We performed a similar dose-response experiment including 2.9
against the new construct and included 2.11 as a control (this molecule is the reverse amide analog
of 2.9, generated by ring-opening of the GMPS inhibitor quinoline analog). First, only 2.9 was
able to label CTPS1, critically showing that the orientation of the amide bridge was absolutely
critical for CTPS1 engagement (Figure 2.18B). Second, although the fluorescent signal intensity
was improved from the FLAG immuno-purification system, we were unable to achieve signal
saturation, indicating that the binding interaction may not be as potent as we originally suspected.
Despite this observation, because we were able to identify a probe molecule that could reliably
label CTPS1, we decided to use this molecule in pulse-chase experiments with our three
generations of compounds to determine whether their antiviral mechanism was CTPS1-dependent.
This would allow us to validate the hypothesis posited by the Feng lab, that CTPS1 is the critical
link between SARS-CoV-2 and IRF3 dysregulation. To test this, we incubated Nsp12-expressing
293T cells with a saturating concentration of all 22 compounds from the three generations of
inhibitors. After a PBS washout, we incubated the cells with 2.9 and evaluated the ability of the
inhibitors to target CTPS1 based on out-competition of binding and a decrease in the fluorescent
signal. To our surprise and dismay, none of the compounds were as effective as expected; only a
few later-generation inhibitors were able to induce even slight inhibition, despite using very high,
saturating concentrations (Figure 2.19). Worse yet, the competition results did not align with the
IFN-B reporter and viral titer assays. Based on these datapoints, we began to suspect that the drugs,
while being clearly effective as antivirals, must be operating through a mechanism independent of
CTPS1 inhibition.
64

Figure 2.19 Validating CTPS1 as mediator of antiviral effects. Cells expressing CTPS1-
TwinStrep were pre-treated with a saturating amount of inhibitors from the three combined panels
described previously. After a washout by PBS, cells were treated with 2 uM 2.9 and harvested for
in-gel fluorescence after CTPS1 enrichment and on-resin CuAAC.

Discovery of Nsp12 as a viral factor targeted by putative antiviral compounds
Because we had previously determined that highly electrophilic inhibitors could interact with
Nsp12, we chose to turn our attention away from host factor interactions and instead ask whether
our panel of antivirals were acting on viral proteins. We transfected 293T cells with a moderate
sample of SARS-CoV-2 non-structural protein constructs, which we culled based on two selection
criteria. The first is their importance in viral replication (Nsp5, main protease) and secondly, their
suspected propensity to bind nucleotide mimics (Nsp7+8+12, replicase complex; Nsp13,
helicase+triphosphatase; and Nsp15, uridine-specific endoribonuclease). CTPS1 was also included
as a positive control. Treatment with probes 2.3, 2.9, and 2.34 revealed intense labeling with
65

CTPS1 and additionally, apparent interactions with Nsp12 and Nsp15 (Figure 2.20B,C). This was
the first indication that our observed antiviral effects might be coming, in part, from inhibition of
viral factors rather than host.

Figure 2.20 Profiles of SARS-CoV-2 viral engagement with activity-based probes derived
from putative antivirals. A) Design principle for functionalizing inhibitor 2.19 with an alkyne
reporter group. Cells expressing a few, select viral proteins were assessed for labeling by inhibitor-
derived probes B) 2.34 and C) 2.34. Probe 2.9 was included in both experiments as a reference
molecule.

Next, we performed a similar competitor validation experiment, using the best antiviral
compounds as pulses against a fluorescent chase with either 2.9 or 2.34. In cells transiently
expressing Nsp12, we saw fairly significant decreases in signal intensity for both 2.9 (Figure 2.21A)
and 2.34 (Figure 2.21B), particularly with competitors 2.23, 2.25, and 2.19, which is very
66

consistent with previous antiviral data. This was a very affirming result, as we were unable to
validate CTPS1 labeling by the inhibitors with this method. We also performed the pulse-chase in
Nsp15-expressing cells, though no competition was observed, indicating that it is not a bona fide
target for any of the putative antivirals (Figure 2.21C). We then performed separate dose-response
competitor titrations for 2.23 (Figure 2.22A), 2.25 (Figure 2.22B), and 2.19 (Figure 2.22C) and
were able to confirm dose-dependent inhibition of Nsp12 by all 3 compounds. The results provide
evidence against CTPS1 as being the primary intervention node by which our antivirals act. The
fact that our competition assay format was not able to provide data in support of CTPS1 inhibition
calls into question its role during immune evasion by SARS-CoV-2. While CTPS1 may play some
part in modulating the IRF3-regulated response of cytokines, our compounds are exerting their
antiviral effect through it. Rather, the data is indicative of direct intervention upon viral proteins
themselves. Based on the fact that Nsp12 appears to be the primary target, we hypothesize that the
molecules are acting as competitive inhibitors, due to being structural analogs of nucleotides, and
inhibiting RdRp-mediated genome replication.

67

Figure 2.21 Antiviral competitor validation against viral proteins labeled by activity-based
probes. Cells expressing the indicated viral protein were pre-treated with saturating concentrations
of antiviral drugs. Target binding site was occupied in Nsp12, as indicated by out-competition of
signal from A) 2.9 and B) 2.34. C) Inhibitors did not appear to target Nsp15.

Figure 2.22 Dose-dependent competitor titrations of best antiviral compounds against Nsp12.
Dose-dependent decrease in probe signal for all three inhibitors tested.

68

Finally, we were intent on having a more rigorous characterization of the Nsp12 binding event.
Our primary motivation was to perform MS/MS analysis to identify the specific residue that is
covalently modified by our electrophilic inhibitors. For this purpose, we incubated Nsp12-
expressing cells with 2.19 and 2.25 and analyzed the adduct by MS/MS fragmentation after tryptic
digest of an SDS-PAGE-resolved gel slice (Figure 2.23A). The results returned indicated several
potential cysteines which may be the site of covalent modification. Based on agreement between
the two inhibitors’ results (and based on the orientation of the cysteine sidechains in the active site
pocket), we narrowed our investigation down to four residues: C396, C564, C800, and C814
(Figure 2.23B,C). Of note, C564 had been previously identified as a covalent reactivity site in our
earlier work with 2.1 and 2.3 quinazoline-based inhibitors.

69

Figure 2.23 MS/MS identification of reactive binding residues in Nsp12. A) SDS-PAGE profile
of Nsp12-expressing cells treated with the indicated inhibitor (10 µM, 1 hour under culture
conditions) after enrichment. Gel slices were digested by trypsin and analyzed by MS/MS
fragmentation. Protein coverage data and cysteines identified as modification sites with a B) 2.25
or C) 2.19 adduct. Promising residues are highlighted in red text.

To validate the MS/MS fragmentation data, we generated alanine mutants for each cysteine-
of-interest. Our intention was to express cysteine-to-alanine mutants in 293T cells perform in situ
inhibitor treatments to see which mutational event was able to abrogate Nsp12 labeling. Our
earliest attempts at this experiment were performed with relatively high concentrations of
inhibitors and in situ incubation times of 1 hour (Figure 2.24). Unfortunately, none of the four
cysteine-to-alanine mutants appeared to compromise 2.9 or 2.34 labeling under these conditions.
We repeated these experiments by reducing the incubation time and concentrations of probes,
suspecting that the high and long incubation conditions were causing non-specific labeling of
70

surface-exposed/accessible cysteine residues. With these refined conditions, we were able to
observe moderate decreases in signaling intensity for all mutants, except C814A, compared to
wild-type Nsp12. Between both 2.9 (Figure 2.25) and 2.34 (Figure 2.26), C564A appeared to have
the greatest effect, suggesting that it may be the primary modification site. Unfortunately, we were
not able to achieve complete ablation of the fluorescent signal. This is most likely due to non-
specific labeling of highly reactive cysteines, despite our tempered conditions. Nonetheless, our
data points to Nsp12 as being the mechanistic point of pharmacological intervention by our panel
of molecules and is suspected to bind covalently with our molecules at C564.

Figure 2.24 Confirmation of MS/MS site ID data through in situ labeling of Nsp12 alanine
mutants. Cells expressing the indicated Nsp12 mutant were treated with relatively high
concentrations of probes. Fluorescence data suggests modest or no decreases in signal as a result
of the alanine mutations, under these conditions.

71

Figure 2.25 Dose titration of 2.9 in Nsp12 mutants at lower concentration ranges. Evaluation
of compromised labeling ability in Nsp12 mutants using a dose-course treatment of 2.9 up to A) 4
µM against C396A, C564A, C800A and B) C814A. C) Reduced range up to 2 µM was included.

Figure 2.26 Dose titration of 2.34 in Nsp12 mutants at lower concentration ranges. Evaluation
of compromised labeling ability in Nsp12 cysteine-to-alanine mutants using a dose-course
treatment of 2.34 up to A) 4 µM and a reduced range up to B) 2 µM. Modest decreases in signal
intensity can be discerned from select mutants.

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2.2.3 Summary
Through the derivatization of nucleotide analogs previously demonstrated to be effective
inhibitors of de novo purine biosynthesis, we have discovered a series of molecules that exhibit
efficacy against SARS-COV-2 infection. Early investigations regarding the putative antivirals’
mechanism of action led to the hypothesis that the activity of a host target factor, CTPS1, was
being modulated. Attempts to validate CTPS1 engagement with our panel of inhibitors were
unfruitful, thereby casting doubt on its role in mediating the observed antiviral effects. We had
recently screened for the reactivity of inhibitors with chloroacetamide warheads towards viral
factor Nsp12 and confirmed formation of a covalent adduct. Accordingly, we turned our attention
to the possibility of a viral target. Exploratory characterization efforts enabled by activity-based
probes derived from our antivirals verified an irreversible modification of the Nsp12 RdRp. This
study has thus culminated in the generation of a library of potential SARS-CoV-2 therapeutics
acting through the bona fide target, Nsp12, identified from forward pharmacological, phenotypic
drug discovery.

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2.3 SARS-CoV-2 Nsp5: 3C-like/main protease

2.3.1 Introduction
Functional role of M
pro
in coronavirus infection and proliferation
The M
pro
protein of SARS-CoV-2 is encoded by the Nsp5 segment of the viral genome. It, in
combination with the other proteolytic enzyme, PL
pro
(catalytic domain of multi-functional Nsp3),
is responsible for the processing of viral polyproteins, pp1a and pp1ab, upon translation of the
viral RNA genome to release the components of the viral replicase complex.
105
Nsp5 operates at
11 cleavage sites on the larger pp1ab substrate, which are characterized by a distinct recognition
sequence of Leu-Gln-X-(Ser, Ala, Gly), where ‘X’ denotes the position of the scissile amide
bond.
106
Intriguingly, there is no documentation of any human protease that recognizes this
cleavage signature.

Structural features of the SARS-CoV-2 main protease (Nsp5)
Crystal structure analyses of the Nsp5 protease were achieved quite rapidly after the initial
outbreak of SARS-CoV-2.
107
All characterization efforts revealed very high degrees of similarity
between the SARS-CoV-2 main protease and its SARS-CoV homolog, which is unsurprising,
given the 96% sequence identity between the two. Indeed, the root mean square deviation (RMSD)
between both orthologs were determined to be roughly half an angstrom for all Cα positions.
108

The catalytically-functional protease exists as a homodimer of two Nsp5 protomers. The individual
monomeric unit consists of three separate domains, which include a chymotrypsin-like and
74

picornavirus 3C protease-like domains I and II, along with a third domain simply referred to as
domain III. Domains I and II are characterized by an antiparallel β-barrel structure which forms
the basis of the catalytic domain while domain III contains an anti-parallel cluster of five α-helices.
A long loop region connects domains II and III to establish the complete monomer structure.
109

Domain III is the primary region involved in facilitating the protomer-protomer contacts
needed for dimerization. The driving dimer-formation interaction is a salt bridge that exists
between a glutamate of one protomer and an arginine of the other – the dimer is asymmetric. In
virtue of this interaction, there exists a contact interface between domains II of the first protomer
(Glu-possessing protomer) and a small number of N-terminal residues (termed the N-finger) on
the second (Arg-possessing protomer). This arrangement forces the two protomers to be oriented
towards each other perpendicularly and shaped such that N-finger residues coordinate with specific
glutamate residues appropriately (Figure 2.27A). The series of these contacts are critical for
shaping the substrate-binding site, specifically the S1 pocket that binds the critical glutamine
residue bearing the scissile amide bond of the substrate. Interestingly, the non-conserved region of
SARS-CoV-2 (compared to predecessor SARS-CoV) resides largely within the dimer interface of
domain III.SARS-CoV M
pro
dimers contain a symmetric hydrogen-bond interaction taking place
between threonine residues that are anchored in place through hydrophobic contacts with a
neighboring isoleucine. These hydrogen-bond productive threonine residues are mutated to alanine
in SARS-CoV-2 Nsp5, which allows tighter dimerization between the protomers, in virtue of their
ability to approach each other more closely.

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Figure 2.27 Structure of the SARS-CoV-2 main protease homodimer. Nsp5, in a catalytically
competent conformation driven by asymmetric dimerization, has an active catalytic dyad. A) One
monomer (shown as an orange cartoon) engages opposite protomer (shown as teal surface
representation) perpendicularly via the domain III interface. B) Key residues in the S1-S3 binding
site (highlighted in green) comprise the catalytic pocket, which houses the catalytic His-Ala dyad
(highlighted in purple. Figure adapted from Kneller, D. W. et. al., Nat. Comm. 2020.
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The catalytic domain exists in a cleft between domains I and II, which houses the substrate
binding site. Nsp5 proteolysis is mediated by a catalytic dyad comprised of His41 and the
nucleophilic Cys145 residue (Figure 2.27B). The S1 binding pocket has an absolute requirement
for a glutamine residue at the P1 position, which grants most of the substrate-selectivity of Nsp5.
111

The S2 subsite is a large hydrophobic pocket that has some tolerance for a range of substrate side
chains at this position, though it appears to prefer leucine residues. There is very little substrate
preference at the third S3 subsite, as it is solvent exposed. Further binding subsites do not
significantly discriminate substrate sidechains. Another important motif within the protease active
site is the oxyanion hole, which is comprised of Gly143, catalytic Cys145, and Ser144. This small
pocket is essential for stabilizing the negatively charged intermediate that forms upon nucleophilic
attack by the catalytic cysteine.
112

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Antiviral strategies targeting the protease
Early efforts to identify SARS-CoV-2 Nsp5 inhibitors were largely guided by the repurposing
of protease inhibitors used for the treatment of closely related viruses. The most well-known
examples that showed some degree of efficacy against COVID-19 include lopinavir, darunavir,
and atazanavir.
113–115
All of these candidate drugs are peptidomimetics, which function by
mimicking the structure of the natural Nsp5 substrate. Despite the rigid requirement of a glutamine
residue at the P1 position of the substrate peptide, none of these drugs feature an isosteric or
isoelectronic side chain at the corresponding position. Nevertheless, at one point these drugs were
all considered as treatment options for COVID-19.
More recently, groups have made pioneering efforts in the design of Nsp5-tailored inhibitors.
Much of the basis for these design programs involves SAR optimization of main protease
inhibitors originally developed as SARS-CoV antivirals. The guiding design principle behind these
inhibitors features the utilization of a scaffold that closely resembles the protease native substrate.
By preserving and optimizing the contact points of the P1-P4 substrate fragments, particularly the
amide sidechain of P1 and a relatively large hydrophobic sidechain in P2, high binding affinities
been enzyme and inhibitors can be achieved. Furthermore, the best inhibitors of this class contain
an electrophilic moiety located within position of the scissile amide bond (Figure 2.28).
116
This
places the warhead in proximity of the nucleophilic cysteine, which can “trap” the enzyme via
covalent inhibition. The best example of this comes from a molecule designed by Pfizer, PF-
00835231, which was originally intended to be used as an antiviral for SARS-CoV .
117,118
Because
SARS-CoV was controlled and contained before widespread propagation of the virus, the molecule
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was shelved and did not see much use. Because of the homology between SARS-CoV and SARS-
CoV-2 main proteases, this molecule served as a launchpad for inhibitor development that rapidly
accelerated the identification of highly effective Nsp5 inhibitors. Currently, this optimized
molecule (discussed in further detail below), in combination with ritonavir, is sold under brand
name Paxlovid.
119
Note that ritonavir does not possess much inherent antiviral activity; it is
included as a booster by inhibiting cytochrome P450 3A4 (CYP3A4), which is known to
metabolize and inactive protease inhibitors.
120
Despite initial excitement over the efficacy of this
new Pfizer drug, some concerns have recently arisen concerning the development of resistance
mutations in SARS-CoV-2.
77,121,122
Thus, it is necessary to continue discovering highly potent
inhibitors that can overcome resistance mechanisms.

Figure 2.28 Design rationale of inhibitors targeting the catalytic residues of SARS-CoV-2
Nsp5. A) Spatial context of the catalytic cysteine targeted by covalent inhibitors. B) Schematic
illustrating productive binding interactions between Nsp5 inhibitors and catalytic residues. A
covalent warhead in place of the scissile amide bond places it in proximity to the catalytic cysteine.
Pyrrolidone is a bio-isostere for the glutamine side chain and maintains P1-S1 interactions. Figure
adapted from Dai, W. et al., Science 2020.
123

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In the following section, we outline our attempts thus far at the discovery of irreversible
covalent inhibitors of Nsp5. Our strategy is built around the exploration of various electrophilic
warheads, with particular attention paid to latent alkyne warheads and their derivatives.

2.3.2 Results and discussion
2.3.2.1 Crudely profiling Nsp5 sensitivity towards covalent electrophilic warheads
Screening quinazoline-based inhibitors for covalent activity towards Nsp5
From our earlier screening efforts against Nsp12 (as described above), we discovered that a
handful of quinazoline-based inhibitors had inhibitory properties and targeted reactive cysteines
within the polymerase binding pocket. The most successful selections of inhibitors bore
chloroacetamide warheads and were irreversibly labeling Nsp12 proteins to great effect. Before a
relative abundance of information regarding Nsp5-targeted inhibition became available, we
wondered whether a similar batch-screen approach with our electrophilic inhibitor cocktails could
reveal information about the druggability of Nsp5, the main protease involved in polyprotein
processing during the viral maturation process. Because Nsp5 contains a highly reactive cysteine
in its catalytic dyad motif, we reasoned that similarly reactive electrophilic compounds could
potentially target Nsp5 within its active site. We therefore pursued the same experimental strategy
applied for Nsp12, wherein cells transiently expressing the target protein were treated with a small
cocktail of electrophilic inhibitors, grouped based on the type of warhead they possess. Covalent
adducts formed could be visualized by in-gel fluorescence of cell lysate after CuAAC with
TAMRA-N3 (Figure 2.5). Our first efforts to this end were largely unsuccessful; from three batches
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of electrophilic inhibitors – containing either chloroacetamide (CA), acrylamide (AA), or
isothiocyanate (ITC) warheads – no sign of Nsp5 labeling could be detected in cells over-
expressing the protein (Figure 2.29A). We repeated the procedure, while including a Streptavidin
enrichment step, to evaluate unequivocally whether any indication of Nsp5 labeling was detectable
(Figure 2.29B). Unfortunately, even with the enrichment, there was no sign (based on our method
of readout) that Nsp5 was being inhibited by any of our quinazoline-based molecules.

Figure 2.29 Attempts to identify Nsp5 binders from quinazoline-based inhibitors. Cells
expressing Nsp5 were treated with a cocktail of quinazoline inhibitors by type of electrophile. CA
= chloroacetamide, AA = acrylamide, ITC = isothiocyanate. Samples were processed and analyzed
by standard CuAAC procedure. Red bracket indicates anticipated Nsp5 location (~33.0 kDa) A)
The indicated cocktail concentrations did not produce discernible labeling signatures in anticipated
Nsp5 positions. B) Enriched samples produced no difference (conducted before adopting on-resin
CuAAC procedure).

These results were ultimately unsurprising; Nsp5 has an active site that is modeled for extreme
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specificity towards its substrate and is unlikely to react indiscriminately with covalent inhibitors.
This is further underscored by the fact that the catalytic dyad (and by extension, the cysteine that
we were interested in targeting) is somewhat embedded in the interior of the protein and is only
accessible by warheads poised for reaction upon proper orientation within the binding pocket.
Given these insights, we abandoned our previously-adopted “shotgun” approach and instead opted
for a more traditional, reverse pharmacological strategy for discovering Nsp5 inhibitors. As
described above, the SARS-CoV-2 Nsp5 active site bears a high degree of homology with the main
protease from its antecedent SARS-CoV predecessor. Accordingly, many Nsp5 drug discovery
programs are founded based on the adaptation of previously designed SARS-CoV antivirals, which
are essentially Nsp5 substrate mimics. In light of this, we set out to improve upon other
peptidomimetic inhibitors in this class by systematic and iterative optimization of covalent
warheads placed near the P1 position, in a location equivalent to the scissile peptide bond.

2.3.2.2 Expression and purification of recombinant Nsp5 from bacterial and mammalian
cells
Bacterial expression of Nsp5-6xHis construct in pDONR223 from BL21
In order to characterize the inhibitory properties of our Nsp5 substrate mimics, we needed to
prepare recombinant Nsp5 protein for use in our in vitro enzymatic assays. We received the Nsp5
construct contained within the pDONR223 bacterial expression vector, which was flanked by an
N-terminal glutathione S-transferase (GST) and C-terminal 6xHis affinity handles. Immediately
downstream of the GST tag is an Nsp5 cleavage site, which is removed upon proper expression of
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the protein by intracellular self-processing via its protease activity. The remaining 6xHis tag is
used for purification by fast protein liquid chromatography (FPLC) by standard affinity
chromatography. Purity of Nsp5 fractions were illustrated from SDS-PAGE profiles and were
pooled for use in future applications (Figure 2.30).

Figure 2.30 Bacterial expression of Nsp5-6xHis in BL21. Nsp5-6xHis construct in pDONR223
vector was expressed and purified by standard protein expression procedures. A) Elution profile
after FPLC purification. B) SDS-PAGE confirmation of protein purity from FPLC fractions. Red
arrow indicates position of Nsp5. Lys = cell lysate, FT = flowthrough, CW = column wash.

Mammalian expression of Nsp5-Twin-Strep-tag® construct in pLVX from HEK293T
Simultaneously, we decided to perform the expression and purification of Nsp5 from
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mammalian cells as an alternative protein source. For this purpose, HEK293T cells were
transiently transfected with a pLVX expression vector containing the Nsp5-Twin-Strep-tag®
construct. Initially, we had difficulties (Figure 2.31A,B) obtaining pure protein after
immunopurification by incubation of cell lysate with Strep-Tactin® resin and chemical elution by
competition with biotin. Based on SDS-PAGE profiles of the eluent, our earliest efforts only
produced very little total Nsp5 with less than 20% purity. This was worsened by heat elution, from
which Nsp5 was almost indetectable/indistinguishable behind the co-eluted background.
Modifications to the transfection and enrichment conditions produced no effect; a second
expression and purification attempt altering these parameters did not improve upon the first trial.
We further modified the enrichment conditions by reducing incubation time with the resin to a half
hour. Furthermore, we wanted to refine the elution buffer components by switching from biotin to
desthiobiotin, which exhibits slightly lower binding affinity to Streptavidin-based resins and their
analogues. These revamped conditions produced stunningly pure Nsp5, which we deemed to be
greater than 99% purity with excellent yield (Figure 2.31C). Purified Nsp5 was prepared for
enzymatic activity assays by removing the elution components via centrifugal filtration with 10K
MWCO filters.
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Figure 2.31 Mammalian expression of Nsp5-TwinStrep in HEK293T. Nsp5-TwinStrep
construct in pLVX vector was expressed in mammalian cells by Lipofectamine2000 transfection.
Protein was purified via StrepTactin enrichment and eluted by competitive desthiobiotin binding
before dialysis. A, B) Excessive incubation with StrepTactin resin and biotin elution attempts were
insufficient to yield Nsp5. C) Reduced enrichment and desthiobiotin elution produced pure protein.

2.3.2.3 Iterative optimization of three generations of covalent Nsp5 inhibitors
First generation of Nsp5 inhibitors: native peptide sequence containing Cbz-capped C-termini
Our earliest efforts to produce enhanced inhibitors of Nsp5 was launched on the premise that
we would simply adopt the native peptide sequence of the Nsp5 substrate as a scaffold and sample
a series of electrophilic covalent warheads placed within proximity of the native scissile bond.
This would allow us to draw systematic conclusions about the influence of the warhead type and
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how it impacts protease inhibition potency. These first-generation peptides were prepared by
standard Bergmann-Zervas methods, which introduces a benzyloxycarbonyl (Cbz) capping group
at the N-terminus of the molecule, intended to preserve the hydrophobic P3-S3 interactions.
124
The
rest of the structure is an exact graft of the substrate with a P2-Leu, P1-Gln motif. We attached our
warheads immediately upstream of the P1’ residue and included seven different types in our first-
generation study, including: benzylated vinyl ester (2.35), vinyl sulfone (2.36), vinyl sulfonamide
(2.37), aldehyde (2.38), α-ketoamide (2.39), terminal alkyne (2.40), and nitrile (2.41). From this
panel, we were particularly interested in exploring the inhibitory properties of the terminal alkyne.
There exists literature precedent for the efficacy of this warhead moiety, specifically in the
irreversible inhibition of a human lysosomal cysteine protease, cathepsin K (CatK). The alkyne
functionality is desirable as a warhead because of favorable pharmacokinetics/pharmacodynamics
profiles and latent reactivity, eliminating risk factors often linked to covalent inhibitors. CatK
inhibitors outfitted with the alkyne display covalent bond formation rates that do not correlate with
intrinsic reactivity of alkynes and instead suggest a mechanism of proximity-driven reactivity.
125

With this in mind, we were keen to explore the promise and potential of applying successful
principles in CatK inhibition to Nsp5.

85

Figure 2.32 First-generation Nsp5 inhibitors and kinetic characterization. Structures of first-
generation Nsp5 inhibitors based on simple substrate mimics with a Cbz C-terminal cap and varied
covalent warheads, highlighted in red. IC50 analysis was performed by FRET-based substrate
cleavage assay at 0.5 µM Nsp5 and 15-minute pre-incubation of inhibitors. Reaction was initiated
upon addition of substrate bearing a DABCYL-EDANS FRET pair and monitored for fluorescence
every 38 seconds for 1 hour.si

86

We performed preliminary assessment of our small panel of inhibitors by monitoring in vitro
enzymatic activity of recombinant Nsp5 upon treatment with the small molecules (Figure 2.32).
Standard experimental conditions were applied following the widely used Förster resonance
energy transfer (FRET)-based substrate cleavage assay. Briefly, an Nsp5 substrate containing an
N- and C-terminal FRET donor-acceptor pair (the substrate we employed had a primary sequence
of KTSAVLQSGFRKME, with an N-terminal DABCYL and C-terminal EDANS) was incubated
with Nsp5 in the presence or absence of inhibitor, introduced 15 minutes prior to reaction initiation.
Nsp5 activity was determined based on the rate of appearance of fluorescence due to diminished
quenching after substrate cleavage and IC50 values for each inhibitor were determined from the
kinetic dose-response curves. From the data, 2.38, 2.39, and 2.41 were shown to be very poor
inhibitors. This was somewhat surprising, considering the intrinsic reactivity of aldehydes and the
precedence of nitriles and α-ketoamide groups as widely utilized reversible covalent warheads. We
noted that our alkyne-containing inhibitor 2.40 was not the worst-performing analogue, hinting at
the viability of this warhead, provided that appropriate scaffold modifications are made to amplify
proximity-driven effects. The most promising set of inhibitors appear to be 2.35, 2.36, and 2.37,
which reached near or exact single-digit IC50 values. Encouraged by these results, we decided to
pursue further studies of these top-performing inhibitors in in cellulo and in situ formats.

Cellular profiling of activity-based probes derived from first-generation inhibitors
Shortly after retrieving kinetics data for our first panel of inhibitors, a study was published
detailing the structures of Nsp5-targeting activity-based probes (ABPs). These molecules were
87

structurally similar to our first-generation inhibitors, save for some variation towards the C-
terminus, including the conjugation of a fluorogenic dye. One interesting application that the
authors’ proposed was the use of these molecules as SARS-CoV-2 diagnostic tools for enzyme
detection.
126
Inspired by this study, we saw an opportunity to repurpose standout molecules from
our first-generation panel and create ABPs that could facilitate in situ characterization of Nsp5
inhibition, amongst other applications. Based on relative potency within the panel and because of
the incorporation of a vinyl sulfone warhead in the previously mentioned study, we selected 2.36
as the model from which an ABP would be designed. The strategy was simple: we merely installed
a terminal alkyne group at the meta-position of the Cbz cap to afford probe 2.42. Additionally, we
synthesized probe 2.43 from sulfonamide-bearing 2.37 as a less-reactive, analogous companion,
fearing that the vinyl sulfone would be overly reactive and react non-specifically with activated
thiols in the proteome (Figure 2.33A).

88

Figure 2.33 Activity-based probes derived from first-generation Nsp5 inhibitors and initial
attempts at in situ labeling. A) Structures of ABPs derived from 2.36 and 2.37. Reporter alkyne
highlighted in blue. B) Standard CuAAC procedure of whole cell lysate after dose-course
treatment of 2.42 (1 hour incubation). Nsp5 expression monitored by immunoblot. C) Inclusion of
positive CuAAC control and D) StrepTactin enrichment after 24 hour in situ probe treatment.

With these two first-generation ABPs, we set out to determine the in situ labeling efficiency
of Nsp5 upon incubation, first with the theoretically-more-reactive analogue, 2.42. Nsp5-
expressing cells were treated with the molecule for 1 hour at 37°C and harvested for CuAAC with
TAMRA-N3, as described above. The whole cell lysate was resolved by SDS-PAGE and in-gel
fluorescence was visualized. Regrettably, we could not detect any labeled Nsp5 under these
89

experimental conditions (Figure 2.33B). The experiment was repeated a second time, including
2.1 as a positive control to ensure that the CuAAC conditions were suitable for producing a signal
(Figure 2.33C). In both attempts, Nsp5 did not yield a fluorescent signal, despite being expressed
successfully in the cells. In an effort to exhaust any simple solutions available, we chose (perhaps,
naively) to increase the in situ incubation time from 1 hour to 24 hours and repeated the same
procedure, We applied on-resin CuAAC to these samples but still not did observe fluorescent
labeling (Figure 2.33D). We reasoned that the probe’s inability to label Nsp5 was due to stability
issues, as the combination of its peptidic nature and reactive warhead may render it susceptible to
hydrolytic degradation or inactivation of the electrophile, respectively. Therefore, we pursued a
comparative experiment, by which we would analyze the differences in labeling success after
either in situ (exposure via ABP-supplemented cell growth medium, followed by on-resin CuAAC)
or on-resin (direct exposure to ABP after enrichment by Strep-Tactin® and sequential CuAAC)
ABP treatment (Figure 2.34). Because of our success in performing on-resin CuAAC (see section
2.2.2.1), we believed that various on-bead chemistries were feasible after isolation of protein. In
agreement with our previous results, we were unable to see in-gel fluorescence after in situ
treatments, bolstering the thought that the peptide may be suffering from stability issues. However,
the on-resin ABP incubation did appear to produce a signal – albeit very weakly – at the highest
concentration tested (Figure 2.34B).

90

Figure 2.34 Detection of Nsp5 engagement on-resin, in vitro by vinyl sulfone ABPs.
Comparison of A) in situ versus B) on-resin (after lysis and enrichment of Nsp5-expressing cells)
treatment of ABP and detection by corresponding CuAAC procedure. Incubation was performed
for 24 hours with the indicated concentration range.

The promise of slight signal detection enticed us to the possibility of visualizing target
engagement through this assay format. We tried adjusting various doses of in situ inhibitor
treatment or incubation time-length and repeated the previous on-resin CuAAC workflow. For
comparison’s sake, we also decided to include the less-reactive analogue, 2.43, for side-by-side
evaluation. Because the reactivity of the vinyl sulfone warhead is relatively high, we speculated
that the warhead might be quenched from hydrolytic inactivation before it can react with Nsp5.
Inclusion of 2.43 should provide some insight regarding the stability of the vinyl sulfone warhead,
as we expect sulfonamide to be relatively inert in an aqueous medium. For all conditions tested,
we were still unable to visualize a signal corresponding to labeled Nsp5 (Figure 2.35). We finally
concluded that the peptide scaffold of our first-generation inhibitors may not be suitable for in situ
analysis, much less drug/therapeutic applications.

91

Figure 2.35 First-generation ABPs fail to label Nsp5 in situ. Comparison of in situ labeling
efficiency between vinyl sulfone 2.42 and vinyl sulfonamide 2.43 after A) 12- versus B) 24-hour
incubation. Poor stability likely contributes to labeling inability.

Second-generation Nsp5 inhibitors: C-terminal indole capping and P1-Gln modifications
Seeing as how our first-generation panel suffered from issues ultimately related to
bioavailability and poor pharmacokinetics, we allowed these considerations to guide our design
for a second-generation series. One strategy to improve pharmacokinetics is to reduce the number
of hydrogen-bond donors (HBDs) present in a drug candidate. Indeed, this has been applied to the
improvement of Nsp5 and Nsp5-related protease inhibitors through cyclization of the P1-
glutamine side chain.
127
Previous enhancement efforts have incorporated an unnatural amino acid
at the P1 position, featuring a pyrrolidone motif that is bio-isosteric with the glutamine amide.
Another scaffold modification that we chose to adopt was inspired by the structure of the original
Pfizer drug (PF-00835231), then developed as an inhibitor of the SARS-CoV main protease.
118
In
place of a Cbz C-terminal cap, this molecule contains a 4-methoxyindole capping group. This
motif is shown to be superior because of additional stabilizing hydrogen-bond interactions with
the backbone of Glu166 that are otherwise non-existent with a Cbz group. Our design philosophy
was to update the substrate scaffold with these two modifications and further explore the alkyne
and nitrile (which has relatively low reactivity and is alkyne-like) warheads. Again, our objective
92

was to investigate the amenability of Nsp5 to CatK-inspired inhibitor design. With these principles,
we synthesized 5 inhibitors, all possessing the 4-methoxyindole cap. Either an alkyne or nitrile
was appended to a scaffold containing the original parent glutamine side chain (2.44 and 2.45,
respectively) or the unnatural pyrrolidone-bearing amino acid (2.47 and 2.48, respectively). We
also included an aldehyde warhead analogue (2.46), intended as a reactive positive control.

Figure 2.36 Second-generation Nsp5 inhibitors and kinetic characterization. Structures of
second-generation Nsp5 inhibitors produced from the cyclization of P1 side chain to pyrrolidone
and replacement of Cbz C-terminus with a 4-methoxyindole group. Covalent warheads selected
for second generation are highlighted in red. Kinetic analysis performed by standard FRET
cleavage assay with 15-minute inhibitor pre-initiation of reaction.

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In vitro enzymatic analysis of this second-generation panel by FRET-based substrate cleavage
assay (described above), produced unsurprising results (Figure 2.36). With a 15-minute pre-
incubation, the most potent molecule was the highly reactive, aldehyde-bearing inhibitor, 2.46
(IC50 = 0.72 ± 0.54 µM). Between the remaining four inhibitors, we found that those containing
the cyclized Gln side chain performed significantly better compared to their natural amino acid
counterparts. Interestingly, there was not much difference between the inhibitors containing
identical scaffolds, i.e., the differing warheads did not contribute much to differential potency,
though we note that the alkyne-containing Gln analogue 2.44 was slightly more potent than the
nitrile-containing 2.45.
This last observation was particularly striking to us and we wished to perform a more careful
dissection of the differences in labeling mechanisms for alkynes and nitriles. As shown in the CatK
inhibition study, among others, alkyne warheads are known to produce irreversible covalent
adducts with their targets, as evinced in crystallographic experiments. On the other hand, nitriles
have milder electrophilic properties, engaging in reversible covalent inhibition. This is oft cited
and harnessed as a desirable quality; indeed, nitriles are present in a vast number antiviral and
antiparasitic cysteine protease inhibitors. In order to achieve what we believe is a duly thorough
characterization of our inhibitors, we wanted to definitively show that Nsp5 inhibitors were
proceeding by irreversible (alkyne) or reversible (nitrile) mechanisms. We repeated our FRET-
based substrate assays, this time modifying the pre-incubation conditions so that differences in
mechanism could be teased out. Specifically, we measured IC50 kinetics with either a 3-hour pre-
treatment of inhibitor or simultaneous, co-administration of inhibitor with substrate (Figure 2.37).
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As the data indicates, there is no significant difference in the IC50 values for nitrile-bearing
inhibitors in either pre-treatment condition. In contrast, the alkyne-bearing inhibitors enjoyed
greater than 10-fold improvements in IC50 with a 3-hour pre-incubation compared to coincident
treatment of inhibitor and substrate. These findings suggest that alkyne-containing inhibitors
exhibit greater degrees of potency upon prolonged treatment times, suggestive of a slow-acting
but irreversible mechanism. Nitrile-containing inhibitors, conversely, are rapidly-acting and do not
benefit from increases in exposure time.

Figure 2.37 Irreversibility versus reversibility reaction kinetics with second-generation
inhibitors containing alkyne and nitrile warheads. Second-generation inhibitors possessing
either alkyne or nitrile warheads were subject to kinetic analysis via FRET cleavage assay. To
evaluate off-rate (reversibility) properties, inhibitor was either pre-incubated with enzyme for 3
hours before substrate addition (black squares) or added synchronously with substrate for
immediate reaction initiation (black circles). Significant differences in IC50 values were observed
for irreversible alkynes (left column) while no significant effects were detected for reversible
nitriles (right column).

95

Nirmatrelvir-inspired analogues: a third-generation panel of Nsp5 inhibitors
Sometime around the completion of our second-generation Nsp5 inhibitors, Pfizer published
the structure of their latest contribution to the SARS-CoV-2 antiviral arms race, which they named
Nirmatrelvir (brand name Paxlovid).
118
The road to FDA-approval for Nirmatrelvir was
dramatically accelerated by riding the successes of the original molecule from which it was
optimized, PF-00835231. As mentioned above, this molecule was designed against the SARS-CoV
main protease and only required a few, slight modifications to achieve potent, selective status
against SARS-CoV-2 Nsp5. Remaining faithful to optimization strategies described above, three
major changes were made to reduce the number of HBDs and improve pharmacokinetics. These
include: 1) the substitution of α-hydroxyketone for a covalent, reversible nitrile warhead, 2)
cyclization of the P2-Leu backbone by addition of a 3-azabicyclo motif fused with a 2,2-
dimethylcyclopropyl ring, and 3) replacement of the C-terminal 4-methoxyindole with a
trifluoroacetamide capping group. Nirmatrelvir is reported to be a potent SARS-CoV-2 antiviral
with excellent bioavailability and off-target selectivity/in vivo safety profiles.
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Incorporation of the nitrile warhead immediately caught our attention. Because we had
previously shown that the alkyne warhead improved upon the nitrile in many ways, we
hypothesized that a simple strategy of supplanting the nitrile in Nirmatrelvir with an alkyne would
yield an enhanced irreversible, covalent inhibitor. To this end, we synthesized three initial
analogues: alkyne-modified Nirmatrelvir (2.49), an amide warhead analog (2.51, intended to
function as a non-reactive control), and an aldehyde warhead analog (2.52, included as a highly
reactive, potential positive reference). We also synthesized Nirmatrelvir in-house, referred as 2.50
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in this manuscript henceforth. Evaluation of inhibitor properties of our third-generation panel was
performed using the same FRET-based substrate cleavage assay as described above, first with the
standard 15-minute inhibitor pre-treatment before substrate addition (Figure 2.38). We found that
the inhibitors behaved exactly as expected. The non-reactive “control” 2.51 was not at all potent,
indicating that the scaffold itself contributes very little to Nsp5 inhibition. This is an important
observation because it highlights the importance of warhead identity in producing effective
inhibitors. Our alkyne analog 2.49 slightly improved upon inhibition compared to 2.50, which is
consistent with the trends observed within our second-generation panel. That is, the alkyne
improves potency subtly, though to an appreciable extent, over the nitrile.

Figure 2.38 Third-generation Nsp5 inhibitors and kinetic characterization. Structures of
third-generation Nsp5 inhibitor based on the Nirmatrelvir scaffold. Parental nitrile was swapped
for an alkyne (2.49) or amide as a negative control (2.51) to compare contributions to inhibitor
potency from non-covalent interactions. IC50 values were measured by standard FRET cleavage
assay with a 15-minute pre-incubation of inhibitor.
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With this data in hand, we wished to continue our inhibitor characterization by performing
similar experiments as done previously to reveal differences in reversibility. Inhibitors were
subject to the same treatment conditions – either a 3-hour pre-treatment or instant, co-
administration with substrate – and analyzed for their IC50 values (Figure 2.39). Compound 2.52
contains an aldehyde, a documented, rapidly-acting reversible warhead, and expectedly did not
produce substantial differences in IC50 between the two conditions. However, like what was
observed in our second-generation molecules, alkyne analog 2.49 displayed dramatic
improvements with a 3-hour pre-incubation, enjoying a near-300-fold improvement in observed
potency. Compound 2.50, in contrast, did not feature any significant differences.

Figure 2.39 Irreversibility versus reversibility kinetics of third-generation Nsp5 inhibitors.
Standard FRET substrate cleavage assay conditions were applied, with a 3-hour pre-incubation of
inhibitor (black squares) or simultaneous addition with substrate (black circles). Reversible
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aldehyde and nitrile warheads exhibited no differences in kinetic behavior between the two
conditions while alkyne electrophile was dramatically more potent with a pre-incubation.

Development of third-generation ABPs and cellular profiling applications
The combination of our observed improvements in potency and purportedly favorable bio-
availability properties conferred by the Nirmatrelvir scaffold prompted us to reattempt ABP-driven
experiments, hopefully to redeem our first-generation failures. We first synthesized third-
generation-inspired ABP, 2.53, which was designed by replacing the P3-tert-butyl group of 2.49
with a fairly isosteric propyne reporter group (Figure 2.40A). Kinetic analysis of 2.53 in vitro
showed that the propyne substitution only diminished potency by about two-fold, compared to the
parent inhibitor 2.49. We were also able to recapitulate irreversible covalent inhibition reaction
kinetics (Figure 2.40B).

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Figure 2.40 Characterization of ABPs derived from third generation inhibitors in vitro. A)
Structure of third generation ABP. Warhead is highlighted in red; reporter group is highlighted in
blue. B) Irreversible reaction kinetics measured by FRET substrate cleavage. IC50 values are
reported for either simultaneous administration of probe and substrate (black circles) or with a 3-
hour pre-incubation (black squares). C) Labeling of recombinant Nsp5 (1 µg protein) in vitro
performed with either the indicated dose-course of 2.53 for 3 hours (left) or indicated time-course
with 10 µM 2.53 (right). Robust labeling is observed.

Before moving into in situ profiling experiments, we first wished to evaluate the ability of
2.53 to form a covalent adduct with recombinant Nsp5 in vitro and produce a detectable fluorescent
signal after CuAAC to TAMRA-N3. We applied separate dose-course and time-course treatments
of 2.53, both of which successfully led to robust signal responses (Figure 2.40C). For our initial
attempts at in situ characterization (Nsp5-expressing cells were incubated with ABP for 3 hours),
we followed an on-resin CuAAC protocol (as described in section 2.2.2.1). To our delight, we were
able to produce a very nice dose-response curve and showed detectable labeling of Nsp5 at a
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concentration as low as 1 µM of 2.53 (Figure 2.41A). Evidently, the improved pharmacokinetics
of the ABP have unlocked the in situ characterization workflow. We noted how clean the post-
enrichment fluorescence signal was and decided to repeat the experiment excluding Strep-
Tactin®-based enrichment (Figure 2.41B). Consistent dose-response curves were obtained but
more intriguingly, the samples displayed strikingly clean labeling of Nsp5, indicating no off-targets
could be detected (Figure 2.41C). We decided to explore this further and performed in situ
treatment of 2.53 in either HEK293T or HeLa cells that were not expressing Nsp5. We reasoned
that this would allow us to detect any off-targets labeled non-specifically by the probe. The labeling
profiles did not produce any signal, which clearly points to its remarkable selectivity (Figure 2.42).
Given that we paneled 2.53 against human cell lines, this result is somewhat unsurprising; the P1-
Gln motif, here captured by the pyrrolidone mimic, is highly unique to the SARS-CoV proteases
and would thus not serve as a suitable substrate or competitive inhibitor against human proteases.
Furthermore, we wanted to confirm that the probe labeling event was specifically dependent upon
engagement with the catalytic cysteine. To test this, we generated a non-nucleophilic mutant
Nsp5
C145A
and compared the ability of 2.53 to label this mutant versus the wild type (Figure 2.41D).
No detection of fluorescence occurred even up to 20 µM 2.53, evincing labeling at the catalytic
cysteine.

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Figure 2.41 In situ characterization of Nsp5 labeling with third-generation ABPs. A) Dose-
course treatment of 2.53 in cells expressing Nsp5 produced successful labeling with an on-resin
CuAAC procedure. Incubation of probe was performed for 3 hours in situ. B) Labeling protocol
did not require an enrichment step; no off-targets could be detected from CuAAC of whole cell
lysate (C). D) Mutation of the catalytic Cys145 to a non-nucleophilic alanine completely abrogates
2.53 binding.

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Figure 2.42 Off-target labeling profile of third-generation ABPs in HEK293T and HeLa.
Cells lacking transient over-expression of Nsp5 were incubated with 2.53 for 3 hours and evaluated
by the standard CuAAC procedure. No targets in mammalian tissue could be detected.

Having established the utility of 2.53 as an effective ABP in situ, we wished to apply it in
assessing the in cellulo inhibitory and reactivity properties of our third-generation inhibitor library.
First, a pilot experiment was performed on recombinant Nsp5 in vitro, wherein Nsp5 was subjected
to either a sequential, dose-course treatment of 2.49 followed by 2.53 (allowing for irreversible
inhibition) or co-treatment of 2.50, with a similar concentration range, and 2.53 (simultaneous
competition for active-site binding by reversible inhibitor). The results show that competitive
binding did take place, with a more pronounced effect observed upon 2.50 treatment. Specifically,
roughly 10 µM 2.49 was required for 50% diminishment of the signal, taken relative to the DMSO-
treated control, compared to slightly below 1 µM for 2.50 (Figure 2.43A). Next, we performed the
analogous experiment in situ opting to first assess co-treatment conditions (Figure 2.43B,C). The
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relative results of the titration were consistent with our in vitro data, that is, effective half-maximal
concentrations were distinct by a factor of 10-fold, with 2.50 being more potent.

Figure 2.43 ABPs enable competitive titration of third-generation inhibitors in vitro and in
situ. A) In vitro inhibitor titration was performed with either 1-hour pre-incubation of 2.49 (at
indicated concentration) followed by 1-hour treatment of 2.53 (left) or simultaneous co-treatment
of both probe and inhibitor or 1 hour (right). Standard CuAAC was performed. In situ treatment
of either B) 2.49 or C) 2.50 was performed in Nsp5-expressing cells for 1 hour (both inhibitors
were applied synchronously with the probe) and visualized by CuAAC.

We envisioned that an ABP would allow us to monitor the residence time of 2.50 in live cells.
Such an experiment could be valuable for confirming a slow off-rate of the covalent inhibitor from
the E-I complex, which was cited as a presumed benefit of covalent inhibition (sustained inhibition
enabling less frequent dosage; described in section 1.2). Briefly, Nsp5-expresing would be treated
with a saturating amount of covalent inhibitor, washed out with PBS, and chased with the ABP
after various time post-washout. Longer residence times would manifest as diminished fluorescent
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signals upon CuAAC with TAMRA-N3 since probe binding would be precluded by the occupancy
of pre-treated inhibitor within the active site. Unfortunately, our earliest attempts to execute this
experiment with a combination of 2.50 and 2.53 were unsuccessful (Figure 2.44). Because our
irreversible, covalent warhead is slow-acting, the required incubation time-length to produce a
sufficient “chase” signal exceeds what we tentatively observe as the lifetime of 2.50 residence.
Repeating this experiment with a probe tuned to be slightly more reactive, and thus rapid-acting,
will enable the determination of inhibitor residence times.

Figure 2.44 Measuring Nirmatrelvir residence time in live cells. Nsp5-expressing cells were
pre-treated with a saturating amount of 2.50 for 1 hour. At variable times after washout of 2.50-
supplemented medium, saturating amounts of probe 2.53 was added for 20 minutes to chase vacant
Nsp5 active sites. In situ probe labeling kinetics were too slow and could not be used to detect
residence time.

Going forward: tuning alkyne functionality and antiviral evaluation of inhibitors
Recognizing the need for a more rapidly-acting probe, we questioned whether we could
augment the reactivity of the terminal alkyne by tuning its electronic properties through the
incorporation of electron-withdrawing groups. This strategy would afford internal alkynes that are
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activated towards nucleophilic substitution by increasing the electro-positive nature of the alkyne
carbon atoms. Recently, a series of ubiquitin-based probes, created for the purpose of profiling
deubiquitinases (DUBs), have been described. The study featuring these probes included a
comparison between two separate alkyne warhead moieties, either of which were outfitted with an
electron-donating methyl group or an electron-withdrawing trifluoromethyl group. Ultimately, the
authors conclude that, despite being essentially isosteric, the trifluoromethyl-alkyne probes were
significantly primed to react with certain DUBs. It appears that this simple modification could
dramatically enhance reactivity properties of the original terminal alkyne while preserving the
overall labeling profile. We borrowed this philosophy and synthesized an additional Nirmatrelvir
analog, 2.54, which contains a trifluoromethylalkyne warhead (Figure 2.45A). Comparative
analysis of a 2.54 titration in vitro and in situ produced similar, consistent results (Figure 2.45B,C).
To our pleasure, the new inhibitor was a significant improvement upon the parental terminal alkyne
analog. Indeed, based on in vitro data, there was roughly a 10-fold increase in potency, elevating
the molecule to rival, and even slightly out-edge, Nirmatrelvir, in terms of competitive binding to
Nsp5 (Figure 2.45B). We believe that this chemical signature paves the way for the development
of a series of alkyne-based inhibitors, wherein a gradient of inhibitor reactivity can be achieved by
increasing or decreasing the extent and type of halogenation of internal alkynes. We plan to pursue
the synthesis and characterization of these covalent inhibitors, in addition to performing
experiments unlocked by the highly reactive trifluoromethylalkyne probe 2.54 (e.g., residence
experiments described in the previous section).

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Figure 2.45 Tuning reactivity of latent alkyne warhead by trifluoromethyl appendage. A)
Structure of Nirmatrelvir containing a latent alkyne with tuned electronic properties. Warhead
highlighted in red. B) Dose-dependent in vitro inhibitor labeling analysis was performed with 1
hour co-treatment of the indicated inhibitor and probe at 30°C. C) Analogous dose-dependent
labeling in situ with a 3-hour co-treatment of inhibitor and probe. Standard CuAAC conditions
applied.

In collaboration with the Cheng lab (UCLA, Department of Microbiology, Immunology, and
Molecular Genetics), we have performed some preliminary analyses on the antiviral effects of our
most-promising molecules across the three generations of inhibitors synthesized. Specifically, we
were interested in profiling the efficacy of all alkyne- and nitrile-containing inhibitors. We also
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included 2.46 and 2.51, as positive and negative controls, respectively. First, cytotoxicity of the
compounds was determined for a range of concentrations up to 100 µM using the Cell Counting
Kit-8 (CCK8) assay, which indicated that our inhibitors were relatively non-toxic; significant
compromise of cell viability was only observed at 100 µM, the highest concentration applied
(Figure 2.46). Next, we evaluated antiviral effects by measuring the abundance of SARS-CoV-2
viral genomic DNA upon inhibitor treatment (Figure 2.47A). Briefly, HeLa cells overexpressing
the ACE2 receptor were infected with the SARS-CoV-2 virus and synchronously treated with 10
µM of each inhibitor. After a 24-hour incubation period, viral DNA detected after each inhibitor
condition was measured by rtPCR and plotted by histogram against a DMSO control. The results
show that the most efficacious antivirals were 2.50 (Nirmatrelvir) and 2.48 (second-generation,
nitrile warhead). Compounds 2.40 (second-generation, aldehyde) and 2.49 (third-generation,
alkyne) were equivalent and trailed shortly behind 2.50. We were optimistic about these results;
our alkyne-bearing analog appeared to be quite comparable to Nirmatrelvir. Deciding to probe
further, we repeated a similar experiment, applying a dose-course treatment of 2.48, 2.40, and 2.49
simultaneously with SARS-CoV-2 infection of HeLa-ACE2 cells (Figure 2.47B). Viral titers were
quantified by plaque assay after a 24-hour inhibitor treatment/viral infection. Somewhat
unsurprisingly, our reactive “positive reference” 2.40 produced the best results as an antiviral.
While the remaining two compounds were generally effective as antivirals, their relative potencies
were not consistent, considering our previously determined IC50 values. Though these initial
results are promising and point to some success as viable therapeutics, further studies must be done.
In particular, the antiviral assays should be performed in combination with a P-glycoprotein
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inhibitor, as recent data indicates that Nirmatrelvir-like scaffolds are effectively jettisoned from
cells via efflux. Preventing this may enhance the extent of an observed antiviral effect.
Nevertheless, the collective data compiled thus far indicates that our third-generation inhibitors
and ABPs are highly potent and may serve as effective tools for both treating and studying SARS-
CoV-2.

Figure 2.46 Cytotoxicity of Nsp5 inhibitors in Vero-E6 cells. Viability of cells was measured by
CCK8 assay in the presence of 10-fold increases of the indicated drug, from 100 nM up to 100µM.

Figure 2.47 Antiviral analysis of Nsp5 inhibitors in SARS-CoV-2-infected HeLA-ACE2 cells.
A) HeLa cells over-expressing the ACE2 receptor were infected with SARS-CoV-2 in the presence
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of 10 µM inhibitor or DMSO. Abundance of viral genomic DNA was determined by rtPCR after
24 hours. A zoomed-in region of most potent drugs 2.48, 2.49, 2.50, and 2.46 is provided. B) Dose-
course inhibitor analysis of viral titer by plaque assay for best-performing drugs.

2.3.3 Summary
Progressive improvement in drug design against the SARS-CoV-2 main protease is crucial for
the development of COVID-19 antivirals. Piggybacking on structural insights gleaned by previous
Nsp5 inhibitor optimization campaigns, we have performed a reverse pharmacological, target-
based study on the effects of various electrophiles appended to peptidomimetic scaffolds. By
tuning the electronic and steric properties of latent alkyne warheads, we have produced highly
potent and specific irreversible inhibitors of Nsp5. Further, we derivatized these molecules and
created activity-based probes that overcame traditional stability and pharmacokinetic
shortcomings associated with peptide-like small molecules. Looking ahead, we envision the
application of these findings to enable in vivo investigations concerning viral infection and the
continuing enhancement of Nsp5 inhibitor potency.

2.4 Experimental details
Plasmid construction
All constructs were prepared using standard molecular cloning techniques unless obtained from
other labs as indicated in text. All mutations were generated using QuikChange Lightning Site-
directed Mutagenesis (Agilent). Detailed cloning information and sequences will be made
available upon request.
110

Plasmid transfection
HEK293T cells were seeded at 7.5x10
5
cells per well in 6-well plates 16 hours before transfection
with Nsp12 or CTPS1 constructs (pLVX-EF1α-2xStrep-IRES) using Lipofectamine 2000 (Life
Technologies).

Whole cell lysate inhibitor “pulse-chases” and Cu-catalyzed azide-alkyne cycloaddition (CuAAC)
HEK293T cells were seeded at 5x10
5
per well in 6-well plates and transfected with appropriate
plasmid next day. At 24 hr post-transfection, the growth media (DMEM supplemented with 10%
FBS) was aspirated off and the cells were treated with fresh media containing various
concentration of probe (1,000× stock solution in DMSO) or vehicle control for the indicated time.
For pulse-chase-style competition experiments, cells were first incubated with the inhibitor at
various concentrations for 1 hour, washed with fresh warm medium three times, and then treated
with probe at the appropriate concentration for another hour. After the probe treatment, the medium
was aspirated off and the cells were washed twice with ice-cold DPBS. The cells were harvested
and the pellet was resuspended in 100 µL of NP40 lysis buffer (50 mM HEPES, pH 7.4, 1% NP-
40, 150 mM NaCl) with protease inhibitor cocktail (Roche). The lysate was incubated on ice for
20 min and fractionated by centrifugation at 18,000 × g for 10 min. The protein concentration was
measured from each of the supernatant sample by BCA assay (Pierce) and normalized to 1 mg/mL.
Click reaction was performed at a final concentration of 25 µΜ TAMRA-azide, 1 mM Tris(2-
carboxyethyl)phosphine (TCEP, Thermo-Scientific), 100 µΜ Tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine (TBTA, TCI), and 1 mM CuSO4 (Sigma-Aldrich) in a total volume of 100 µL.
111

The reaction was performed at room temperature for 1 hour in the dark before termination by
addition of 40 µL of 4X Laemmli sample buffer (Bio-Rad) and boiling for 5 minutes. 30 µL of the
samples were loaded and resolved on a 4- 20% SDS-PAGE before visualization at 532 nm for
excitation and 610 nm for emission on a Typhoon 9400 Variable Mode Imager (GE Healthcare).

Inhibitor treatment in situ
The cells were treated with various concentrations of compounds for various times at 37°C. After
compound treatment, the media was removed, and cells were washed twice with ice-cold D-PBS.
The cells were harvested, and the pellet was resuspended in 50 µL of NP40 lysis buffer (50 mM
HEPES, pH 7.4, 1% NP-40, 150 mM NaCl), supplemented with 5 mg/mL protease inhibitor
cocktail (Roche). The lysate was incubated on ice for 20 min and fractionated by centrifugation at
18,000 x g for 10 min at 4°C. The protein concentrations were measured from the supernatant by
BCA assay (Pierce).

Twin-Strep-tag® : StrepTactin® enrichment
The lysate was adjusted to 2 mg/mL and 100 µg total protein was incubated with StrepTactin®
sepharose beads (Cytiva), pre-washed twice with 1% NP40, for 20 min at 4°C. Following protein
binding, the beads were triply washed with 5x column volume of 1% NP40.

On-resin Cu-catalyzed azide-alkyne cycloaddition (CuAAC)
Beads were resuspended in PBS and subjected to on-resin CuAAC at a final concentration of 25
112

µM TAMRA-azide (Click Chemistry Tools), 1 mM tris(2-carboxyethyl)phosphine (TCEP,
Thermo-Scientific), 100 µM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, TCI), and
1 mM CuSO4 (Sigma-Aldrich) in a total volume of 15 µL. The reaction was performed at RT for
1 hr in the dark before termination by addition of 15 µL of 2X Laemmli sample buffer (Bio-Rad)
and boiled for 15 min. The entire protein sample was resolved by 4-20% SDS-PAGE (Bio-Rad)
and visualized at 532 nm excitation/610 nm emission by ChemiDoc MP imaging (Bio-Rad).

Silver Stain
Following visualization of fluorescence, protein gels were stained by Silver Stain (ThermoFisher
Scientific). Briefly, gels were first washed twice for 5 minutes in ultrapure water. They were then
incubated in a fixing solution (30% ethanol:10% acetic acid) twice for 15 min at RT before washes
with twice 10% ethanol and twice ultrapure water for 5 minutes each. Gels were incubated in
Sensitizer Working Solution (0.2% Sensitizer in ultrapure water) for 1 minute, followed by 2 brief
washes with ultrapure water. Gel was stained in Stain Working Solution (2% Enhancer in Silver
Stain Solution) at RT for 20 min. After two 20 second washes with ultrapure water, gel was
developed in Developer Working Solution (2% Enhancer in Developer Solution) until bands were
visible. Staining was terminated by addition of 5% acetic acid and protein bands were visualized
by ChemiDoc MP imaging (Bio-Rad).

Cells and Viral infections
HEK293T (ATCC, cat. no. ACS-4500), HCT116 (ATCC, cat. no. CCL-247), mouse embryonic
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fibroblasts (MEFs) (isolated from the fetuses of different mouse strains) and Vero E6-hACE2 (a
kind gift from Dr. Jae Jung, Cleveland Clinic Foundation, Cleveland, OH) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 10% fetal bovine
serum (FBS; Gibco), penicillin (100 U/mL) and streptomycin (100 μg/mL). Caco-2 (ATCC, cat.
no. HTB-37) and Calu-3 (ATCC, cat. no. HTB-55) were cultured in Minimum Essential Medium
supplemented with 10% FBS and antibiotics. Normal human bronchial epithelial (NHBE) cells
(ATCC, cat. no. PCS-300-010) were cultured in Airway Epithelial Cell Basal Medium (ATCC)
supplemented with Bronchial Epithelial Cell Growth factors (ATCC) and antibiotics. Sendai Virus
(Cantell strain) was purchased from Charles River (cat. no. 10100774).

qRT-PCR
qRT-PCR was performed by standard methods. Total RNA was extracted using TRIzol reagent
(Invitrogen). Complementary DNA (cDNA) was synthesized from total RNA using reverse
transcriptase (Invitrogen). cDNA was diluted and analyzed by qRT-PCR using SYBR Green
Master Mix (Applied Biosystems) with CFX Connect PCR instrument (Bio-Rad Lab.). Relative
mRNA expression for each target gene was calculated by the 2-ΔΔCt method using β-actin or
GAPDH as an internal control.

On-bead tryptic digest and MS/MS analysis
Samples to be subjected to MS/MS analysis were subjected to conditions outlined above for
inhibitor treatment and enrichment. Bead-bound proteins were denatured and reduced at 37 °C for
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30 min and after being brought to room temperature, alkylated in the dark with 3 mM
iodoacetamide for 45 min and quenched with 3 mM DTT for 10 min. Proteins were then incubated
at 37 °C, initially for 4 h with 1.5 μl trypsin (0.5 μg/μl; Promega) and then another 1–2 h with
0.5 μl additional trypsin. To offset evaporation, 15 μl 50 mM Tris-HCl, pH 8.0 were added before
trypsin digestion. All steps were performed with constant shaking at 1,100 rpm. Resulting peptides
were combined with 50 μl 50 mM Tris-HCl, pH 8.0 used to rinse beads and acidified with
trifluoroacetic acid (0.5% final, pH < 2.0). Acidified peptides were desalted by ZipTip
(ThermoFisher) for MS analysis.

Dual-Luciferase Reporter Assay
HEK293T cells in 24-well plates (~50% cell density) were transfected with reporter plasmid
cocktail containing 50 ng luciferase reporter plasmids (IFN-β-luc), 5 ng TK-renilla luciferase
reporter (control vector) and the indicated expression plasmids by calcium phosphate precipitation.
Whole cell lysates were prepared at 24 – 30 h post-transfection and used for dual luciferase assay
according to the manufacturer’s instruction (Promega).

SARS-CoV-2 Propagation and Plaque Assay
T175 flaks of Vero E6-hACE2 cells were infected with SARS-CoV-2 at an MOI of 0.005 for 72
hours. Supernatants were collected after centrifuging at 2500 rpm for 5 min, aliquoted and stored
at -80°C. Virus titer was determined by plaque assay. Briefly, Vero E6-hACE2 cells were infected
in duplicate or triplicate with serial dilutions of virus for 45 min in serum-free DMEM, overlaid
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with 1× DMEM/1% agarose, and incubated at 37°C for 3 days. Cells were fixed with 4%
paraformaldehyde for 1 h. Overlay was removed, and plates were stained with 0.2% crystal violet
and washed with water.

FRET-based substrate cleavage assay
A fluorescent substrate harboring the cleavage site of SARS- CoV-2 Mpro (Dabcyl-
KTSA VLQSGFRKM-E(Edans)-NH2; GL Biochem) and buffer composed of 20 mM Tris, 100 mM
NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4 was used for the inhibition assay. In the FRET-based
cleavage assay, the fluorescence signal was monitored at an emission wavelength of 475 nm with
excitation at 340 nm, using a SpectralMax iD5 multi-mode microplate readers (Molecular Devices)
or at emission wavelength of 470 nm with excitation at 340 nm, using Cytation 5 cell imaging
multimode reader (BioTek). Stock solutions of Nsp5 inhibitors were prepared with 100% DMSO.
Initially, 5 μL of the SARS-CoV-2 or SARS-CoV Nsp5 at the final concentration of 0.5 μM was
pipetted into a 96-well plate containing pre-pipetted 44 μL reaction buffer and 1 uL of drug at
different final concentrations varied from 0.039 to 40 μM (0.039, 0.078, 0.16, 0.31, 0.63, 1.25, 2.5,
5, 10, 20, 40 μM). Incubation was done at 30 deg. C for 0h, 15 mins, or 3h. Afterwards, the reaction
was initiated by addition of 50 μL of the substrate dissolved in the reaction buffer to 100 μL final
volume at 20 uM. Reactions were monitored every 38 seconds for one hour. The initial velocities,
calculated by linear regression, were plotted against various concentrations of Nsp5 inhibitors by
the GraphPad Prism 9.0 software for the calculation of the IC50 values. Measurements of
inhibitory activities of the compounds were performed in duplicate and are presented as the mean
116

± SD.

2.5 References
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(2) Zaki, A. M.; van Boheemen, S.; Bestebroer, T. M.; Osterhaus, A. D. M. E.; Fouchier, R. A. M.
Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. N Engl J Med 2012,
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H.; Ng, T. K.; Yuen, K. Y . Coronavirus as a Possible Cause of Severe Acute Respiratory. THE
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(4) Tan, W.; Zhao, X.; Ma, X.; Wang, W.; Niu, P.; Xu, W.; Gao, G. F.; Wu, G. A Novel Coronavirus
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(5) Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu,
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(6) Zhou, P.; Yang, X.-L.; Wang, X.-G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.-R.; Zhu, Y .; Li, B.;
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Chapter 3: A methodological platform for chemical-genetic interrogation of RAF inhibitor
polypharmacology and biology

3.1 Introduction
3.1.1 Protein kinases in signal transduction
Cellular signaling is a tightly regulated process that enables cells to respond adequately to
their environment. In the presence of certain stimuli, cells are equipped with mechanistic
machinery that translates extracellular signals (e.g., pathogens, hormones, damage markers, inter
alia) to the appropriate internal response.
1,2
In many instances, this is often a change in gene
expression that reprograms the cell towards a state that meets the demand of the stimulus. This
intracellular process (in contrast to intercellular signaling, whereby individual cells communicate
to one other) is achieved through a cascade of biochemical reactions, termed signal transduction.
3

In short, various effector molecules within a cell may participate in a chain of reactions that result
in the alteration of a signaling molecule’s size, shape, or charge. It is through these molecular
“deviations” and a series of corresponding molecular “responses” that lead to momentary or
persisting changes in transcription/translation. Given the wide range of activities that a cell must
perform in order to survive and function, it is unsurprising that several signaling axes within a cell
may be firing simultaneously, such that a high degree of complexity is ever-present.
4
However,
this complexity can be reduced to some unifying principles, one of which is that the signal is
mediated by the covalent modification of proteins (serving as messengers of the transduction) by
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small chemical motifs after they have been translated.
5
Some of the most well-studied and
frequently occurring examples of protein post-translational modifications (PTMs) include
phosphorylation, acylation, and glycosylation.
Perhaps the most important – at least, the most prevalent – PTM is protein phosphorylation,
which is carried out by protein kinases (Figure 3.1).
6
This family of enzymes is one of the largest
in the human genome, being comprised of 538 distinct members that account for approximately 2-
3% of all human gene products.
7
The act of phosphorylation entails reversible attachment of a
negatively charged phosphate group (sourced from the γ-phosphate of ATP) to hydroxyl side
chains of neutral, polar amino acid residues such as serine, threonine, and tyrosine. Although less
common, some non-canonical phospho-sites have now been documented on residues including
arginine, histidine, lysine, aspartate, glutamate, and cysteine.
8,9
The major net consequence of this
PTM is charge introduction, which carries the potential to alter the stability and/or structure,
subcellular location, and enzymatic activity of the phosphorylated substrate through a variety of
mechanisms.
10

Figure 3.1 Protein phosphorylation by kinases. Schematic representation of kinase-mediated
phosphorylation of canonical substrate residues. Modified proteins proceed to induce an effect by
signal transduction.

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Protein kinases operate across a vast spectrum of distinct signaling pathways that have very
diverse functional outputs including cellular proliferation, differentiation, induction of apoptosis,
and cell motility, to name a few.
11
One natural consequence of this ubiquitous functional presence
is an extensive scope of protein substrates. Indeed, it is estimated that one third of all human
proteins are phosphorylated within a cell at any given moment, with over 230,000 documented
phosphorylation sites existing in humans.
12
The protein phosphorylation landscape is increasingly
convoluted (within the context of signal transduction) by a non-linear regulatory and feedback
network. For example, it is not appropriate to characterize a signaling event as proceeding merely
through a linear causal chain such that protein A phosphorylates protein B, protein B
phosphorylates protein C, et sic porro. Rather, phosphorylation events may loop back onto itself
within a single signaling axis (e.g., protein C phosphorylates protein A, potentially at a distinct
phospho-site) or they may create intersections between distinct signaling axes, in which case a
single substrate may serve as a node conjoining multiple pathways. Furthermore, phosphate
moieties may be removed from proteins by a hydrolytic reaction carried out about protein
phosphatases.
13
The action of phosphatases introduces an on/off switch that renders the
phosphorylation status of a substrate subject to spatial and temporal regulation (i.e., where is a
protein localized when it is phosphorylated and how long does it carry the phosphate marker?)
14

These features indicate a cellular signaling environment that is exceedingly complex, governed by
four-dimensional regulation.
15

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3.1.2 Mitogen-activated protein kinase signaling pathway
The RAS-RAF-MEK-ERK pathway (alternatively known as the mitogen-activated protein
kinase / extracellular signal-regulated kinase: MAPK/ERK pathway) is a major signal transduction
pathway that relays extracellular signals from cell surface receptors to the nucleus to regulate
transcription of genes critical for cell growth, differentiation, and survival (Figure 3.2).
16,17

Initiation of the signal cascade is triggered by ligand binding to receptor tyrosine kinases (RTKs).
Classically, growth hormones capable of acting as mitogens (e.g., epidermal growth factor, platelet
derived growth factor, etc.) feed into the MAPK pathway by binding to their cognate receptors
(epidermal growth factor receptor, platelet derived growth factor receptor, etc.)
18
Upon ligand
binding, the cytoplasmic domains of RTK dimers undergo a conformational change, becoming
reoriented and primed for auto-phosphorylation in trans by their intracellular kinase domains.
Newly-formed phospho-tyrosine residues then serve as binding sites for adaptor proteins (e.g.,
growth factor receptor-bound protein 2, GRB2) containing SH2 domains, which themselves recruit
guanine nucleotide exchange factors (e.g., Son of Sevenless, SOS) to the activated receptor.
19
SOS
promotes the removal of GDP from RAS, enabling it to bind GTP and exist in an active “on” state.
In the GTP-bound conformation, RAS exhibits high affinity for RAF via its Ras-binding domain
(RBD) located within its N-terminal domain (NTD).
20
RAF is thereby translocated to the
membrane where it undergoes full activation by priming phosphorylation events, enabling
homo/heterodimerization through its kinase domain. Fully activated RAF then sets in motion a
kinase cascade, first phosphorylating its substrate, dual specificity mitogen-activated protein
kinase 1/2 (MEK1/2), which in in turn phosphorylates extracellular signal-regulated kinase 1/2
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(ERK1/2).
21,22
Phosphorylated ERK can effect various signaling events, though the most common
fate for ERK-family proteins involve its translocation into the nucleus to modulate the activity of
nuclear transcription factors.
23

Figure 3.2 RAS/MAPK signaling. Growth factor (GF) stimulation of RTKs trigger RAS-GTP
activation and rouse downstream phosphorylation of MAPK effectors. Figure adapted from V oskas,
D. et. al., J. Cell . Physiol. 2014.
24

3.1.3 Regulation of RAF activity and dimerization
The RAF family of proteins is widely considered as one of the most critical, core components
of the MAPK pathway. The family members are encoded by three homologous genes (ARAF,
BRAF, and CRAF), which adopt typical kinase domain architecture featuring a highly conserved
active site housed within a cleft between two asymmetric lobes (Figure 3.3).
25
The N-terminal lobe
(N-lobe) is generally the smaller of the two, comprising roughly 80 amino acids in a five-stranded
β-sheet and one α-helix (C-helix), while the larger C-terminal lobe (C-lobe) comprises up to 200
amino acids arranged as α-helices and loops. Catalytically important residues – such as those of
the nucleotide (ATP or ADP) binding pocket, the magnesium binding site (DFG motif), and the
139

phospho-acceptor site (activation loop) – are found at the interface between the two lobes, which
are connected by a short and flexible conserved loop known as the hinge region. Formation of the
enzyme-substrate complex and release of the catalytic product depend on lobe motion and are
regulated by hinge region conformation.
26

In their inactive state, kinases can sample a wide range of conformational space, reflecting the
high degree by which they may be regulated. However, catalytically active kinases adopt a specific
conformation, characterized by the proper alignment of two hydrophobic “spines” that connect the
N- and C-lobes. Assembly of the regulatory spine (R-spine) is a hallmark signature of a
catalytically competent kinase and is driven by a regulated set of events (including PTMs – largely
phosphorylation – along the activation loop). Catalytic spine (C-spine) assembly is achieved upon
ATP-binding, allowing the two kinase lobes to close, poising the kinase for catalysis. When the
kinase is in a closed conformation, the C-helix of the N-lobe becomes fixed in an inward position,
precluding the proper folding of the activation loop and causing it to adopt an unfolded, disordered
state (Figure 3.3).
27
Upon this positioning, the catalytic residues are brought into productive
distance and orientation for transfer of the phosphate group from ATP to the substrate.

140

Figure 3.3 Conformational features of RAF activation. Active BRAF protomer (left)
participating in back-to-back dimerization features a disordered activation segment and an αC-IN
conformation. Inactive monomer (right) contains a highly ordered, helical activation segment and
αC-OUT conformation. Figure adapted from Karoulia, Z. et al., Nat. Rev. Cancer 2017.
27

Recent efforts in RAF structural biology have revealed key features of its dimerization patterns
and how conformational transitions occurring during dimer formation can drive/regulate kinase
activation.
28
The solution of a crystal structure for monomeric BRAF indicates that cytosolic RAF
adopts an auto-inhibited state due to an intramolecular interaction between its N-terminal
regulatory domain and C-terminal kinase domain.
29
In this conformation, the N- and C-lobes of
the kinase domain are configured in an open arrangement, which is counterproductive for catalysis.
Additionally, the C-helix is positioned outwards and is stabilized by residues of a folded activation
loop. A separate crystal structure of dimeric BRAF bound to its substrate, MEK, shows the C-helix
fully shifted inwards and engaging in productive enzyme-substrate interactions, which appear to
be enabled by an unstructured activation loop.
30
Key to the RAF dimerization interface is a small
region of the C-helix of each protomer, apparently critical for maintaining dimer integrity.
31

141

Previous mutagenesis experiments have shown that the specific interaction between R509 residues
of each protomer is essential for dimer formation.
32
The above structural studies clearly illustrate
an interconnected structural network that exists amongst the C-helix, activation loop, and N/C-
lobes and demonstrate a mechanism by which RAF dimerization facilitates kinase activity.

3.1.4 Dysregulation of RAF in disease and therapeutic strategies
Aberrant signaling along the MAPK axis is associated with cancer; instances of hyper-
activating mutations in the RAS-RAF-MEK-ERK core have been found in human tumors.
33
These
mutations drive unchecked activation of MAPK-regulated effectors and can lead to uncontrolled
cellular proliferation.
34
Several decades of research have pointed to frequent occurrences of
activating mutations in genes encoding for the RAS family of proteins, accounting for or present
in 15-30% of cancer incidences.
35
Unfortunately, RAS is notoriously difficult to drug by small-
molecule inhibition and has been somewhat overlooked in favor of its downstream kinases as
viable therapeutic targets.
36
Amongst those kinases – RAF, MEK, and ERK – RAF has traditionally
been the focal point in anti-oncogenic drug development programs. This is in large part due to the
high mutation frequency of BRAF: a mutation in the BRAF gene is found in 8% of all human
tumors, including melanoma (50% occurrence rate), papillary thyroid cancer (45%), colon cancer
(10%), and non-small-cell lung cancers (NSCLCs) (10%).
37–40
One particularly malignant and
omnipresent mutation is the 1799T>A substitution that results in an amino acid change of valine
(V) to glutamate (E) in the activation loop (V600E).
41
This missense mutation converts a
hydrophobic isopropyl group found in the wild-type protein to a negatively charged carboxylate.
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Functionally, this produces a phosphomimetic effect, as it simulates an activating phosphorylation
event that occurs on nearby T599 and S602 residues. The V600E mutant thus generates an artificial
salt bridge that forms between E600 and K507, a residue that resides near the C-terminal end of
the C-helix.
42
This provides a positive interaction that encourages the kinase to become locked in
a constitutively active closed conformation, even in the absence of upstream activation or
stimulation.
28
Therefore, BRAF
V600E
bypasses regulatory signals and induces downstream
signaling well beyond levels typically found in a healthy cell.
Early efforts to discover selective ATP-competitive inhibitors of mutant BRAF resulted in the
production of vemurafenib and dabrafenib.
43,44
Both molecules contain nitrogen heterocycles
intended to mimic the structural and electronic properties of the adenine ring of ATP and possess
a sulfonamide specificity element that was designed to enhance discriminative binding of RAF
kinases. The two inhibitors yielded excellent clinical results and were granted Food and Drug
Administration (FDA) approval for the treatment of BRAF
V600E
-driven melanoma in the early
2010s. However, vemurafenib and dabrafenib efficacy was limited; although monotherapy with
either molecule prolonged expected patient survival, neither were curative and were marred by
eventual drug resistance development and, ultimately, tumor relapse.
45,46
These observations led
clinicians to prescribe combinations of RAF inhibitors and MEK inhibitors (motivated by pre-
clinical evidence) in the hopes of more potently diminishing ERK signaling output.
47
Combination
therapies of vemurafenib and cobimetinib (Roche/Genentech) and dabrafenib and trametinib
(Novartis) are considered the new standard of care for RAF-driven metastatic melanoma today.
48–
50
Despite clinical efficacy, resistance mechanisms still develop and thus incite researchers to
143

develop more potent MAPK inhibitors that can produce longer lasting and more durable responses.

3.1.5 Paradoxical activation: a transactivation model for inhibitor-induced RAF activity
RAF inhibitors have become somewhat infamous for displaying strange biochemical
properties. “Traditional” kinase inhibitors can be classified in up to 6 different types, which differ
based on their binding mode and the consequent structural effect on the drug-enzyme complex
(Figure 3.4).
51
Type I and II inhibitors are both ATP-competitive, occupying regions of the adenine
binding pocket while establishing hydrogen bonds with the hinge region. They differ in the
resulting catalytic competence of the enzyme: type I inhibitors engage in a kinase active (DFG in,
C-helix-IN) conformation while type II inhibitors bind to an inactive (DFG-out, C-helix-IN)
conformation. Other types of kinase inhibitors bind allosterically to a region adjacent to the ATP-
binding pocket (type III) or to a location entirely distinct from the active site (type IV).
52,53
They
may also be bivalent in nature, binding simultaneously to two regions of the kinase domain (type
V) or may bind through a covalent mechanism (type VI).
54,55
RAF inhibitors, such as vemurafenib
and dabrafenib, fall into a distinct category (type I
1/2
), which can be described as kinase-competent
(DFG-in, C-helix-OUT), but with C-helix displacement of varying severity depending on the
chemical properties of the inhibitor.

144

Figure 3.4 Types of kinase inhibitors. Classifications of inhibitors are based on catalytic impact
or binding mode. Figure adapted from Martinez, R. et al., Next Gener. Kinase Inhib. 2020.
51

Type I
1/2
inhibition yields puzzling physiological consequences. While most kinase inhibitors
suppress enzymatic activity upon binding in all types of cells, RAF inhibitors selectively suppress
MAPK signaling only in cells expressing mutant BRAF.
56
In tumors and normal cells expressing
wild-type BRAF, there is an apparent increase in MAPK output as a direct result of RAF inhibitor
treatment.
57
This phenomenon has been linked as a potential mechanism of drug resistance,
wherein RAF inhibitors appear to paradoxically activate RAF-dependent signaling.
Through detailed analyses of crystallographic data and carefully designed, cell-based
biochemical assays, researchers have proposed a transactivation model to account for the
aforementioned paradoxical observations (Figure 3.5).
58
In summary, an initial binding event
between a type I
1/2
inhibitor and a single RAF protomer places the C-helix in an outward, displaced
position. Such an orientation is not sterically allowed for both protomers within the RAF dimer.
Thus, the inhibitor can impact the opposing protomer by allosteric means: stabilizing the inhibitor-
bound C-helix in the outwards orientation forces the opposite protomer to adopt a C-helix-IN
conformation. This has two consequences. First, the second protomer – by virtue of adopting a
forced C-helix-IN position – has reduced affinity for binding a C-helix-OUT type inhibitor. It
145

simply cannot accommodate the spatial requirements for such a binding event. Second, in cells
containing high levels of activated RAS-GTP (e.g., non-BRAF mutant or wild-type BRAF-
expressing cells), the affinity between the second RAF protomer and RAS increases.
59
The forced
C-helix-IN conformation resembles RAF in a state primed for both RAF-RAF dimerization and
RAS-GTP binding/activation. Overall, the effective outcome of type I
1/2
inhibition is a second,
inhibitor-unoccupied protomer which cannot be bound by a drug and is instead promoted to
interact with RAS-GTP, thereby increasing active RAF dimer.

Figure 3.5 Transactivation model of type I
1/2
inhibitor-induced RAF dimerization. Inhibitor
(shown in purple) binding to one monomer facilitates formation of the dimer complex, leading to
transactivation (+) of the second RAF protomer. Kinase components: C-helix (green), N-lobe
(blue), C-lobe (brown).

Efforts to better understand inhibitor effects on RAF dimerization have produced several
hypotheses concerning the impact of C-helix positioning on protomer activation. While early
models of transactivation held that the entire C-helix was relevant in its allosteric influence,
analysis of RAF priming by C-helix-IN inhibitors revealed that only the positioning of a single
critical R506 residue was pertinent.
42
According to this newer model, C-helix-IN inhibitors (which
were observed to promote RAF-RAS-GTP interactions) induced RAF dimerization because of
R506 displacement to an inward position. Investigation of vemurafenib and dabrafenib co-crystal
146

structures with BRAF later affirmed that despite stabilizing a C-helix-OUT conformation overall,
R506 was oriented inwardly. These findings eventually informed the development of next-
generation inhibitors (e.g., PLX7904, PLX8394, etc.) which are able to stabilize the entire C-helix
– including the R506 residue – in an out position, avoiding paradoxical activation.
60
Although
these so-called paradox breakers, in principle, represent a class of RAF inhibitors that may
circumvent resistance mechanisms currently seen in the clinic, there is still much left to be
understood about their detailed mechanisms of action. For example, it is not clear whether paradox
breakers are able to exert their dimer-breaking effects through binding of a single BRAF protomer
alone, a single CRAF protomer alone, both protomers of a dimer pair, or through some other mode
of engagement.
61,62

3.1.6 Perspectives and motivations
In order to inform future inhibitor design, there is a need to achieve a greater level of
understanding of the mechanistic underpinnings of the transactivation model. There are many
considerations that are left unexplored and thus leave gaps in our models accounting for
paradoxical activation. Three such factors are immediately pressing and demand a methodological
platform that addresses them.
Firstly, it has already been shown that the specific chemical properties of an inhibitor can
dramatically influence the extent to which transactivation occurs. Of the type I
1/2
inhibitors,
dabrafenib and vemurafenib appear to exert varying degrees of C-helix displacement, to the effect
that minor helical perturbances only impart a blunted negative allosteric effect. Despite
147

dabrafenib’s classification as a type I
1/2
kinase inhibitor, crystal structures have been produced that
reveal dual occupancy of both protomers in the BRAF dimer, likely due to a lesser C-helix-OUT
configuration adopted by dabrafenib-bound RAF.
63
On this same note, other types of kinase
inhibitors, including type I inhibitors GDC0879 and SB590885, appear to robustly steer RAF
monomers towards dimerization and RAS-GTP activation, despite lacking any obvious structural
features that would be suggestive of this phenomenon.
64,65
Experimental approaches to assess RAF
transactivation must therefore be sensitive to the binding properties of the inhibitors used;
methodologies must be able to account for subtle protomer-occupancy characteristics of the
inhibitors used and control the extent and nature of protomer engagement.
Secondly, inhibitor-induced RAF dimerization is subject to nuanced differences in their
observed arrangements and permutations. While the earliest documentations of RAS-activated
RAF dimerization indicate BRAF-CRAF heterodimerization as the default signaling mode,
various other combinations are accessible pending the regulatory environment of the cell in
question. For example, in-frame deletions in BRAF (e.g., L485-P490 and N486-P490) have been
observed in certain tumors and were determined to be signaling via homodimerization.
66
A recent
study has also depicted KRAS mutation scenarios in which MAPK signaling is dependent on
CRAF activation and heterodimerization with ARAF.
67
In fact, many of these sorts of examples
abound and evince a RAF dimerization climate in-cell that is becoming increasingly obscure.
Various possible dimer modes exist and are regulated by complex mechanisms. At present, there
is no systematic study available that adequately profiles the transactivation phenomenon paying
mind to the – in principle – 9 RAF dimer permutations possible between the 3 extant RAF isoforms.
148

The last factor in consideration is an extension of the previous point: mutant RAF alleles
exhibit high levels of diversity in their properties and can manifest as very different dimerization
behaviors. Of the 200 or so mutant alleles identified in human tumors for BRAF alone, three
classes of mutations emerge that can be distinguished based on their levels of kinase activity, RAS-
dependency for activity, and dimer-dependency for activity.
68
The RAF mutational landscape is
certainly rife and rich with a mixed assortment of mutations, point or otherwise, that contribute to
vast diversity in dimerization properties. Successful attempts to dissect RAF transactivation should
be able to trace an observed dimerization effect to a possible mutation-dependent cause.
To better understand paradoxical RAF transactivation, we envision the application of an allele-
specific chemical genetics approach that utilizes covalent complementation between specific RAF
isoforms and cognate small-molecule probes tailored to mirror properties of various kinase
inhibitor types. This methodological platform will allow for the profiling of RAF transactivation
events in a manner that is sensitive to the RAF dimerization mode (hetero vs. homo) and the
participant isoforms (Figure 3.6). Moreover, these effects can be related to specific and controlled
inhibitor/probe occupancy patterns, as enabled by a bio-orthogonal interaction between an
engineered RAF isoform and a small-molecule ligand.
149

Figure 3.6 Allele-specific chemical genetic engineering of isoform-selective RAF inhibitors.
Conventional RAF inhibitors cannot distinguish between highly homologous kinase domains (left).
Isoform-selectivity imparted through orthogonal Ele-Cys pair (right).

3.2 Engineering covalent complementation to probe RAF kinase activity and biology
3.2.1 Design and development of isoform-selective RAF inhibitors
Previous efforts to establish allele-specific chemical genetics platforms for studying kinases
have been successful in identifying selective, irreversible inhibitors for the RTK, Ephrin type-B
receptor 3 (EphB3).
69
The method relies on the use of inhibitors bearing a 4-anilinoquinazoline
scaffold with reactive electrophilic warheads that covalently target an artificially-introduced
cysteine residue located in the hinge region. Through sequence alignment analyses, we have
identified the homologous residue in BRAF (S535) and mutagenized the protein to introduce an
analogous cysteine. This is performed in the hopes of applying similar quinazoline-based inhibitors
for selective covalent modification of the engineered BRAF
S535C
. Hereafter, achieving covalent
complementation by reaction between an electrophilic warhead and an engineered cysteine will be
referred to as the “Ele-Cys” method.
Our interest in the 4-anilinoquinazoline scaffold is born from its success as a high-binding-
150

affinity pharmacophore in EGFR antagonists.
70,71
Given the high degree of similarity between
RTK and RAF family kinase domains, we intended to modify the scaffold in a manner that would
decrease its affinity for EGFR and increase binding selectively to RAF. Based on structural
information about EGFR-inhibitor complexes, it is known that the aniline ring extends deeply into
a small, hydrophobic pocket located within the kinase active site.
72,73
Our strategy was to modify
this aniline moiety and attach additional functional groups that would preclude well-fit binding to
EGFR. Our intention was to graft certain specificity elements from parent compounds used as RAF
drugs (GDC0879, vemurafenib, and paradox breaker PLX7409) onto the aniline ring. This would
impart chemical similarities to the newly-designed inhibitors such that they may be used as proxies
for drawing conclusions about drug-induced effects on RAF behavior. Following these design
principles, a panel of RAF inhibitors was synthesized. The inhibitor, 3.1, possesses a 2-chloro-5-
hydroxyphenyl ring that was utilized to enhance potency and selectivity in EphB3 inhibitors.
Although this moiety does not exhibit any structural analogy to GDC-0879, the two molecules
were determined to have similar functional properties. On the other hand, inhibitors 3.2 and 3.3
were appended with propylsulfonamide and N-ethyl-N-methylsulfamide groups that are directly
borrowed from vemurafenib and PLX7904, respectively (Figure 3.7).
151

Figure 3.7 RAF inhibitors and quinazoline-based derivatives. Specificity motifs are
highlighted to indicate preservation of functionality between drugs and Ele-Cys covalent inhibitors.

3.2.2 Functional assessment of Ele-Cys platform
In order to assess the efficacy of our inhibitors as BRAF-targeting molecules in live cells, we
set out to monitor their ability to modulate downstream signaling in the MAPK pathway after
transient transfection of inhibitor-reactive BRAF in HEK293T cells (Figure 3.8B). To simplify the
analysis, a triple mutant of BRAF (BRAF
TM
) was generated which contains the hyper-activating
oncogenic mutation V600E, the reactive cysteine mutation S535C, and a dimer-disrupting
mutation R509H. The decision to include these three mutations was to have a system with
monomeric BRAF that could induce MAPK signaling activity while exhibiting similar sensitivity
to inhibition by vemurafenib. MAPK signaling output is measured by immunoblot detection of
phosphorylated MEK and ERK (pMEK/pERK). The data indicates that all three inhibitors were
effective in diminishing phosphorylation events downstream of RAF, compared to a control
molecule possessing a naked phenyl ring (lacking any specificity elements, Figure 3.8A). The most
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potent inhibitor against monomeric, hyper-activated BRAF was the vemurafenib analogue, 3.2,
which is in line with clinical and experimental data. This experiment proves definitively that our
electrophilic inhibitors can be used to target engineered RAF proteins and can provide information
about inhibitor-induced effects on MAPK signaling in cells.

Figure 3.8 Potent and selective inhibition of engineered BRAF
TM
by quinazoline inhibitors.
A) Structure of control molecule lacking specificity elements. B) Demonstration of methodology
in cells was monitored by phospho-immunoblot of downstream RAF signaling.

Next, we were interested in exploring the extent of information that could be derived from our
platform. Our initial ambition was to develop a methodology that could provide information
relating an inhibitor’s structural features to active site occupancy within RAF dimers of controlled
protomer identity. We conducted a pilot experiment, set up in a manner that we reasoned would
representatively address all these facets. HEK293T cells were co-transfected with CRAF
WT

containing a V5 epitope tag and BRAF
S535C/V600E
-FLAG and exposed to a dose-course treatment
of inhibitors 3.1, 3.2, and 3.3 (Figure 3.9A). The results indicate that in co-expression conditions
conducive to BRAF-CRAF heterodimer formation, our GDC0879 analog 3.1 was ineffective in
inhibiting MAPK signaling. The BRAF isoform bears the reactive cysteine – thus, we can infer
that 3,1 occupancy of the BRAF protomer induces transactivation through the kinase-active
153

CRAF
WT
. In contrast to this, both 3.2 and 3.3 were able to disrupt RAF signaling, presumably by
preventing dimerization and subsequent transactivation. Importantly, depending on the appended
specificity elements, the inhibitors exert unequal effects. As expected, the PLX7904 analog 3.3
was most effective in reducing dimer formation, while slight resistance was observed for the
vemurafenib analog 3.2. We also explored the differences in single vs. dual occupancy of
heterodimers and how that manifests in the context of MAPK downstream signaling (Figure 3.9B).
Co-transfections of BRAF
V600E
-CRAF
WT
were performed in a set such that both, neither, or one of
each isoform bears the reactive cysteine handle. After a dose-course treatment of 3.1, we show that
only dual occupancy is effective in inhibition of MEK phosphorylation. This lends support to the
idea that GDC0879-like inhibitors induce transactivation through the unbound protomer and that
overall kinase output is only compromised when both protomers are occupied.
74
The two selections
described above demonstrate that our system can impart a rich amount of variable-specific
information and allows for systematic analysis of its individual components, depending on the
researcher’s needs and interests.

154

Figure 3.9 Effects of isoform-selective RAF inhibitors on MAPK signaling. A) Dose-response
effects in various BRAF-CRAF dimer contexts. B) Single vs. dual RAF occupancy and variable
signaling responses to GDC-0879 mimic.

Finally, in addition to signal transduction (measured by phosphorylation of downstream
substrates), we were interested in reading out effects specifically concerning RAF dimerization
events. In particular, we would like to be able to retrieve information about how inhibitor type and
inhibitor occupancy influence the ability of different dimers to form, depending on the nature of
the participant protomers. Experimentally, 293T cells were co-transfected to enable formation of
155

BRAF
V600E
-CRAF
WT
heterodimers or BRAF
V600E
-BRAF
V600E
homodimers in which either a single
or both constituent protomers are engineered with a reactive cysteine. The cells were then exposed
to a dose-course treatment of 3.1, 3.2, and 3.3 and analyzed for dimerization by co-
immunoprecipitation. Briefly, the results indicate that paradox-disruptor mimic 3.3 – true to its
name – is effective in interrupting the dimerization event in all contexts of single vs. dual
occupancy, irrespective of protomer identity. Strikingly, vemurafenib analog 3.2 had relatively
modest inhibitory effects on dimerization, except during dual occupancy of BRAF
V600E
-
BRAF
V600E
homodimers, in which it potently prevented dimerization. Compound 3.1 was
ineffective in all contexts though it appeared to aggressively stimulate transactivation during single
occupancy of BRAF
V600E
-BRAF
V600E
homodimers. These results demonstrate that this system is
indeed able to provide a stunning degree of control, which allows the user to garner isoform-
specific/inhibitor-specific information about RAF dimer perturbation.

156

Figure 3.10 Dimer formation effects in response to varied RAF inhibitors in multiple contexts.
Dose-response effects arising from RAF inhibitors enabling single vs. dual occupancy in homo-
and hetero-dimers. A) Single-site heterodimers, B) dual-site heterodimers, C) single-site
homodimers, and D) dual-site homodimers.

3.2.3 Inhibitor-derived chemical probes to monitor RAF occupancy
Because electrophilic compounds are intrinsically reactive in biological systems, we
determined it was necessary to synthesize probe analogues of these inhibitors to assess proteomic
selectivity and RAF-specific target occupancy. This is to ensure that any phenotypes observed as
a result of inhibitor treatment can be justifiably pinned to RAF inhibition and not to non-
specific/off-target inhibition. These molecules are outfitted with terminal alkyne moieties that can
be used as reactive handles upon copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)
reactions between themselves and azide-bearing reporter molecules (e.g., fluorophores, pull-
down/enrichment tags, etc.) We produced one such probe molecule, 3.4, after appending a
propargyloxy group to the 6-position of the quinazoline core found in 3.2 (Figure 3.11A).
157

Probe 3.4 was shown to label BRAF
DM
(possessing V600E and S535C mutations)
successfully in live cells in a dose-dependent manner after visualization of BRAF-bound probe by
CuAAC with fluorogenic TAMRA-N3 (Figure 3.11B). The molecule also retained its ability to
inhibit MAPK signaling in BRAF
TM
-expressing cells (Figure 3.11C). Finally, BRAF target
engagement by 3.1, 3.2, and 3.3 was validated in a pulse-chase experiment. Briefly, cells
expressing BRAF
DM
were “pulsed” with a dose-course treatment of the inhibitors, washed out, and
finally “chased” by a moderately high concentration of 3.4. The decrease in fluorescent signal as
a function of increasing inhibitor treatment indicates that the active site of BRAF
DM
is irreversibly
occupied by the pre-treatment molecule (Figure 3.11D).

Figure 3.11 RAF quinazoline-based probe directly labels BRAF. Synthesis of a CuAAC-
enabled probe derived from 3.2 features a propargyloxy group at the 6-position of the quinazoline
core (A). B) Probe labels BRAF in cells, C) retains inhibitory effects on MAPK signaling, and D)
can be used as a probe to monitor target engagement of quinazoline inhibitors.

158

3.2.4 Visions for this methodology
The findings outlined above establish a baseline system that can be used as an interrogative
tool for probing aspects of RAF biology. Previous results demonstrate that the inhibitors can indeed
display isoform-selective effects, notably by decreasing MAPK signaling after RAF active site
occupancy. Furthermore, the system was shown to illuminate key properties of RAF kinase
activation via dimerization and provided key insights about how said process is influenced by
chemical signatures in drug molecules. Specifically, dimerization appears to be dependent on the
key functional groups acting as specificity elements, the type of isoforms participating in the RAF
dimer, and the nature of site occupancy by inhibitors. Though the platform has undeniable utility,
there are some key concerns about the details of its operation and the breadth of its applicability
to other RAF systems. In the following section, I will first attempt to provide a more thorough
charcterization of the Ele-Cys platform. I will then describe efforts undertaken to explore its
potential for shedding light on under-investigated questions in RAF biology.

3.3 Results and discussion
3.3.1 In-depth characterization of covalent RAF inhibitors in Ele-Cys chemical-genetic
platform
Kinetics of RAF engagement by inhibitors/probes in situ
We have presented some exemplary applications of our chemical genetic platform in which it
provides unprecedented control over specific experimental variables. One of the major advantages
of a chemical genetics approach over traditional classical genetics probing methods is the timescale
159

on which it operates. That is, in order to appropriately monitor rapid, dynamic biological processes
(such as the phosphorylation events constituting signal transduction), kinetics of the small-
molecule interrogation must be commensurate. We attempt to provide in situ characterization of
covalent labeling kinetics by our inhibitors in cells transiently expressing BRAF mutants with
engineered cysteines. For this purpose, we reasoned that our CuAAC-reactive probe 3.4 could
provide a convenient model for inhibitor engagement kinetics, assuming reasonably equivalent
properties between probe and parental inhibitors. We performed a simple time-course analysis, in
which BRAF
DM
was treated with 3.4 at timescales reaching up to 2 hours (Figure 3.12A). After
the indicated time had elapsed, cells were harvested and lysate was subjected to CuAAC with a
TAMRA-N3 reporter group. The labeling kinetics were read out by in-gel fluorescence of the
BRAF
DM
-3.4 covalent adduct. The results show that engagement between the engineered pair was
detected at the earliest timepoint selected, which was 5 minutes. Although the signal steadily
increased through all the timepoints include, we reasoned these kinetic properties were sufficient
for monitoring most biological processes.
Next, because the previous experiment is an indirect measure of in situ labeling kinetics –
wherein parental inhibitor kinetics are inferred by the probe results – we decided to perform a
pulse-chase experiment with a time-course treatment of 3.2 (Figure 3.12B). Cells expressing
BRAF
DM
were treated with 3.2 for varying amounts of time up to 2 hours, washed out with PBS,
and then chased by a saturating amount of 3.4 for 30 minutes. Decrease in in-gel fluorescent signal
could be used to measure the amount of time it takes for inhibitor 3.2 to saturate the BRAF
DM

binding pocket. Following this strategy, we observed results that were consistent with our previous
160

probe time-course experiment: engagement of BRAF
DM
was detected as early as 5 minutes and
appeared to have saturated over 50% of vacant enzyme in situ, indicative of much faster labeling
by 3.2 compared to the probe 3.4. These observations suggest that our system features inhibitors
with kinetic properties suitable for rapid modulation of protein function.

Figure 3.12 Time-course engagement of 3.4 to BRAF
DM
. A) Direct vs, B) indirect evaluation of
in situ 3.4 labeling kinetics. Indirect monitoring was achieved by treatment of 3.2 for varying
timepoints, followed by a saturating amount of 3.4 for 30 minutes. Transfection was performed
with 250 ng of plasmid DNA.

Qualifying differences in MAPK signaling output after RAF inhibition based on RAF abundance
One of the major concerns within the domain of RAF therapeutic discovery is the apparent
acquired drug resistance in BRAF
V600E
tumors. Other studies have shown that resistance
mechanisms can involve RAF dimerization events which would preclude active site occupancy by
161

inhibitors due to unfavorable conformational rearrangements. In cases where this phenomenon is
inhibitor-induced via binding to a single protomer, negative allostery is invoked as the driving
mechanism. It is therefore critical that we control our system to avoid forcing homodimerization
in cells transiently over-expressing an allele-of-interest. Such an occurrence would confound
interpretation of our results due to the possibility of artificial homodimerization, especially in cases
where the allele-of-interest under study is intended to be monomeric. Additionally, at high levels
of overexpression, we may be observing artificial resistance, arising merely due to an exceptional
overabundance of protein. To address this concern, we set out to optimize the transfection
procedure and titrate plasmid DNA used for engineered BRAF expression. We wanted to compare
MAPK signaling output with increasing amounts of BRAF
V600E
or BRAF
DM
treated with their
corresponding inhibitors (e.g., vemurafenib and 3.2 or PLX7904 and 3.3) and determine the cells’
sensitivity.
Cells were transfected with up to 1 µg of BRAF
V600E
or BRAF
DM
DNA and downstream
phosphorylation was monitored in the presence or absence of vemurafenib/PLX7904 and 3.2/3.3,
respectively. The 3.3/PLX7904 panel of data showed that they were quite effective at completely
abrogating MEK phosphorylation at low concentrations of DNA, with the engineered pair
exhibiting slightly greater potency. However, at very high levels of DNA, pMEK and pERK was
gradually restored, indicating a level of resistance that tracks with the amount of protein present.
These observations were essentially mirrored in the 3.2/vemurafenib pair, with the engineered pair
again displaying a more pronounced effect in diminishing downstream signaling. Inhibitor
resistance prominently appeared at higher concentrations of transfected DNA and appeared to have
162

the most significant resistant effect against vemurafenib.

Figure 3.13 BRAF DNA titration and influence of protein abundance on inhibition. Increasing
amounts of plasmid DNA for BRAF
DM
or BRAF
V600E
were used for transfection before treatment
by A) 3.3, B) PLX7904, C) 3.2, or D) Vemurafenib. E) A BRAF mutant containing a dimer-
disruptor mutation was utilized to investigate effects of dimer-based vs. stoichiometric resistance.

At this point, we wondered whether the observed resistance and restoration of downstream
phosphorylation was due to artificially forced dimerization by exceedingly high levels of protein
or to a simple stoichiometric excess of non-dimerized/monomeric protein such that the inhibitors
are limiting reagents. To test this, we repeated the same DNA titration setup using a double mutant
BRAF construct containing the V600E hyperactivating mutation and R509H, a dimer-disrupting
mutation that prevents C-helices from engaging in dimer-productive interactions. Cells treated
with or without vemurafenib produced immune-phosphoblot data that showed very poor
163

restoration of downstream signaling, even at the highest concentrations of plasmid DNA used for
transfection. Our interpretation is that this BRAF mutant is necessarily forced into a monomeric
state and cannot exhibit resistance against inhibitor binding from dimerization. Because MAPK
signaling upon inhibitor treatment at high levels of BRAF
R509H/V600E
is negligible, we concluded
that any observed resistance is due to artificial dimerization upon over-expression of RAF that
exceeds normal physiological levels. From these investigations, we learn that valid conclusions
can only be drawn in scenarios where protein is expressed at near-physiological levels. This is
especially important for our platform, given that it relies heavily on the exogenous over-expression
of a target allele. In order to address this concern, we generated transgenic C57BL/6 mice
containing genomic knock-ins of serine-to-cysteine BRAF and CRAF mutants (described in
section 3.3.4).

3.3.2 Broadly applying the Ele-Cys method to probing under-investigated problems in RAF
biology
Truncated p61-BRAF
V600E
mutants
Thus far, we have only explored a relatively limited set of problems within RAF biology,
which include transactivation among pairs of B:B and B:C homo-/hetero-dimers and the V600E
hyper-activating allele. While our results have been consistent with observations and reports in the
literature, we wondered just how broadly applicable this system truly is. Some of the under-
explored areas of RAF biology could benefit from this platform, assuming that it is not limited in
application to well-behaving and well-studied RAF systems. Therefore, we turned our attention
164

first to an alternatively spliced BRAF
V600E
isoform named p61-BRAF
V600E
. This protein is a
truncated mutant of the better-studied hyperactivating allele missing the RAS-binding domain
located near the N-terminal region of the full-length BRAF protein.
75
The absence of the RBD
drives p61-BRAF
V600E
towards constitutive dimerization and sustained downstream
phosphorylation. Its observed kinase activity is essentially monomeric and independent of
stimulation events including upstream RAS-induced activation and transactivation by vemurafenib
binding.
76
Although the truncated mutant represents a legitimate resistance mechanism, it has not
yet been reported in the clinic. However, from an academic perspective, these mutants provide an
interesting opportunity to study RAF-induced (dimer-dependent) signaling, as these variants form
constitutive dimers in cellulo without any driving factors.
77
We were therefore intent on performing
inhibitor titrations against p61-BRAF
V600E
mutants containing Ele-Cys-enabling mutations. By
comparing MAPK signaling between full-length and truncated BRAF treated with different types
of inhibitors, we hoped to observe whether there are differing intrinsic responses.
To begin, we compared expression efficiencies of the p61-BRAF
DM
and full-length BRAF
DM

in HEK293T cells (Figure 3.14A). For fair comparison, we needed to achieve equivalent levels of
the two types of protein in cells, which evidently requires roughly a 4:1 ratio of full-length BRAF
to truncated mutant.
Using 200 ng of full-length BRAF DNA and 50 ng of the p61 variant, we performed
comparisons of dose-response treatments with either 3.2 or 3.3 and determined whether there were
differences in response between the two alleles (Figure 3.14B,C). For both types of inhibitors,
there was a more prominent inhibitory effect observed against the full-length proteins. In fact, 3.2
165

was roughly 3-fold more potent against full-length BRAF than against the p61 variant. This
discrepancy was even more exaggerated by 3.3, which appeared to induce a 10-fold greater
response from full-length BRAF. As expected, the paradox-breaker-inspired analog 3.3 fared better
in inhibiting pMEK compared to 3.2. We noted that any qualitative comparisons might be slightly
obscured because MEK phosphorylation did not display a neat dose-response relationship. We
hypothesized that this might have been due to an excessive amount of protein over-expressed
above (see discussion from above). If an excess of BRAF – either full-length or truncated – is
present, we might be seeing irregular PPIs that drive rogue phosphorylation activity. We therefore
repeated the experiments with reduced levels of transfected DNA, at a new ratio of 2:1 full-
length:truncated mutant, hoping to simulate endogenous physiology more faithfully (Figure
3.14D,E). Interestingly, even under the reduced transfection conditions, our results were fairly
consistent with the previous attempt. Although 3.2 had a more pronounced inhibitory effect against
the full-length allele compared to the truncated mutant, the overall dose relationship was nearly
identical to the elevated protein conditions. Most puzzlingly, the dose-response of the p61 variant
to 3.3 was reproducible, characterized by a sigmoidal relationship which sharply dropped off upon
a threshold level of inhibitor. Regardless, from the data produced, we were able to discern certain
relationships from the system. Specifically, dimer-mediated resistance to drug treatment appeared
to be strongest when constitutive BRAF dimers are formed. This is reflected by the fact that p61
variants are generally more resistant than their full-length counterparts. These findings have
important implications for signaling generated by inhibitor-induced versus constitutive RAF
dimerization.
166

Figure 3.14 Inhibitor titration against p61-BRAF
DM
and analysis of downstream signaling.
A) Titration of plasmid DNA indicates 4:1 transfection efficiency. Dose-dependent effects of
inhibitors B) 3.2, and C) 3.3 after applying a transfection ratio of 200 ng of full-length BRAF
DM

to 50 ng p61-BRAF
DM
. Reduced transfection ratio of 50 ng of full-length BRAF
DM
to 25 ng p61-
BRAF
DM
shown against D) 3.2 and E) 3.3.

Non-canonical BRAF mutants
Hyper-activating mutations in the RAF alleles take on many forms, though the V600E point
mutation by far takes center stage due to its frequency of occurrence in patients. Thanks to rigorous
chemical and structural analysis, the V600E activating mechanism is now relatively well
understood. Nevertheless, other instances and classes of hyper-activating mutations exist and have
comparatively mysterious mechanisms of action. Recent efforts to stratify the various types of
documented RAF mutations have been instrumental in providing a framework for the
167

demystification of these mutants. The V600E mutation – simply put – falls under the class 1
designation: they are RAS-independent, dimer-independent, hyperactive mutants.
78
A variety of
other non-canonical mutants have been described and differentiated based on their dependencies
to RAS-activation or RAF-dimerization for aberrant kinase activity (which notably, can be either
elevated or diminished).
68
We decided to launch an attempt to profile non-canonical BRAF mutants
by introducing the S535C mutations into BRAF
G469A
and BRAF
D594N
, which we take as
representative examples of class 2 and 3 mutants, respectively. Briefly, class 2 mutants are RAS-
independent and hyperactively signal as homodimers; class 3 mutants are RAS-dependent and
hypoactively signal as heterodimers.
79–81
We wished to determine how amenable these alternate
mutant classes would be to our ASCG strategy and hopefully glean novel information about their
responses to various types of kinase inhibitors.

168

Figure 3.15 Ele-Cys platform and its application in non-canonical BRAF mutants. A)
Demonstration of 3.4 engagement in class 1, 2, and 3 BRAF mutants; cells were transfected with
1 µg of corresponding plasmid DNA. Global survey of inhibitor effects against sensitized and
insensitive B) class 2 mutant and C) class 3 mutants using 100 nM of 3.2 and 500 nM of 3.1/3.3.
Dose-dependent effects against D) class 2 mutants and E) class 3 mutants after transfection with
50 and 200 ng plasmid DNA, respectively.

After producing inhibitor-sensitized S535C mutants – one representative from each class of
BRAF mutation – we performed a simple labeling experiment to see if they were susceptible to
covalent modification by 3.4 (Figure 3.15A). Parental alleles lacking the exogenous cysteine
mutation were included as controls. The results show that we could achieve robust labeling of the
engineered BRAF mutants selectively, as there was no significant background labeling detected in
the single-mutant controls. With the confirmation of successful non-canonical mutant sensitization,
we proceeded to globally survey inhibitor effects on class 2 and class 3 BRAF mutant-driven
MAPK signaling using 3.1, 3.2, and 3.3 (Figure 3.15B,C). We chose to include two sets of DNA
169

transfection conditions – a relatively low set versus a high set – to determine the extent of
sensitivity of the system to abundance-related confounding effects. Reassuringly, there weren’t
any detectable changes in downstream signaling after inhibitor incubation with cells expressing
class 2 mutants lacking cysteine-enabled sensitization. On the other hand, class 2 double mutants
were, in fact, inhibited by the compounds, with minor decreases in pMEK/pERK for 3.1 and near
total depletion by 3.2 and 3.3. An analogous global survey against class 3 BRAF mutants had
similar results, though we noted some slightly confusing nuances. While paradox-breaker-inspired
3.3 was able to reduce pMEK/pERK in the class double mutants, 3.1 and 3.2 promoted an increase.
Dose-response analysis of each panel produced results that were consistent with our initial findings
(Figure 3.15D,E). Class 2 mutant-driven signaling was effectively inhibited by 3.2 and 3.3, even
at the lowest concentrations applied. Compound 3.1 also eventually extinguished pMEK/pERK,
though it required doses greater than 10-fold more for the other two inhibitors. In similar fashion,
the dose-response curves for the class 3 double mutants point to activation by 3.1 and 3.2. Some
slight differences can be teased out: 3.1 appears to reasonably sustain a basal level of pMEK
stimulation at all doses tested while the signal from 3.2 peters out in a dose-dependent manner. We
speculate that these distinctions may be related to the propensity of each mutant class to
dimerization and their unique dependence on the cellular signaling environment (e.g., RAS
activation state, upstream activation, etc.) Even so, these experiments show that alternative classes
of BRAF mutations are amenable to our chemical genetic approach, which can uncover distinct
response patterns that are dependent upon inhibitor type.

170

ARAF
Of the three RAS effectors (within the context of MAPK signaling) – ARAF, BRAF, and
CRAF – relatively little is known about the ARAF paralog. By far the most well-studied isoform
is BRAF, likely due to the prevalence of its mutations in many types of human tumors. This has
provided clinical impetus for pursuing BRAF research and developing therapeutics. It has the
highest basal kinase activity compared to the other two family members, with CRAF trailing only
shortly behind.
82
The BRAF and CRAF genes encode active kinases and appear to play critical
roles in the normal development of cells. Previous studies have shown that wholesale BRAF and
CRAF knock-out is associated with embryonic lethality; conditional ablation of these two genes
suggest varying kinase-dependent and -independent responsibilities in normal cell development
and stem cell self-renewal. In contrast, ARAF has been recorded as exhibiting very low levels of
endogenous kinase activity and do not appear crucial for cell viability and/or development.
83

Knock-out of ARAF appears to have few or no effects on ERK signaling and does not carry any
immediate lethal consequence.
84
However, ARAF-knockout mice do show tendencies towards
developing neurological and intestinal disorders.
85
Functionally speaking, studies have suggested
that ARAF is resigned to mere scaffolding roles within the MAPK pathway, providing a supportive
effect in stabilizing BRAF-CRAF heterodimers and catalytic output.
86
Recent studies are now
beginning to uncover cell-dependent contexts wherein ARAF may act as an obligatory kinase in
the absence of BRAF or CRAF activity.
67
Although the occurrence rate of ARAF mutation in
tumors is not nearly as high as it is for BRAF, there have been reports of its appearance in certain
human cancers.
87

171

The net body of ARAF knowledge that we collectively possess is quite lackluster. Though
BRAF and CRAF are understandably more attractive research subjects, a comprehensive
understanding of MAPK signaling (and the subsequent implications for medicine) demands a more
rigorous analysis of ARAF. Because ARAF plays a much more nuanced and subtle part in MAPK-
related physiology, it is difficult to apply methodologies that are appropriately sensitive. The
studies cited above suggest that ARAF biology is very dependent upon its environment and behave
differently within varying mutational landscapes. These observations invite a methodological
platform that offers high tunability and control of the system-of-study’s variables. We therefore
wished to investigate the applicability of our Ele-Cys approach as an avenue into mechanistic
studies on ARAF.
First, we applied site-directed mutagenesis to produce an ARAF mutant containing a serine-
to-cysteine mutation at the homologous position of BRAF-S535. In addition to this mutant,
ARAF
S388C
, we also chose to include a similar mutant, ARAF
389C
, as a control to determine the
sensitivity of our covalent inhibitors to subtle positional differences of the engineered cysteine.
Our first objective was to determine if we could simply observe in situ target engagement of our
ARAF mutants by covalent probe 3.4 (Figure 3.16A). Since this probe is highly chemically similar
to 3.2, we reasoned that it could give us an indication of active site complementarity. After transient
transfection of ARAF mutants and in situ incubation with 3.4, we harvested cells and measured in-
gel fluorescence after CuAAC of lysate with TAMRA-N3. BRAF
DM
was included as a positive
control and showed strong labeling. However, to our surprise and frustration, neither of the ARAF
alleles appeared to be receptive towards 3.4 binding. While BRAF and CRAF have greater than
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80% sequence homology within their kinase domains, ARAF has a slightly lower degree of
similarity. We presumed that this was preventing a binding interaction between ARAF and 3.4.

Figure 3.16 Ele-Cys platform and inhibitor-sensitized ARAF. Investigation of downstream
signaling output after ARAF sensitization towards inhibition. Concentrations of inhibitor include
100 nM 3.2 and 500 nM 3.1/3.3, unless otherwise indicated. A) In-gel fluorescence
measurements after transfection with 1 µg plasmid DNA of ARAF mutants. Effects on MAPK
signaling were measured in conditions 500 ng total transfected DNA for B) ARAF
S388C
, C)
ARAF
S388C/D447N
+ ARAF
WT
, and D) ARAF
S388C/D447N
+ BRAF
WT
. E) Global dose-response
effects of inhibitors against ARAF homodimers. F) Signaling output monitored after inhibitor
pre-treatment was chased by 3.1 incubation.

In spite of our failure to produce a fluorescent signal upon ARAF-binding, we wanted to check
whether inhibitors 3.1, 3.2, and 3.3 could induce inhibitor- and allele-specific differences in
downstream MAPK signaling. We performed a simple comparison between cells over-expressing
ARAF
WT
and ARAF
S388C
and treated them separately with our three inhibitors before monitoring
pMEK/pERK (Figure 3.16B). Although downstream phosphorylation was weak overall, there
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were detectable differences between our samples. Specifically, a prominent increase in pMEK was
observed upon treatment of 3.1 with engineered ARAF
S388C
compared to all other conditions. This
effect was also slightly observed upon 3.2 treatment, though to a far lesser extent. We wondered
whether pMEK/pERK visualization was being hampered by a lack of kinase output, despite
apparent activation caused by these two inhibitors (endogenous amounts of BRAF and CRAF may
not be sufficient to transmit signaling robust enough for our detection methods). We thus repeated
the previous experiment with a co-transfection of ARAF
S388C/D447N
and ARAF
WT
, again compared
to a condition of ARAF
WT
alone (Figure 3.16C). The inclusion of the double mutant was to probe
whether the increase in pMEK/pERK was due to dimerization (either hetero- or homo-) since the
catalytic activity of the inhibitor-occupied mutant would be deleted by the DFG motif mutation.
This could allow us to conclude that inhibitor-binding stimulates an effect akin to transactivation.
Under these conditions, 3.1 produced an intensely strong pMEK signal. This was also shown to be
dose-dependent on 3.1, as increasing amounts of the inhibitor increases pMEK output (3.16E).
Interestingly this effect is only observed when the co-transfection is performed. Additionally, we
questioned whether this activation was dependent upon the type of dimerization. In other words,
since these conditions presumably lead to ARAF
S388C/D447N
-ARAF
WT
homodimerization, we
wondered whether pMEK amplification could be observed through BRAF
WT
as well. These
conditions yielded similar results as the homodimerization, indicating that inhibitor-bound
engineered ARAF requires a kinase-active dimer partner for transmission of activity, but does not
discriminate paralog identity (Figure 3.16D). Notably, GDC0879, from which 3.1 is based, has
been reported to induce RAF dimerization across a wide range of contexts through the stabilization
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of a closed, compact kinase domain. We were pleased to see that our system could reproduce these
phenomena, which suggests that a wide range of dimer scenarios could be systematically assessed.
We questioned the inability of vemurafenib- and PLX7904-derived molecules to produce a
downstream MAPK signal (3.2, 3.3) and engage ARAF
S388C
(3.4). In an effort to experimentally
uncouple these two related but distinct phenomena, we performed an experiment where cells co-
expressing ARAF
S388C/D447N
and ARAF
WT
were pre-treated with 3.2 or 3.3 and subsequently treated
by 3.1 following PBS washout (Figure 3.16F). Because 3.1 reliably induces a strong pMEK signal,
active site occupancy during the pre-treatment with either 3.2 or 3.3 should lead to a decrease in
pMEK output. This would signify that target occupancy occurs, but in a manner that is non-
productive for kinase activation. However, if the signal is unaffected, it is likely that the inhibitors
are not able to bind ARAF. The data reveal slightly mixed results. Compound 3.2 did not appear
to appreciably alter the signal intensity; therefore, 3.2 is most likely not able to bind ARAF. On
the other hand, 3.3 treatment produced a slight decrease of pMEK. We conclude that 3.3 is able to
bind ARAF but it does not transactivate a dimerized kinase (most probably due to its paradox-
breaking properties). In summary, despite diverging binding competencies, combinations of our
inhibitors can be utilized to infer mechanistic information about ARAF biology.

3.3.3 Combining Ele-Cys with novel assay formats to probe dimer-dependent paradoxical
activation
Desthiobiotin-ATP probes as competitors to read out dimer-dependent kinase activity

175

Our earliest ambition for the Ele-Cys chemical genetic method was to have a method that
could provide precise and near-quantitative measurements of transactivation. At its most bare,
the transactivation model involves inhibitor occupancy at one protomer, followed by dimerization
and presumed amplification of kinase activity at a second, inhibitor-unoccupied protomer. Because
the phosphorylation reaction is ATP-dependent, we envisioned the use of a desthiobiotin-
conjugated ATP probe, which would allow us to enrich ATP-bound/kinase-competent
transactivation partners.
88
The particular desthiobiotin-ATP probe we imagined employing
operates by covalent transfer of the desthiobiotin enrichment handle to a conserved lysine that sits
near the ATP-binding pocket.
89
The overall idea would be that augmented kinase activity via
transactivation leads to RAF proteins exhibiting greater affinity for the ATP cofactor such that
“capturing” the ATP binding event and enriching biotin-tagged kinases could allow us to
qualitatively and quantitatively profile transactivation occurring in RAF dimers. Even further,
when coupled with Ele-Cys, we would be able to comprehensively profile all possible dimer
permutations while controlling inhibitor occupancy.
To establish this system, we first assessed whether recombinant BRAF could be enriched after
treatment with the desthiobiotin-ATP probe. Recombinant BRAF
V600E
-6xHis expressed from sf-9
insect cells was incubated with desthiobiotin-ATP in either the absence of presence of an
equivalent amount of vemurafenib. The samples were enriched by Streptavidin pulldown and
analyzed by immunoblot. We were able to enrich a significant amount of BRAF treated with the
probe and could show that vemurafenib partially out-competes desthiobiotin-ATP binding (Figure
3.17A). We repeated a dose-course variation on this experiment using two sets of conditions:
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BRAF
V600E
-6xHis treated with vemurafenib and BRAF
DM
-6xHis treated with 3.2 (Figure 3.17B).
The results indicate that in the absence of inhibitor, BRAF could be reliably enriched using the
desthiobiotin-ATP probe. Moreover, both types of inhibitors were able to partially out-compete the
probe before BRAF enrichment, with 3.2 slightly edging out vemurafenib. Interestingly, neither of
the inhibitors were able to completely out-compete desthiobiotin-ATP probe binding, even at
equivalent concentrations.

Figure 3.17 In vitro labeling of recombinant BRAF with a desthiobiotin-ATP probe. Reaction
conditions included BRAF at a final concentration of 0.3 µM. A) Monitoring probe labeling
efficiency and B) dose-dependent titration of inhibitor (vemurafenib or 3.2) in BRAF
V600E
or
BRAF
DM
, respectively.

Having shown that the desthiobiotin-ATP probe was functional in an in vitro format, we moved
into performing similar experiments in situ. At the most basic level, we first wished to see whether
the desthibiotin-ATP probe could be used to readout inhibitor binding of BRAF
DM
transiently
expressed in 293T cells. Our workflow would proceed by performing the in situ incubation of 3.2
with BRAF
DM
-expressing cells, followed by treatment of the lysate with the desthiobiotin-ATP
probe and subsequent Streptavidin enrichment. In the absence of inhibitor, BRAF
DM
was nicely
enriched and produced a prominent signal by immunoblot, indicating that we could pulldown
recombinant protein from 293T cells (Figure 3.18A). Moreover, dose-course treatment of 3.2
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returned a very nice dose-response curve, gradually out-competing desthiobiotin-ATP probe
binding to completion, in virtue of irreversible active site occupancy. Essentially total ablation of
enrichment efficiency was ablated by 0.03 µM 3.2. We attempted to repeat the analogous
experiment for ARAF
S388C/D447N
given the success we saw of 3.1-induced binding and activation
in previous results (Figure 3.18B). Unsurprisingly, no ARAF could be enriched, since binding of
the ATP analog probe was precluded by mutation of the DFG motif, critical in mediating ATP
binding interactions. We thus repeated the experiment and included ARAF
S388C
and evaluated
pulldown efficiency against the ARAF
S388C/D447N
double mutant in the presence or absence of pre-
treatment with 3.1 (Figure 3.18C). These results were quite promising; a robust immunoblot signal
was produced in the ARAF
S388C
, DMSO-treated control condition and was out-competed upon pre-
incubation with 3.1. Before proceeding, we wished to confirm the role of the DFG motif in
influencing readout by the desthiobiotin-ATP probe in BRAF. We included analogous BRAF
mutational conditions as in the ARAF experiment, inhibitor-sensitization and/or inactivated-DFG
mutants and confirmed that the kinase-dead point mutation ablates desthiobiotin-ATP engagement
(Figure 3.18D). Only partial inhibition of the inhibitor-sensitized single mutant was observed upon
treatment 3.2, which we rationalized was due to the low concentration of inhibitor utilized. These
results indicate that in situ profiling of RAF-inhibitor interactions is possible with our
desthiobiotin-ATP probe.

178

Figure 3.18 Competitive labeling of transiently expressed RAF by desthiobiotin-ATP probe.
Inhibitor treatment was performed in situ after application of the corresponding transfection
conditions of either 300 ng BRAF (A, D) or 1.5 µg ARAF (B,C) plasmid. Desthiobiotin-ATP
reactions were performed after cell lysis and analyzed by immunoblot.

Next, we wished to probe into transactivation events occurring in situ in both homo- and
hetero- RAF dimer contexts. Previously, it has been proposed that RAF transactivation might
proceed through persisting conformational changes in the inhibitor-unbound, activated protomer.
The rationale was that the inhibitor-bound protomer interfaces with the second protomer in a way
that produces “significant, lasting alterations to the enzyme,” ultimately increasing the ATP-
affinity of said activated RAF protomer.
90
We wanted to challenge this hypothesis and test whether
inhibitor-binding events in situ could truly translate into enhanced, prolonged ATP binding. Our
vision was to achieve the formation of specific RAF dimers in situ by co-transfection and observe
the effects of inhibitor treatment on ATP affinity using the desthiobiotin-ATP probe. Our initial
foray involved two heterodimer systems, wherein inhibitor-sensitized BRAF-FLAG was intended
to either homodimerize with BRAF
WT
-V5 or heterodimerize with CRAF
WT
-V5 (Figure 3.19). The
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idea was to apply inhibitor 3.2 or DMSO and observe any differences in desthiobiotin-ATP-
mediated enrichment of the inhibitor-insensitive, wild-type protomer. We recognized the potential
obstacle we would face in controlling dimer events in situ and thought that we could tune this
through adjustment of plasmid DNA ratios in the co-transfection. Unfortunately, within both dimer
contexts, we saw results that were both inconsistent with our previous characterizations and overly
convoluted. The homodimer experiment showed essentially equivalent enrichment of both FLAG-
tagged and V5-tagged proteins in all conditions, which seems to indicate that inhibitor treatment
is unable to induce any differences in ATP affinity. Based on previous co-immunoprecipitation
data, we expected 3.2 to play some part in disrupting the ability of the dimers to form. More
confusingly, in the heterodimer context, we saw variations in the produced effect – either no
difference, enhancement, or a decrease – of ATP-binding as a function of the dimer ratio. Unable
to rationalize these findings, we were urged to adopt additional methodologies to achieve highly
controlled dimerization events in situ.

180

Figure 3.19 Monitoring in situ dimer events and their effects on enhancing ATP-binding
affinity. Co-transfection conditions were performed using either 200 ng or 100 ng for the indicated
plasmid ratios. In situ inhibitor treatment was followed by in vitro reaction with desthiobiotion-
ATP. Reaction product was immunopurified by Streptavidin and analyzed by Western blot.

Utilizing SpyCatcher/SpyTag technology to access controlled, irreversible RAF associations
Very recently, groups have reported the design and development of a technology that enables
users to genetically encode rapid, near-irreversible interactions into macromolecular systems-of-
interest.
91
It is based on a complementary peptide-protein pair modeled after a highly ordered
region of the immunoglobulin-like collagen adhesion (CnaB2) domain found in Streptococcus
pyogenes fibronectin binding protein (FbaB).
92
Stability of the CnaB2 domain is mediated by the
formation of an isopeptide linkage via an intramolecular amidation reaction between Lys31 and
Asp117 residues.
93
Researchers have split this domain into two separate entities – with each
individual part possessing either the reactive Lys or Asp – and have shown that spontaneous
isopeptide bond formation is extremely rapid, essentially bound by the diffusion limit only. The
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constituent peptide and protein components are named SpyTag and SpyCatcher, respectively. Our
ambition was to overcome our inability to coordinate in situ RAF dimer dynamics by leveraging
this technology. We set out to create SpyCatcher/SpyTag-RAF fusion proteins, which would enable
tightly controlled RAF pairings, so long as the desired dimer is composed of protomers containing
the appropriate tag element.
To begin, we needed to create a small library of representative RAF fusion proteins which
would allow us to explore the applicability of the SpyCatcher system to addressing our
dimerization woes. In lieu of performing our pilot experiments with full-length RAF molecules,
we decided to utilize a truncated variant of CRAF (abbreviated as catC), which is a stripped version
composed solely of the catalytic domain.
31
We reasoned that this would simplify our studies and
remove excessive variables, as catC is known to be a catalytically active and dimer-competent
variant that, importantly, does not rely on activation by upstream RAS-GTP. Since the report of
the earliest SpyCatcher/SpyTag pair, there have since been optimizations in the primary sequence
of each part to dramatically improve the kinetics of association. First, we ordered gBlock gene
fragments for the most-optimized versions, SpyCatcher003 and SpyTag003, and sub-cloned them
immediately upstream of catC in pcDNA3.1. We produced four plasmids expressing four separate
catC fusion proteins for our preliminary studies: SpyCatcher003-catC
S427C
-FLAG (A3*),
SpyTag003-catC
S427C
-FLAG (B3*), SpyCatcher003-catC
WT
-V5 (C3), and SpyTag003-catC
WT
-V5
(D3), (refer to appendix A for detailed information and naming convention used henceforth). Note
that wild-type versions of the FLAG-tagged constructs were unavailable at this time and, therefore,
not produced.
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The SpyCatcher system is only useful for us insofar as it is able to provide specific RAF
dimers in situ. To test this, we performed a DNA dose titration of complementary plasmid pairs
and assessed RAF dimerization by immunoblot. For our first experiments, we selected plasmids
that would lead to the expression of SpyCatcher/SpyTag pairs with different epitope tags. For both
A3*-D3 and B3*-C3 co-transfection experiments, we were able to show that expression of the
individual fusion proteins was quite robust (Figure 3.20A,B). Moreover, upon increasing amounts
of total plasmid DNA at a 1:1 ratio of each component, we observed a corresponding increase in
dimer formation, determined by a gel-shifted band roughly equivalent to the sum of individual
protomers. This effect was most cleanly observed for A3*-D3, as the mass-shifted FLAG band
intensity for B3*-C3 plateaued at 200 ng for each plasmid. Next, we set up an analysis to allow
for an exploration of all possible dimer combinations using 300 ng of each plasmid, given the
observations from our previous experiment. The results show that dimers were nicely formed from
all transfection scenarios, with the most intense dimer signals arising from co-transfections of
partners sharing the same epitope. However, despite our success in flat-out dimer formation, we
noted that the actual efficiency was quite low. We qualitatively evaluated this by comparing the
amount of monomer remaining in co-transfection lanes, taken as an indicator of the availability of
dimer ingredients that have been unable to participate in dimerization. Under such lines of thinking,
“perfect” dimer efficiency would be reflected by a complete lack of monomer; in fact, our
experimental setup seems to represent, at best, 50% dimer formation efficiency.

183

Figure 3.20 DNA dose titration of SpyCatcher-Tag-induced RAF dimerization in situ. Co-
transfection DNA amounts are indicated in the blot annotation. Fusion proteins used are A)
SpyCatcher003–catC
S427C
–FLAG (A3*) and SpyTag003–catC
WT
–V5 (D3) and B) SpyTag003–
catC
S427C
–FLAG (B3*) and SpyCatcher003–catC
WT
–V5 (C3). C) Comparison of efficiency for all
dimer contexts.

In light of these observations, we next wished to address the tunability of dimer formation. As
mentioned above, a small portion of residues within the C-helix are critical for maintaining
productive back-to-back dimer contacts, thereby facilitating successful RAF-RAF interactions.
So-called dimer-disrupting point mutations can be applied to these critical arginine residues and
be used to counteract the ability of RAF to align fruitfully. Common examples of dimer-disrupting
point mutations include – for CRAF - R401A and R401H, thought to function mechanistically
184

through either electronic or steric perturbance. Accordingly, we generated dimer-disrupting
mutants from our existing panel of SpyCatcher plasmids and evaluated their effects on
dimerization by immunoblot. Briefly, 293T cells were co-transfected with dimer pairings such that
either of the protomers (either the SpyCatcher-component or SpyTag-component) possesses the
R401 mutant. For our first set of investigations, we chose to utilize the alanine mutation, which is
considered a milder, moderate dimer-disruptor, often preventing dimerization to an intermediate,
but not complete, extent (Figure 3.21A,B). To our dismay, visualization by FLAG was rather poor;
for both A3-D3 and B3-C3 combinations, we could not produce high quality immunoblots.
Nevertheless, visualization of V5 revealed somewhat disappointing results. Again, for both dimer
types, it appears as if introduction of the dimer-disruptor mutation into either single protomer was
insufficient to bear any observable effect. We then decided to generate the more-severe R401H
mutations for each plasmid and performed a more comprehensive repeat of the previous
experiment (Figure 3.21C). In this second attempt, we only evaluated A3-D3 derivatives and
compared effects of R401A to R401H on dimerization. While some subtle dimer-breaking effects
were observed upon introduction of the R401H mutation, this result was hardly significant and
suggests against the possibility of tuning dimerization. We hypothesized that the optimized
SpyCatcher003/SpyTag003 pair featured binding properties that were overly potent and could thus
override contributions from R401 mutations.
185

Figure 3.21 Tuning in situ SpyCatcher-SpyTag engagement through dimer-disrupting
mutations. Readout of dimerization efficiency after introduction of dimer-disrupting mutants A,
B) R401A or C) R401H (indicated through a terminal A or H in the naming convention).

Still, we were intent on comprehensively profiling the SpyCatcher/SpyTag interaction.
Therefore, to circumvent what appeared to be overly robust engagement between the SpyCatcher
components, we turned to earlier generations of the technology, pre-optimization that led to the
current “003” pair. We retrieved the primary sequences from SpyCatcher/SpyTag 001 and 002
186

series, which have been shown to exhibit slowly poorer engagement kinetics in vitro. Our hope
was that we could take advantage of the weaker interactions present in earlier generations and
produce a “Goldilocks” system that would be suitable for our needs. The 002 and 001 gene
fragments were sub-cloned into pcDNA3.1 containing catC to produce library member sets of A1,
B1, C1, D1 and A2, B2, C2, D2. Note that during this time, we performed site-directed mutagenesis
to generate the wild-type analogs for A and B plasmids. Furthermore, in anticipation of future
experiments, we also performed several additional rounds of mutagenesis to produce the R401A
and R401H mutants for each of the four plasmids across all 3 generations of SpyCatcher and
SpyTag.

Figure 3.22 Tuning in situ SpyCatcher-SpyTag engagement through exploration of less-
optimized peptide tags. A, B) Comparison of dimerization efficiency between generations 001-
003. Amounts of transfected DNA for individual plasmids are indicated. C, D) DNA titration of
generation 002 and 001 SpyCatcher/Tag pairs using indicated DNA ratios.
187

With our fully complete SpyCatcher plasmid library, we started with a simple evaluation
comparing the dimerization of the 3 generations. Our results were consistent with what is reported
in the literature: we observed increasing dimerization proficiencies as we ascended through the
generations (Figure 3.22A,B). This was particularly pronounced in the V5 immunoblots, which
show steady decreases in the abundance of dimer from generation 003 to 002 to 001 and a
corresponding decrease in the SpyCatcher-fused proteins, C3, C2, and C1. We note that the SpyTag
components appear to be better expressed in cells and were in vast excess such that the
dimerization event was limited by abundance of SpyCatcher component only. Given the promise
of V5 visualization, we continued with a plasmid titration of the C2-D2 and C1-D1 sets (Figure
3.22C,D). Briefly, we transfected cells with a fixed amount of SpyTag-catC (either D2 or D1) and
included am increasing gradient of the SpyCatcher component. Unfortunately, protein expression
appeared to be unresponsive to the varying DNA amounts used during transfection, with the
exception of C2 in the co-transfection conditions. Perhaps relatedly then, we were also unable to
observe any DNA dose-dependent effects on dimerization; that is, the ratio of C:D did not
significantly influence dimerization. We wished to return to our earlier efforts to compromise RAF
dimerization via inclusion of dimer-disrupting mutants, this time in earlier-generation
SpyCatcher/SpyTag fusion proteins (Figure 3.23). Our hope was that the apparent weaker
interactions in these earlier generations could now be overcome by R401 mutations. Although the
differences in signal intensity are subtle, we can see that R401H mutations can lead to a decrease
in observed dimer formation. This is most evident in the generation 001 mutants, when comparing
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the R401H set to the A1-B1 control condition. Though there does appear to be some influence
manifested in relative dimer abundance, we note that overall efficiency of dimerization is still quite
low across all conditions tested. Further characterization experiments need to be performed to
achieve a thorough understanding of the relationship between SpyCatcher generations and
intermediate to severe dimer disrupting mutations. Fortunately, we have successfully produced a
comprehensive library of these fusion proteins that will facilitate a systematic investigation of their
properties.

Figure 3.23 Introduction of dimer-disrupting mutations into early-generation SpyCatcher-
Tag-RAF dimers. Comparison of A) generation 002 and B) generation 001 Spycatcher/Tag-
mediated dimerization with R401A or R401H mutations.

3.3.4 Investigating RAF modulation for regulating embryonic stem cell self-renewal
BRAF and CRAF have diverging roles in MAPK-mediated maintenance of ESC pluripotency
Regenerative medicine is one of the most recent branches of medical science that aims to
achieve functional restoration of specific tissues that have been compromised, either through
chronic disease or acute damage.
94
Stem cells provide the foundation for the genesis of all cells in
an organism through and exist in four tiers, based on trans-differentiation potential: unipotent,
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multipotent, pluripotent, and totipotent.
95
The ability to harness stem cell differentiation as a
therapeutic modality thus underlies the major basis of regenerative medical strategies. In order to
advance our stem cell technologies, researchers must necessarily be able to control their self-
renewal properties ex vivo and their differentiation potential.
Embryonic stem cells (ESCs) are a type of pluripotent stem cell (PSC) that have an unlimited
capacity for self-renewal and can differentiate into any type of somatic cell.
96
Consequently, they
have received a lot of attention within various therapeutic applications. Maintenance of ESCs in
vitro requires a certain set of culture conditions that alter signaling pathways in a way that is
conducive to promoting self-renewal. Several combinations of inhibitors have been discovered
that act on various proteins within manifold signal transduction pathways to this effect. One of the
earliest examples of this being successfully applied comes from a study in which ESCs derived
from the inner cell mass (ICM) of a mouse blastocyst were stably cultured in serum-free medium
supplemented with leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4).
97

Some time later, two small-molecule inhibitors, CHIR99021 and PD0325901, acting on glycogen
synthase kinase 3 (GSK3) and MEK, respectively, were utilized for in vitro ESC maintenance.
98

These culture conditions are referred to as 2i and provide evidence for the importance of the Wnt/β-
catenin and MAPK signaling in stem cell maintenance. Further support for the significance of
MAPK signaling has recently been affirmed by the discovery of additional self-renewal small
molecules acting through the MAPK pathway, including BRAF inhibitor SB590885.
99
Because of
these developments and other related findings, we wished to apply our Ele-Cys method and shed
light on the mechanistic details of RAF involvement in ESC self-renewal.
190

First, we wanted to provide direct evidence in support of RAF being a critical regulator of
ESC pluripotency. We also wanted to begin investigations towards understanding allele-specific
effects within the family (i.e., are BRAF and CRAF redundant, distinct, complementary, etc. in
ESC renewal?). To this end, in collaboration with the Ying lab (USC Stem Cell Biology and
Regenerative Medicine), we utilized CRISPR/Cas9 gene-editing technology to generate mouse
ESCs (mESCs) containing genetic knockouts of BRAF, CRAF, and BRAF+CRAF (double
knockout).
100
Two pairs of sgRNAs targeting the BRAF and CRAF loci of the E14TG2a mESC
cell line, which stably expresses Cas9, were designed and infected by lentivirus. The sgRNA
creates a double-stranded break (DSB) within the BRAF and CRAF genes, which prompts DNA
repair machinery to introduce indels, effectively disrupting expression of the gene. After drug
selection and colony expansion, clones were analyzed by immunoblot to verify knockout of BRAF
and CRAF. BRAF- and CRAF-specific antibodies reveal successful production of the single and
double knockouts (Figure 3.24A).
Next, we wished to evaluate the consequence of isoform-specific knockout on ESC
differentiation. Individual cell clones were cultured under either standard 2i conditions or in
medium supplemented with CHIR99021 only, by withdrawal of PD0325901 (this marks day=0 of
the CHIR99021-only condition). Cells in 2i-supplemented medium remained in a self-renewal
state, as evinced in their retention of undifferentiated ESC morphology. However, five days after
removal of the MEK inhibitor, the BRAF-KO mESCs began to resemble the wild-type. Of note,
the CRAF-KO and BRAF+CRAF double knockouts remained in a pluripotent state. These results
suggest that BRAF and CRAF have differing, non-redundant responsibilities in maintaining stem
191

cell pluripotency (Figure 3.24B).

Figure 3.24 Characterization of RAF knockout in mouse embryonic stem cells. A) Western
blot confirmation of successful knockout by CRISPR/Cas9 in mESC lysate. B) Knockout cells
show differential cell morphology by microscopy 5 days after removal of MEK inhibitor,
PD0325901.

Utilizing Ele-Cys to appraise pharmacological RAF intervention in ESC self-renewal
As emphasized in previous sections, genetic knockouts are useful in perturbing biological
function though they can only provide crude and limited information. Teasing apart cellular
phenotypes as resulting from catalytic function or structural interactions require careful dissection
and is more readily achieved through pharmacological means. We intend to pursue an Ele-Cys
study on ESC self-renewal to complement the results obtained from the knockout experiments. To
this end, we introduced serine-to-cysteine mutations through CRISPR-Cas9-mediated knock-in in
C57BL/6 mice. Guide RNAs (gRNAs) were designed to target the exon corresponding to the
desired mutational position within BRAF or CRAF loci in the C57BL/6 mouse genome. Based on
the gRNA sequence, we designed single-stranded oligodeoxynucleotides (ssODNs) that would
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serve as the DNA template during homology-directed repair (HDR) to introduce the single
nucleotide polymorphism (SNP).
101
Pronuclear microinjection of CRISPR components into mouse
zygotes led to the birth of founder mice which were screened by Sanger sequencing to identify
positive knock-in pups.
102
Further breeding with wild-type mice produced F1 heterozygous pups,
which were further crossbred until we obtained homozygous mice expressing either BRAF or
CRAF mutant (Figure 3.25).

Figure 3.25 Sanger chromatograms of CRISPR/Cas9 knock-in mice. Genotyping results
indicate successful generation of knock-in mice B) heterozygous and C) homozygous for reactive
cysteine allele compared to A) wild-type mice.

From our batch of transgenic mice, we performed ESC isolation for the purpose of generating
a mESC line stably expressing RAF serine-to-cysteine mutants (Figure 3.26A). Briefly, surgery
193

was performed on mice five days post-fertilization to retrieve the inner cell mass of blastocysts
from embryonic tissue. The cells were transferred with mouse embryonic fibroblast (MEF) feeder
and cultured in 2i medium supplemented with LIF indefinitely. We were interested in seeing
whether our results from the knock-out experiments could be recapitulated upon small-molecule
inhibition of RAF. To test this, cells were passaged in medium supplemented with a variety of
inhibitor combinations (Figure 3.26B). We included non-supplemented and 2i media as negative
and positive controls, respectively, followed by treatment of CHIR99021 only or CHIR99021
combined with either 3.1, 3.2, or 3.3. Unfortunately, the preliminary results were disappointing.
Like our knock-out data, CHIR99021-only conditions were conducive to stem cell differentiation
while 2i-supplemented medium retained cell stemness. However, inhibitor concentrations at sub-
toxic levels were ineffective in producing meaningful, detectable changes in phenotype as
determined by morphology. Across all inhibitor types, the resultant cell morphology uniformly
resembles the CHIR99021-only-treated cells. Moreover, there did not seem to be any discernible
differences within our experimental parameters between the BRAF and CRAF conditions. Our
earlier knock-out experiments suggested that there might be isoform-dependent variations in the
self-renewal response. Because this effect was not produced, pharmacological perturbance of RAF
may not be sufficient. Tentatively, our interpretation of this data is that RAF is not a viable node
within the MAPK pathway that can modulate stem cell differentiation fate, at least by small-
molecule inhibition. Instead, it appears that productive intervention must occur further downstream,
either precisely at or following the MEK junction. Notwithstanding these early negative results,
we have successfully produced transgenic mice expressing inhibitor-sensitized BRAF and CRAF,
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which can be used as organismic models for Ele-Cys-enabled experiments in physiologically
relevant settings. Furthermore, mESCs expressing these RAF variants have been isolated from our
transgenic mice and are stably maintained in culture. We plan to further refine our experimental
setup so that we may establish a pharmacological map of RAF function – or rule out its
involvement – in ESC self-renewal.

Figure 3.26 Monitoring mouse embryonic stem cell self-renewal after small-molecule
inhibition of RAF. A) Blastocysts isolated from post-fertilized CRISPR knock-in mice were
maintained in 2i medium supplemented with LIF. B) BRAF(-/-) and CRAF (-/-) mESC
morphology was evaluated in the presence of MEK inhibitor and 1 µM 3.1, 3.2, or 3.3. Images are
taken after 5 passages with inhibitor.

3.4 Summary
The Ele-Cys platform is an invaluable extension of chemical genetics that can facilitate allele-
specific deconvolution of problems in RAF biology. Our work shows that several somewhat
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untraditional, under-explored areas of RAF physiology are amenable to our approach, including
truncated BRAF variants, the lesser-understood ARAF paralog, RAF involvement in stem cell self-
renewal, and non-canonical classes of BRAF mutants emerging in the clinic. Through thorough
efforts in characterizing the parameters of our method, we report a robust means for illumination
of RAF-centered nuance within the MAPK signaling pathway.
In particular, we were interested in the demystification of inhibitor-induced dimerization,
considered the driving force behind drug resistance in several BRAF mutant alleles. The
commonly proposed transactivation model for this phenomenon suffers from a lack of control at
the level of active site occupancy and permutation of dimer arrangement. We demonstrate that our
methodology represents an avenue into addressing both of these concerns, supplemented by
technologies that enable the measurement of ATP binding affinity and protomer interface
specificity. At the crux of the matter is the successful utilization of SpyCatcher/SpyTag fusion
proteins to direct in situ dimer formation. While we have shown that specific dimer modes can be
accessed upon transfection in cells, tuning their properties and achieving control can be difficult.
Though much work remains to be done, we provide the groundwork for such future studies.

3.5 Experimental details
Plasmid construction
All constructs were prepared using standard molecular cloning techniques unless obtained from
other labs as indicated in text. All mutations were generated using QuikChange Lightning Site-
directed Mutagenesis (Agilent). Detailed cloning information and sequences will be made
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available upon request.

Cell culture and transfection
HEK293T cells were maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM,
Corning) containing 4.5 g/L glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, with 10%
fetal bovine serum. The cells were seeded at 5x10
5
cells per well in 6- well plates 24 hours before
transfection with appropriate DNA plasmid and Lipofectamine 2000 (Life Technologies).

Inhibitor treatment, cell lysis, and Western blot
At 24 hours post-transfection, the media was aspirated and replaced with fresh DMEM containing
various concentrations of the inhibitor (or mock) for 1 hour at 37 °C. The growth media was
aspirated off, and the cells were washed twice with cold DPBS and then harvested using a cell
scraper. The cell suspensions were centrifuged at 4°C for 5 min at 3,000 × g. The cell pellets were
added NP40 lysis buffer (50 mM HEPES, 1% NP40, and 150 mM NaCl at pH 7.4) supplemented
with protease inhibitor cocktail (Roche) and phosphatase inhibitor (Roche). After removing
insoluble cell debris by centrifugation at 20,000 × g for 10 min at 4°C, the soluble fraction was
resolved by gel electrophoresis and transferred to PVDF membrane. The membrane was incubated
with antibody according to manufacturer’s instruction and developed using ECL substrate (Bio-
Rad) and imaged with ChemiDoc XRS+ molecular imager (Bio-Rad). The following antibodies
were used: anti-pMEK (Cell Signaling), anti-pERK (Cell Signaling), anti-MEK (Cell Signaling),
anti-ERK1 (Santa Cruz Biotechnology), anti-myc tag (Santa Cruz Biotechnology), anti-V5 tag
197

(Invitrogen), anti-Flag tag (Sigma), anti-actin (Cell Signaling). Anti-rabbit secondary antibody was
obtained from Abcam (Cambridge) and anti-mouse secondary antibody was purchased from
Jackson Immuno Research Laboratories (West Grove, PA, USA).

Probe treatment and in-gel fluorescence imaging
HEK293T cells were seeded at 5x10
5
per well in 6-well plates and transfected with appropriate
plasmid next day. At 24 hr post-transfection, the growth media (DMEM supplemented with 10%
FBS) was aspirated off and the cells were treated with fresh media containing various
concentration of probe (1,000× stock solution in DMSO) or vehicle control for the indicated time.
For pulse-chase-style competition experiments, cells were first incubated with the inhibitor at
various concentrations for 1 hour, washed with fresh warm medium three times, and then treated
with probe at the appropriate concentration for another hour. After the probe treatment, the medium
was aspirated off and the cells were washed twice with ice-cold DPBS. The cells were harvested
and the pellet was resuspended in 100 µL of NP40 lysis buffer (50 mM HEPES, pH 7.4, 1% NP-
40, 150 mM NaCl) with protease inhibitor cocktail (Roche). The lysate was incubated on ice for
20 min and fractionated by centrifugation at 18,000 × g for 10 min. The protein concentration was
measured from each of the supernatant sample by BCA assay (Pierce) and normalized to 1 mg/mL.
Click reaction was performed at a final concentration of 25 µΜ TAMRA-azide, 1 mM Tris(2-
carboxyethyl)phosphine (TCEP, Thermo-Scientific), 100 µΜ Tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine (TBTA, TCI), and 1 mM CuSO4 (Sigma-Aldrich) in a total volume of 100 µL.
The reaction was performed at room temperature for 1 hour in the dark before termination by
198

addition of 40 µL of 4X Laemmli sample buffer (Bio-Rad) and boiling for 5 minutes. 30 µL of the
samples were loaded and resolved on a 4- 20% SDS-PAGE before visualization at 532 nm for
excitation and 610 nm for emission on a Typhoon 9400 Variable Mode Imager (GE Healthcare).
After fluorescence scanning, proteins were transferred to PVDF membranes for immunoblotting
with appropriate antibodies.

Co-immunoprecipitation
Following inhibitor treatment and cell harvesting, as described above, cells were lysed in lysis
buffer (20 mM Tris, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, pH 7.5) supplemented with
phosphatase and protease inhibitor cocktail (Roche). The harvested cells were centrifuged at 4 °C
for 5 min at 10,000 x g. The protein concentration was measured from each of the supernatant
sample by BCA assay (Pierce) and normalized to 1 mg/mL. The sample was incubated with anti-
Flag agarose for 1 h, followed by five washes with lysis buffer. Samples were analyzed by western
blot, as described above.

Desthiobiotin-ATP labeling and Streptavidin enrichment
Procedure was performed by standard experimental protocol of Kinase Enrichment and ActivX™
(Pierce). After cell treatment and lysis, as described above, lysate buffer exchange was performed.
Zeba spin desalting columns were utilized; columns were centrifuged at 1000 x g for 2 minutes at
room temperature to remove storage solution. Lysate was diluted with reaction buffer to 2 mg/mL
and transferred to a new microcentrifuge tube. 10 µL 1 M Mgl2 was added to sample and incubated
199

for 1 minutes at room temperature. ATPase active-site inhibitor was added to sample, mixed, and
incubated for 10 minutes at room temperature. Equilibrated desthiobiotin-ATP or -ADP reagent at
room temperature was treated with reconstituted reagent by adding 40 μL of ultrapure water to
make a 0.25mM stock solution and 10μL of desthiobiotin-ATP was added to each sample and
incubated for 10 minutes at room temperature. 50% High Capacity Streptavidin Agarose resin
slurry was added to each digested sample and incubated for 1 hour at room temperature with
constant mixing on a rotator. Centrifuge conditions of 1000 × g for 1 minute was applied to pellet
resin. Resin was washed three times with 500μL of Pierce IP Lysis Buffer. Resin was then washed
four more times with 500μL of PBS. Finally, resin was washed four times with 500μL of LCMS-
grade water. Peptide were eluted by adding 75μL of Elution Buffer and incubating sample for 3
minutes. Lyophilized proteins were resuspended in 2X Laemmli sample buffer (Bio-Rad) and
resolved by SDS-PAGE.

mESC isolation and effects of inhibitors on morphology
Surgery was performed to isolate the uterus and 3.5 mouse blastocysts were flushed using N2B27
medium (ThermoFisher). Uterus was transferred to PBS and washed. Blastocysts were flushed
with a 27-gauge needle using N2B27 medium. Blastocysts were then washed using 2 drops of
acidic Tyrode’s solution. Once the zona pellucida has dissolved, blastocysts were immediately
washed with 2 drops of N2B27 medium. Blastocysts were transferred to plates with mouse
embryonic fibroblasts (MEF) feeder in 2i+LIF medium. After 4-5 days, outgrowth of blastocyst
was digested using 500ul 0.025% (wt/vol) trypsin-EDTA solution and incubated at 37 for 3min.
200

1ml of MEF medium was added to neutralize digestion. Cells were centrifuged at 200 g for 3
minutes and supernatant was aspirated. Cell pellet was resuspended in 2i medium and transferred
to MEF feeder in 2i+LIF medium. Cells were maintained at 37°C, 5% CO2. After stable B/C-RAF
knock-in mESC cell line was constructed. They were treated with different RAF inhibitors at
various concentrations in CHIR/N2B27 medium, CHIR/PD03/N2B27 medium as the control. Cell
morphology was monitored by microscopy.

CRISPR/Cas9 knock-in mice genotype confirmation
Knock-in mice were received from Applied StemCell. Husbandry and cross-breeding were
maintained by USC Stem Cell and Regenerative Medicine staff. For mice 10-15 days of age,
animals were restrained and tails were snipped with a disposable blade. Hemostatis achieved by
heat cautery. Samples were digested by Proteinase K in tail buffer (100 mM Tris-HCl, pH 8.5, 5
mM EDTA, 200 mM, NaCl, 0.2% SDS) and incubated overnight under constant shaking at 500-
600 rpm at 56°C. Samples were centrifuged at 18,000 x g for 15 minutes at 10°C and supernatant
was collected. DNA precipitated by addition of 500 μL isopropanol and spun at 18,000 x g for 20
minutes at 10°C. DNA pellet was resuspended in water and amplified by PCR. Amplicons were
visualized by gel electrophoresis and gel slices were collected, purified, and sequenced.

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Appendix: Chapter 3 SpyCatcher/SpyTag information

Naming convention for SpyCatcher/SpyTag catC plasmids
A3 SpyCatcher003 – WT – FLAG A2A SpyCatcher002 – R401A – FLAG
B3 SpyTag003 – WT – FLAG B2A SpyTag002 – R401A – FLAG
C3 SpyCatcher003 – WT –V5 C2A SpyCatcher002 – R401A –V5
D3 SpyTag003 – WT – V5 D2A SpyTag002 – R401A – V5
A3* SpyCatcher003 – S427C – FLAG

B3* SpyTag003 – S427C – FLAG A2H SpyCatcher002 – R401H – FLAG
B2H SpyTag002 – R401H – FLAG
A3A SpyCatcher003 – R401A – FLAG C2H SpyCatcher002 – R401H –V5
B3A SpyTag003 – R401A – FLAG D2H SpyTag002 – R401H – V5
C3A SpyCatcher003 – R401A –V5
D3A SpyTag003 – R401A – V5 A1 SpyCatcher001 – WT– FLAG
A3A* SpyCatcher003 – R401A / S427C – FLAG B1 SpyTag001 – WT – FLAG
B3A* SpyTag003 – R401A / S427C – FLAG C1 SpyCatcher001 – WT –V5
D1 SpyTag001 – WT– V5
A3H SpyCatcher003 – R401H – FLAG
B3H SpyTag003 – R401H – FLAG A1A SpyCatcher001 – R401A – FLAG
C3H SpyCatcher003 – R401H –V5 B1A SpyTag001 – R401A – FLAG
D3H SpyTag003 – R401H – V5 C1A SpyCatcher001 – R401A –V5
A3H* SpyCatcher003 – R401H / S427C – FLAG D1A SpyTag001 – R401A – V5

A2 SpyCatcher002 – WT – FLAG A1H SpyCatcher001 – R401H – FLAG
B2 SpyTag002 – WT – FLAG B1H SpyTag001 – R401H – FLAG
C2 SpyCatcher002 – WT –V5 C1H SpyCatcher001 – R401H –V5
D2 SpyTag002 – WT – V5 D1H SpyTag001 – R401H – V5

Sequences of SpyCatcher/SpyTag peptides

*Changes between 001 and 002 denoted in red; changes between 002 and 003 denoted in green.

Abstract (if available)

Abstract Covalent target modification has re-emerged in recent years as a promising modality for therapeutic development and mechanistic biochemical analysis. Although slightly colored by some decades’ worth of residual skepticism, current trends suggest a paradigmatic shift towards near-centerpiece status for covalent drugs and probes within medicinal chemistry and chemical biology. Recognizing the benefits that covalent modulators confer, we sought to address two pressing clinical needs through methodologies reliant on small molecules acting through covalent mechanisms. This dissertation details our efforts in the discovery of antiviral compounds for combating the SARS-CoV-2 global pandemic and the demystification of RAF kinase physiology.
Chapter one contains an expository account of the history of covalent drugs and discusses some of the advantages and disadvantages for their use. We describe some of the current innovations and perspectives that possibly influence the trajectory of covalent drug development in years to come. The chapter concludes with a discussion of alternative utilities for targeted covalent protein modification by small molecules in the domain of chemical genetics. We describe the scientific necessity of a chemical genetic approach to biology and biochemistry as a response to more classic genetic methods.
In chapter two, we discuss two inverse, yet complementary, approaches to designing covalent small-molecule inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (Nsp12) and main protease (Nsp5). Of the former, we performed a forward pharmacological, phenotypic screen of inhibitors and selected for those successful in preventing/mitigating SARS-CoV-2 viral infection. From our most potent lead compounds, we designed activity-based probes and identified viral factor Nsp12 as the bona fide pharmacological target presumably responsible for mediating antiviral effects upon covalent inhibition. These drug candidates have verified in vivo efficacy and provide a launchpad for the design of future potent and specific polymerase inhibitors. Of the latter, we executed a reverse pharmacological, target-based optimization of peptidomimetic scaffolds shown to exhibit efficacy in inhibiting Nsp5. Specifically, we pursued a systematic evaluation of covalent warheads and assessed their in vitro and in situ labeling properties across three generations of ligand structures. The culmination of this work has been the characterization of an irreversible, latent alkyne warhead that can be sterically and electronically tuned to adjust reactivity and off-rate kinetics, providing groundwork for the synthesis of appropriately derivatized activity-based probes for in situ and in vivo applications. Additionally, inhibitors bearing the parental, electrophilic alkyne show marked improvement over current FDA-approved drugs targeting Nsp5 and thus represent a class of warheads with verifiable clinical potential.
Finally, chapter three documents our efforts to thoroughly characterize and broadly apply a chemical genetic platform based on covalent complementation for the illumination of various RAF-centered nuances within the MAPK signaling pathway. This so-called Ele-Cys methodology utilizes isoform-specific covalent inhibitors that engage with a cognate, sensitized RAF protein, enabling users to probe into the mechanistic subtleties of RAF kinase biochemistry. While we demonstrate that several under-explored niches in RAF research are amenable to our approach, we ultimately envision our system providing an avenue into unraveling some of the mysteries of inhibitor-induced RAF dimerization and subsequent drug resistance observed in RAF-implicated lesions. Carefully deconstructing the “transactivation” model that has been proposed to account for this phenomenon requires exquisite control over inhibitor occupancy and dimer permutation – both of which we attempt to navigate with Ele-Cys supplemented by the appropriate biochemical technologies.

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Asset Metadata

Creator Feng, Joshua J. (author)

Core Title Leveraging covalent modification for diverse applications in antiviral discovery and kinase mechanism deconvolution

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2022-12

Publication Date 09/23/2022

Defense Date 08/24/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag covalent drugs,nonstructural protein 12,nonstructural protein 5,OAI-PMH Harvest,protein dimerization,Raf inhibitors,Raf kinases,severe acute respiratory syndrome coronavirus 2

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zhang, Chao (committee chair), Chen, Lin (committee member), White, Kate (committee member)

Creator Email fengjosh@usc.edu,jshxfng@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC112023832

Unique identifier UC112023832

Legacy Identifier etd-FengJoshua-11245

Document Type Dissertation

Format application/pdf (imt)

Rights Feng, Joshua J.

Internet Media Type application/pdf

Type texts

Source 20220925-usctheses-batch-984 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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