Design and discovery of small molecules inhibiting the interaction of cellular LEDGF/p75 and HIV-1 integrase

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Design and discovery of small molecules inhibiting the interaction of cellular LEDGF/p75 and HIV-1 integrase

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Content DESIGN AND DISCOVERY OF SMALL MOLECULES INHIBITING THE
INTERACTION OF CELLULAR LEDGF/P75 AND HIV-1 INTEGRASE

by

Tino Wilson Sanchez

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
(PHARMACEUTICAL SCIENCES)

May 2012

Copyright 2012 Tino Wilson Sanchez
ii
Dedication

This thesis is in dedication to my mother, grandmother, father, and godfather; may they
live for all time in my heart.

iii
Acknowledgments
Thank you to my advisor Dr. Nouri Neamati and the rest of my committee
members, my committee chair Dr. Ian Haworth, Dr. Stan Louie, and Dr. Kyung Jung, for
their support and guidance. I have learned a great deal from them, I am proud of their
confirmation, and thank the time they gave to combat my defense. My advisor, Professor
Nouri Neamati, a gifted medicinal chemist, thank you for giving me the opportunity to
make a difference in the fight against viral infection and HIV. I would also like to thank
Dr. Raveendra Dayam, Dr. Nancy Deng, Dr. Zahrah Zawahir, and Dr. Laith Al-Mawsawi
for their scientific support and contributions. Finally I would like to express my gratitude
for all those who believed in me through the years: my mother Anna Maria Sanchez, my
godfather Fortino Sanchez, and mentors and good friends, Dr. Krishnan Nambiar, Dr.
Eugene Major, Dr. Roberta Brinton, Dr. Rodney Cole, Dr. John Davis and the MedCOR
program, Dr. Junming Wang, Dr. Clay Wang, Dr. Gabriel Linares, Erin McKibben,
Christopher James Sanchez, Teresa Nicole Sanchez, Joanna Flores, and Jacob Freed.
To my community of friends and mentors that I have met through the years, thank
you for the added bits of your personalities to my life; my success would not be as
complete without your influences. I'd also like to acknowledge the process, commitment,
and numerous encounters that led me to be the scientist I am today. My story, like many
others, probably begins generations past where the common fruits from the ground, water
from the mountains, and seasonal agricultural migrations led to the strong-willed and
freedom-loving Mexican American family I grew to love. Yet my story is more. It is one
of an American family, a melting pot of different personalities and breeds, with a longing
to share the same plentiful hope and dream for opportunity.
iv
My ancestral lineage follows a pioneer's pattern, a migration for new sights, new
terrain to conquer, and new ideas to accomplish. My grandmother, Cecilia Garcia, the
eldest of her siblings and most stricken with responsibility of mother, father, soother, and
disciplinarian was born in Redlands, California in the late twenties. My grandmother
took care of her siblings, cleaned house, cooked the food, while also earning meager but
steady wage in the fields. Her education did not pass the second grade, yet even at ninety
years of age, she still serves as a key source for knowledge and enthusiasm in my life;
part of the "great generation" of this country indeed. As life is intriguing and joyous so
can it be mysterious and sad; my grandfather Fortino Sanchez was deported from
Southern California during a immigrant sweep when my mother was a young child,
leaving my grandmother alone to raise three children.
Born in the era of the baby boom, my mother, Anna Maria Sanchez, is the oldest
of her siblings, and developed a strong will in a similarly difficult upbringing as my
grandmother. After high school my mother enlisted into the armed services during the
escalation period of the Vietnam War. After her tour in Vietnam, my mother used the GI
Bill to attend California State University in San Francisco and later attend the University
of California San Francisco to acquire a license in physical therapy. Shortly thereafter,
my mother met my father, James Edward Wilson, in Sausalito, California. My father had
been a sea scout and contractor with a love for the ocean. Like my Sanchez and Garcia
heritage from the south, my ancestral consciousness also has a mixture of migrants from
Sweden, and Germany or Scotland before that. My grandparents settled in the Dakotas,
and my father headed west to the San Francisco bay area before the age of 18. I was first
to attend a graduate school in both my families and only the second person in my family
v
to graduate from college since my mother. In her honor, I aspire to help others, combat
innate and pathogenic disease, and make the evolutionary leap for my family by example.
My academic studies and the observations I have made in science and in people
have shown me that even with the knowledge and foresight to act on change, many fall
subject to repeat the same patterns. The collective environments and experience of our
mothers and our mother's mothers or fathers continue until societal leaps can be made. I
grew up in Los Angeles, California, and I would also like to give a special thanks to the
surrounding communities and the collective culture that roots this city and my ambition
for success. I grew up in the 1980s, during an era of peace and hope within the United
States, and although I could mention abuse of power, fear in citizens, and the political
empowerment movements that ensued, my childhood was filled with hope and a self-
recognition to burden some of the weight from society. I chose to fight HIV infection.
In the 1980s, I used to watch Lakers basketball games on a small black and white
TV at my grandmother's home and I was there waiting for my mother to get home when I
saw Magic Johnson's announcement that he was HIV positive. The shock was felt when
he no longer played for the Lakers, giving attention and a lasting connection to the
epidemic. In 1991, with an interest in the medical field, I watched the HBO documentary
"And the Band Played On" which peaked my interest and self-fulfillment to aid society
by tackling the greatest infection known to plague the world. My new focus was to train
as a chemist and learn to attack HIV and the infection from a molecular point of view. I
migrated to Northern California to earn my bachelor of science in Chemistry from the
University of California in Davis on the heels of new reports publishing multi-drug
resistant strains were arising from HAART treated patients.
vi
Immediately after college in 2002, I returned to Los Angeles and joined Dr. Nouri
Neamati's Molecular Informatics Drug Design laboratory to combat HIV, before reports
published the failure of the first HIV-1 IN inhibitor, S1360, in clinical trials. The failure
"steam-filled" our engines; Dr. Raveendra Dayam, Professor Nouri Neamati, and I
worked tirelessly establishing the best publishing trio-tandem in the world for HIV-1 IN
inhibition. After acquiring my Master's degree and before Dr. Dayam's departure, we
began the foundation for an important project to combat HIV. A cellular cofactor named
LEDGF/p75 was identified to be important in viral replication and to bind to the IN
enzyme at a different site from the metal-chelating catalytic active site. Two pre-doctoral
grants were awarded which led to the direct funding and work present in this dissertation.
I thank the University of Southern California for its existence and dedication to serving
local communities. Thank you to Luis Arechiga and Pedro Dominguez for their after
school help in purifying recombinant LEDGF/p75 protein. Lastly, I thank all of my
students for their interests and scientific contributions to HIV and cancer research. They
are the shining example of hope for their generation and the future of scientific
innovation and combating disease: Luis Arechiga, Pedro Dominguez, Olga Shimunova,
Shangida Ahsan, Giselle Ignacio, Cherelene Pereira, Raven Burrell, Mane Hovhannisyan,
Anush Magarian, Alan Gutierrez, Hayk Zeytuntsyan, Kenneth Taing, Gabriella Gonzalez.

vii
Table of Contents

Dedication ii

Acknowledgments iii

List of Tables ix

List of Figures x
Abstract xii
Chapter 1. Introduction 1
1.1 The Human Immunodeficiency Virus 1
1.2 HIV Nucleoprotein Complexes 5
1.3 HIV-1 IN 6
1.4 Viral Recruitment of Cellular Cofactor Proteins 7
1.5 Cellular Transcription Co-activator LEDGF/p75 9
1.6 LEDGF/p75-IN Protein-Protein Interaction 10
1.7 Human LEDGF/p75 Role in Cancer 12
1.8 Therapeutic Intervention 13
1.8.1 AIDS Vaccine 14
1.8.2 Stem Cell Therapeutics 16
1.8.3 HIV HAART 16
1.8.4 Raltegravir, Elvitegravir, and "Metoogravir" IN Inhibitors 18
1.8.5 Allosteric IN Inhibitors 21
1.8.6 Design of Inhibitors Disrupting LEDGF/p75-IN Interaction 23

Chapter 2. Acylhydrazone-based Small Molecules as LEDGF/p75-IN Inhibitors 25
2.1 Methodology 26
2.1.1 Selection and Preparation of Compounds 26
2.1.2 Molecular Docking 27
2.1.3 Preparation of Recombinant Proteins 29
2.1.4 LEDGF/p75-IN AlphaScreen Proximity Luminescent Assay 30
2.1.5 AlphaScreen Counterscreen 31
2.1.6 Integrase Catalytic Assay 32
2.1.7 Cytotoxicity Assays 33
2.2 Results and Discussion 33
2.2.1 LEDGF/p75-IN Interaction-based 3D-Pharmacophore Model 33
2.2.2 Filtering Drug-like Compounds for Experimental Analysis 36
2.2.3 Pharmacophore Search and Acylhydrazone-based Inhibitors 37
2.2.4 Protein-ligand Interaction 41
2.2.5 Inhibition of IN Enzymatic Function 43
2.2.6 Implications for Drug Design 43

viii
Chapter 3. Second Generation Acylhydrazone-based LEDGF/p75-IN Inhibitors 44
3.1 Methodology 45
3.1.1 Selection and Preparation of Compounds 45
3.1.2 AlphaScreen Assays 46
3.1.3 Integrase Catalytic Assay 47
3.1.4 Cytotoxicity Assays 47
3.1.5 GOLD Molecular Docking 47
3.2 Results and Discussion 48
3.2.1 IN and LEDGF/p75 Interaction Inhibition with Acylhydrazones 48
3.2.2 Hydrazines, Diazenes, and Their Derivatives 55
3.2.3 GOLD Docking and Molecular Modeling 60
3.2.4 Cytotoxicity Profile 63
3.2.5 Implications for Drug Design 65

Chapter 4. Pyrazole Small Molecules as LEDGF/p75-IN Inhibitors 67
4.1 Methodology 67
4.1.1 Selection and Preparation of Compounds 67
4.1.2 Biological Assays 67
4.2 Results and Discussion 68
4.2.1 5-oxo-pyrazoles 68
4.2.2 Second Generation 5-oxo-pyrazole-based IN Inhibitors 71

Chapter 5. Rhodanine Small Molecules as LEDGF/p75-IN Inhibitors 75
5.1 Methodology 75
5.1.1 Selection and Preparation of Compounds 75
5.1.2 Biological Assays 75
5.2 Results and Discussion 76
5.2.1 2-imino-thiazolidin-4-ones 76
5.2.2 (Z)-5-benzylidene-4-oxo-2-thioxothiazolidin-3-carboxylic acids 79
5.2.3 Implications for Drug Design 83

Chapter 6. New Potential Lead LEDGF/p75-IN Inhibitors 83
6.1 Methodology 83
6.1.1 Selection of Compounds 83
6.2 Results and Discussion 84
6.2.1 Inhibitors from SAR, Fragment Hopping, and Structure Similarity 84
6.2.2 LEDGF/p75-IN Inhibitors Identified from Random 90
6.2.3 Implications for Drug Design 95

Conclusion 95

Future 96

Bibliography 98

ix
List of Tables
Table 1 LEDGF/p75-IN Inhibitors from the First Pharmacophore Search 38
Table 2 LEDGF/p75-IN Inhibitors from Optimization of Initial Hits 40
Table 3 Pyridine, Pyrazine, and Pyrazole LEDGF/p75-IN Lead Inhibitors 48
Table 4 LEDGF/p75-IN Inhibitors with a Hydrazine and Diazene Linker 55
Table 5 LEDGF/p75-IN Inhibitors with the Hydrazine in a Cyclic Ring 58
Table 6 Cytoxicity Profile against HCT116 p53 +/+ and -/- 64
Table 7 LEDGF/p75-IN Inhibitors with a 5-oxo-pyrazole Moiety 69
Table 8 Second Generation 5-oxo-pyrazoles LEDGF/p75-IN Inhibitors 72
Table 9 LEDGF/p75-IN Inhibitors with a 2-iminothiazolidin-4-one 77
Table 10 LEDGF/p75-IN Inhibitors with a Thioxothiazolidin Moiety 80
Table 11 LEDGF/p75-IN Inhibitors with 1,3-dioxoisoindolines or Indole 85
Table 12 LEDGF/p75-IN Inhibitors with a 2,3-dihydroxybenzamide 88
Table 13 LEDGF/p75-IN Inhibitors with an Indene-1,3-dione 90
Table 14 LEDGF/p75-IN Inhibitors Containing Pyrrole Propanoic Acids 91
Table 15 Uninvestigated Potential Lead LEDGF/p75-IN Inhibitors 94

x
List of Figures
Figure 1 HIV Viral Particle 2
Figure 2 HIV Genome 3
Figure 3 HIV Replication Life Cycle 4
Figure 4 HIV Particles Attaching to CD4+ T Lymphocytes in the Blood 5
Figure 5 Reverse Transcription Complex 5
Figure 6 Pre-integration Complex 5
Figure 7 HIV-1 Integrase Structural Domain 7
Figure 8 List of Cellular Cofactor Complexes 8
Figure 9 Cellular Transcription Co-activator LEDGF/p75 10
Figure 10 LEDGF/p75-IN Protein-Protein Interaction 11
Figure 11 HAART Timeline 14
Figure 12 IN Inhibitors that Entered Clinical Trials 19
Figure 13 Raltegravir, Elvitegravir, and "Metoo-gravir" 20
Figure 14 Published LEDGF/p75-IN Inhibitors 23
Figure 15 Lowest Energy Docking Pose of Compound 1 24
Figure 16 HIV-1 IN Plasmid Map 30
Figure 17 Human LEDGF/p75 Plasmid Map 30
Figure 18 AlphaScreen Methodology for LEDGF/p75-IN Interaction Assay 31
Figure 19 AlphaScreen Counterscreen 32
Figure 20 LEDGF/p75 KID-IN Dimer-dimer Interaction 34
Figure 21 LEDGF/p75 KID Residue-based Pharmacophoore Models 35
Figure 22 Cartoon Scheme of Filtering Compounds 37
xi
Figure 23 Compound 1 Mode of Binding in IN Dimer-dimer Pocket 42
Figure 24 Antituberculosis Compounds 45
Figure 25 Docking Pose of Acylhydrazone-containing Compound 21 53
Figure 26 Merging of LEDGF/p75-IN Inhibitor 976 with 2B4J Pocket 61
Figure 27 Low Energy Docking of LEDGF/p75-IN Inhibitors 33, 56, and 74 62
Figure 28 Lowest Energy Docking of Isoniazid, an Inactive Analogue 62
Figure 29 Acylhydrazone and Hydrazine Metabolic Products 66

xii
Abstract

Cellular transcription co-activator p75, also known as lens epithelial-derived
growth factor (LEDGF/p75), plays an essential role in HIV-1 IN-led integration of viral
DNA into the human genome. Designing compounds to disrupt LEDGF/p75-IN
complexes serves as a novel mechanistic approach different from current antiviral
therapies. The evolution of HIV has afforded the viral species to adapt to external
therapeutic pressure and raltegravir-resistant viral strains have been well documented,
increasing the need for new IN inhibitors with a different mechanism of action.
Mechanistic studies show the majority of reported IN inhibitors effectively chelate with a
magnesium ion in the catalytic active site, a region topologically different from the
LEDGF/p75 binding site. Thus, key LEDGF/p75 K364 I365 D366 amino acid residues
from the IN binding domain laid a foundation for ligand-based pharmacophore models.
Herein we report funding from the National Institute of Allergies and Infectious Diseases
and the California HIV/AIDS Research Program led to the discovery of more than 100
novel small molecules that successfully inhibited LEDGF/p75-IN complex formation
with IC
50
values below 50 µM. A small molecule database of 365,000 was searched and
compounds that had fitness values above 2.0 were selected for prescreening in an in vitro
luminescent proximity assay. Lead inhibitors expanded into training sets using
substructure similarity tools and ADMET filters led to the discovery of four novel classes
of LEDGF/p75-IN inhibitors and additional potential lead inhibitors. Seven antiviral
LEDGF/p75-IN inhibitors were identified from this study.

1
Chapter 1. Introduction
In the late twentieth century, the AIDS epidemic transformed into a global
pandemic. Since the discovery of the human immunodeficiency virus (HIV) as the
pathogenic agent causing AIDS, the goal to eradicate HIV experienced many setbacks.
In all, 60 million people worldwide have been infected, while 33 million currently live
with HIV. At the turn of the century, progress with highly active antiretroviral therapy
(HAART) was stymied by the emergence of multi-drug resistant strains.
128
Transmitted
drug resistant strains is reported to be as high as 22% in many HIV-infected
communities, where 13% of new infections in San Francisco, CA, are new drug-resistant
strains. HIV evolution models show that 60% of the circulating antiretroviral-resistant
strains in San Francisco can lead to new epidemics.
124
The rate of resistance is
significant, and San Francisco, CA, is touted as an indicator of future worldwide trends of
infection. Thus, new research focused on diversifying new antiviral inhibitors is
ongoing.
1.1 The Human Immunodeficiency Virus
The balance of nature and the human host stems from the origins of life where
prokaryotes, eukaryotes, and viruses, evolving over eons, have diversified into the
complexity of organisms we observe today. HIV is a retrovirus from the genus lentivirae
and has a complex multi-stage life cycle that promotes genetic variation in all viral
progeny (Figure 1). During infection, millions of viral particles enter the blood stream
and specifically target the immune system preventing the systemic response required to
combat the viral infection. HIV and other lentivirae, characterized with long incubation
periods, uniquely infect and replicate in both dividing and non-dividing cells.
64, 70, 78, 137

2
The HIV life cycle begins with an enveloped viral RNA genome (Figure 2) and
culminates as a provirus integrated into the host cellular chromosomes. The lysogenic
cycle can exist for years before changing to a lytic cycle which explains the reemergence
of HIV in patients years after therapeutic suppression.

Figure 1. A single HIV-1 viral particle has a gp120/gp41 receptor complex infused in an
outer bilayer lipid membrane. The receptor complex has outer trimeric gp120 subunits
for initial attachment and gp41 subunits extending as the receptor stabilizer and
transmembrane domain. Matrix structural proteins enclose another protective layer,
named the capsid where viral sensitive components are protected, namely two RNA
molecules, IN, and RT.

Matrix
Integrase
Capsid
2 RNA
molecules
RT
gp41 Viral
lipid
bilayer
membran
e
gp120

3

Figure 2. The HIV genome is encoded in an RNA molecule with three major encoding
domains. The gag region encodes for structural matrix, capsid, and nucleocapsid
proteins. The pol region encodes for viral enzymes protease (PR), RT, and IN, while the
env region encodes glycoprotein receptors responsible for HIV attachment to target host.
The viral life cycle occurs in stages: entry/fusion, reverse transcription, nuclear
import, integration, early/late transcription, and viral particle assembly and budding
(Figure 3). Inside the blood stream, millions of viral particles attach to circulating CD4
T-leukocytes or T-lymphocytes and macrophages in local lymphatic drainage nodes
(Figure 4).
41
The HIV envelop (env) gp120/41 complex initially binds to CD4 receptors
via gp120 trimeric proteins causing a conformational change to unmask pockets for
binding with cellular co-receptors CXCR4 or CCR5. The co-receptor binding to gp120
homotrimers enables gp41 transmembrane subunits to facilitate viral-host membrane
fusion and cellular entry.
39, 122
Next, the encapsulated viral RNA genome is inserted into
the cellular cytoplasm and the outer protective group-associated antigen (gag) proteins
diffuse and uncoat. Post cellular entry, the HIV RNA genome undergoes reverse
transcription and the resulting viral DNA products migrate to the nucleus for an
irreversible viral-cellular genomic integration reaction.
37

vif vpr tat
rev

nef

GAG POL
M
a
t
r
i
x
Promoter/
Enhancer
nucleocapsid
P6
PR
RT
IN

vpu gp120
gp41
capsid
ENV
3’
LTR
5’
LTR

4

Figure 3. The HIV-1 replication cycle initiates with viral attachment and the fusion of
viral and host membranes. The replication cycle is next characterized by reverse
transcription of viral genetic RNA, the migration of PIC towards the nucleus, and the
incorporation of proviral DNA into host genome. Next the host cell transcribes,
translates, and modifies viral protein for cleavage and self-assembly. New viral offspring
finally lyse the cell as they bud off.
The HIV genome consists of long terminal repeats (LTR) that act as the lone
promoter region to initiate for transcription of provirus DNA.
69
Encoded from a single
primary transcript, the HIV genome consists of nine genes: gag, pol, vif, vpr, tat, rev,
vpu, env, and nef. The HIV-1 pol gene encodes three enzymes responsible for catalytic
preparation of the viral genome and cleavage of the final protein products for self-
assembly: reverse transcriptase (RT), integrase (IN), and protease (PR). The remaining
accessory gene products are signaling, migration, and regulatory viral proteins to improve
probability for viral survival.
8, 44

5

Figure 4. Cartoon of HIV in the blood stream with
circulating red and white blood cells after viral infection.
HIV viral particles attaching to circulating CD4+ T
lymphocytes.

1.2 HIV Nucleoprotein Complexes

The reverse transcription of viral RNA into viral cDNA occurs on a reverse
transcription complex (RTC) that lacks a proofreading system and generates many
mistakes that can lead to a diverse pool of potential viral transcripts (Figure 5).
109
At this
point each viral particle becomes dozens of disassembled genetic remnants capable of
making numerous beachheads on active chromosomal transcription units once inside the
nucleus.
116
However, prior to migrating to the nucleus, nuclear entry, and the
incorporation of viral cDNA, formation of a pre-integration complex (PIC) is required
(Figure 6). The PIC serves as a nucleoprotein shuttle that docks with other protein-
protein or nucleic acid-protein complexes to recruit host cellular proteins.
73, 81

Figure 5. Nucleoprotein RTC provides stability
for reverse transcription on microfilaments.
Figure 6. Nucleoprotein PIC recruits
cellular cofactor on route for nuclear
import.

6
As remnants of the RTC, RT viral proteins take a supporting role while the PIC
localizes to the nucleus using intracellular trafficking routes of transport.
91, 96
RT
enhances structural stability of the PIC, and together with IN, recruits cellular proteins to
evade innate intracellular proteolytic and nuclease activity as well as completing reverse
transcription, nuclear translocation, nuclear entry, chromosomal association, and provirus
integration.
72, 93, 98, 107, 134, 136
As the PIC migrates to the nucleus, IN displays
endonuclease activity and hydrolytically catalyses the viral DNA phosphodiester
backbone by excising a -GT dinucleotide from the 3' ends, termed as 3' processing (3'P).
The resulting hydroxyl groups of the intasome will serve as nucleophiles to attach the
viral cDNA to host chromosome DNA segments undergoing active transcription for
successful viral integration or strand transfer (ST).
66, 94

1.3 HIV-1 IN
The first catalytic stage of HIV-1 IN requires a dimer at both viral 3' ends to
conduct 3'P and at least a tetramer for concerted viral genetic integration.
85
Interaction
between HIV-1 IN and viral substrate DNA uses three acidic residues coordinating with
two divalent metal magnesium ions within the active site pocket of IN catalytic core
domain.
83
The active site DD(35)E motif recognizes the viral 3' terminal -CAGT and
with the aid of an E152 on a flexible loop, the terminal -GT is cleaved exposing hydroxyl
groups for DNA integration. The second stage requires a higher order of IN where an IN
tetramer covalently links the viral DNA to target cellular DNA, a process known as
integration or strand transfer.
15, 47

The IN structural domain consists of three important domains, the catalytic core
domain, the C-terminal domain responsible for nonspecific polynucleotide binding,
14, 31

7
and an N-terminal domain that promotes and stabilizes multimerization (Figure 7).
132, 140

All three domains form dimers and the full length IN protein readily aggregates into
higher order multimers for concerted integration.
48
Moreover, an induced oligomeric
shift of an IN dimer to a tetramer using cellular cofactor peptide fragments revealed HIV-
1 IN inability to conduct 3'P in the higher order form.
51

Figure 7. HIV-1 genome. IN recognizes terminal CAGT sequences in the 3’ end of both
LTR U5 subdomains. The HIV-1 pol polyprotein domain encodes PR, RT, p15, and IN.
The IN N-terminus (1 – 50) has a conserved zinc-finger motif at H12, H16, C40, and C43
positions, while the catalytic core (51 – 212) has a DD(35)E acidic motif. The C-
terminal domain (213 – 288) is a nonspecific DNA binding domain with a conserved
tryptophan.

1.4 Viral Recruitment of Cellular Cofactor Proteins
The interaction of viral proteins with cellular cofactors play a significant role in
all stages of the viral life cycle (Figure 8).
9
HIV-1 IN uses metal cofactors for catalytic
function and also interacts with a series of beneficiary cellular cofactor proteins
throughout the pre-integration process. In the RTC, IN recruits cellular proteins such as
Gemin2a (sip1) to improve RT-RNA stability and augment reverse transcription.
46

Although IN and other viral proteins have nuclear localization signals, nuclear migration
of the PIC uses IN to interact with several cellular cofactors including microtubule

8
associated proteins to drive the PIC to the microtubule-organization centre near the
nucleus.
27
Lentiviral IN and capsid both interact with transportin-SR2 (tnpo3) to
facilitate docking the PIC with a nuclear pore complex for nuclear import.
126
Other cell
importin proteins have also been identified to play a role in PIC nuclear import.
52, 62, 139

Cellular cofactor Method Role in HIV infection Role in normal cells
Gemin2α, SIP1 Yeast,
siRNA
Augments reverse
transcription via IN
interaction by enhancing
RT assembly with viral
RNA
Assists in formation of
spliceosomal small nuclear
ribonucleoprotein snRNP.
Microtubule-
associated protein
(Dyn2p in yeast)
Yeast Important role driving
IN toward microtubule-
organization centre
(MTOC) near nucleus
Dynein light chain protein
associated with
cytoplasmic transport
Microtubule-
associated protein
(Stu2p in yeast)
Yeast Important role driving
IN toward MTOC,
located close to nucleus
Centrosomal protein
associated with cargo
transport
SET complex
(NM23H1,
TREX1, APE1)
siRNA Protects against auto-
integration
DNA repair complex
mobilized to nucleus,
responds oxidative stress
Transportin-SR2
importin-β
family, TNPO3
siRNA Cargoes IN-led PIC to
nuclear envelop for
nuclear import
Docks with nucleoporin,
shuttles cargo for nuclear
transport and import
Lens epithelial
derived growth
factor/p75, PSIP1
Crystal
2b4j,
3f9k
Tethers PIC to site of
integration on cellular
chromosomes
Regulatory mechanism in
transcriptional activation
Figure 8. List of select cellular cofactor complexes important in viral replication, their
method of discovery, role in HIV infection, and role under normal conditions.

Before catalytic integration and nuclear import, HIV-1 IN interacts with a cellular
transcription factor chaperone, lens epithelial-derived growth factor p75 (LEDGF/p75),
to protect the PIC from proteolytic degradation.
79
Also, the chaperone and chromosome
tethering properties of LEDGF/p75 are utilized by the IN-led PIC to bind to active
transcription units.
42
Furthermore, LEDGF/p75 recombinant mutants,
111
siRNA and

9
shRNA knockdown,
130
and LEDGF/p75 knockout cells
121
significantly impairs
interaction with IN and thus HIV genomic integration and viral infectivity.
The recruitment of LEDGF/p75 and other cellular cofactors enables HIV to
covalently integrate its viral genomic DNA into active transcription units leads to
transcription of the provirus by cellular RNA polymerase II machinery. Both full length
and smaller excised RNA molecules are produced for mRNA production and eventual
translation into new viral proteins. Next, RNA alternative splicing and gag-pol frame-
shifts of the viral genome provides HIV another inherent method to improve survival and
generate mutant viral progeny.
2, 29, 88, 101
Thus, preventing HIV genomic integration into
cell chromosomes represents a last stand to evade infection and help prevent the
establishment of a foothold reservoir of infected cells for future replication.
114

1.5 Cellular Transcription Co-activator LEDGF/p75
Human LEDGF/p75 is a transcription factor adaptor with chaperone and
chromosome docking properties. LEDGF/p75 has several functional domains: an N-
terminal PWWP domain that associates with DNA-bound chromatin, A/T hooks that bind
to chromosomal DNA, highly charged regions (CR domains) that bind to polynucleotidyl
enzymes like RNA polymerase II, and the IN binding domain (IBD) at the C-terminus
which shuttles cargo (Figure 9), such as cellular transcription factors under normal
conditions and the IN-led PIC to the site of genomic integration under viral duress.
80

10

Figure 9. Cellular LEDGF/p75 binding domains consist of an N-terminal chromatin
binding region and IBD that binds to cargo such as the IN-led PIC.

Under conditions of environmental stress, LEDGF/p75 acts as a pro-survival
protein that activates the transcription of stress-related anti-apoptotic proteins to promote
cell growth,
38
thus enhancing HIV’s ability to evade innate stress-induced cell death in
addition to promoting HIV genomic integration. The interaction between cellular
LEDGF/p75 and IN induces an oligomeric shift from a dimer to an IN tetramer,
triggering a change from the IN endonuclease conformation to a polynucleotide
transferase activity.
51
The IN tetramer is stabilized through salt bridges between the NTD
and CCD in the presence of viral DNA, and brings the DDE flexible loop closer to the
other catalytic triad residues. Formation of an tetramer also brings adjacent CCD active
sites into a closer proximity of 18 angstroms, an ideal distance for joining processed viral
strands to chromosomal DNA 5 bases apart during concerted integration.
47

1.6 LEDGF/p75-IN Protein-Protein Interaction
HIV-1 IN and other viral proteins recruit an array of cellular cofactors to serve as
chaperones and protein adaptors to important nucleoprotein hubs. The LEDGF/p75-IN
protein-protein interaction is an essential interaction for a variety of crucial stages.
Cellular protein LEDGF/p75 helps protect IN and PIC from proteasomal degradation
during the nuclear migration inside the cytoplasm and helps transition IN from the first

11
stage of viral DNA catalysis (3'P) to the second stage ST conformation. The ability of
LEDGF/p75 to anchor the PIC to host chromosomes relies on key residues at the IN
dimer-dimer interface, an intact IBD, and the A/T hook domain.
16
The PWWP domain
and CR regions improve viral genomic integration kinetics.
6, 43
Crystallography and
mutagenesis studies elucidate a small number of IBD residues (K364, I365, D366, F406,
V409) protruding into the IN dimer-dimer interface, a region topologically distant from
the IN catalytic active site, are responsible for the interaction (Figure 10).
12, 30
The
LEDGF/p75 IBD interacts with a hydrophobic pocket (residues 128 – 135) on one IN
monomer and a flexible loop linker between the α-4 chain and α-5 chain (residues 165 –
173) on another.
49

Figure 10. LEDGF/p75-IN interaction. Two LEDGF/p75 IBD monomers bind to IN
catalytic core within the IN dimer-dimer pocket, a region topologically distant from the
DD(35)E active site shown in gold.

Non-conserved lentiviral IN CCD residues, such as HIV-1 E170 and H171,
suggest an evolutionary role these residues undertook during human infection.
57
HIV-1
IN dimer-dimer interface stems the full CCD and NTD, however, the LEDGF/p75
binding site is a novel small molecule allosteric binding pocket for HIV-1 IN inhibition.
5

12
Also, small peptides derived from the LEDGF/p75 IBD region inhibit the interaction
between LEDGF/p75 and IN, prevent viral DNA integration, and abolish viral infection
by shifting the IN oligomerization equilibrium to an inactive state.
4, 50
These findings
elucidate the dimer-dimer pocket is capable of accommodating small molecules that
could bind and inhibit LEDGF/p75-IN complex formation.
1.7 Human LEDGF/p75 Role in Cancer
LEDGF/p75 acts as a chromosomal adaptor for many different transcription
factors including positive cofactor-4, TFIIF, and RNA polymerase II, cofactors known to
be important in pre-initiation complex assembly, transcription initiation, elongation, and
re-initiation stages.
40, 87
In addition to interacting with IN, the LEDGF/p75 IBD interacts
with JPO2 and menin/MLL histone methyl transferase, a protein complex that arises in
leukemia. In cancer, MLL fusion proteins or nucleoporin (Nup98) fusion proteins bind to
LEDGF/p75 for transcription-led misexpression. Furthermore, the LEDGF/p75-
menin/MLL oncogenic fusion protein complex targets HOX genes during leukemia
pathogenesis.
10, 138

Under environmental duress such as radiation, reactive oxygen species, and
pathogenic infection, cellular LEDGF/p75 is up-regulated by stress-related and heat
shock proteins to increase cellular survival and resistance to stress.
67
In the role cancer,
this up-regulation aids in the delivery of pro-survival and anti-apoptotic transcription
factors to induce favorable oncogenic expression, and cancer growth signals.
19, 59
For
instance, the LEDGF/p75 interaction with transcription factors activates pro-survival
protein Hsp90 and antioxidant protein 2, and anti-apoptotic proteins Hsp27 and αβ-
crystallin to interfere with caspase apoptotic activation.
35
Moreover, the up-regulation of

13
LEDGF/p75 during cancer pathogenesis stems from BCL2, an anti-apoptotic protein.
Also, the BCL2 up-regulation of ERK-1 and -2 MAP kinases leads to the up-regulation
of LEDGF/p75, where inhibition of these kinases removes BCL2 regulation of
LEDGF/p75.
36
The LEDGF/p75 chromosomal association during RNA polymerase II
transcription makes it an ideal component for HIV genomic integration, and thus a
perfect candidate for novel therapeutic intervention.
1.8 Therapeutic Intervention
The inherent HIV life cycle has a high mutation rate and lacks a correction system
to counter the mistakes made from reverse transcription base pair missense, mismatches,
frameshifts, deletions, and insertions.
2
Sustained therapeutic pressure can lead to a
number of viral mutations resulting in emergent drug-resistant strains.
33
Available
antiretroviral drugs are limited when mutant strains develop. In the 25 years since the
first antiretroviral drug, AZT, was approved for treatment of HIV/AIDS, nearly 20
successive antiretroviral drugs approved by the U.S. FDA are associated with primary
and secondary drug-resistant mutations (Figure 11).
65
Considering limitations of
therapeutic options once viral strains emerge, new drugs targeting different stages of the
viral life cycle with unique mechanics is essential.

14

Figure 11. Important dates of HIV infection and antiretroviral therapeutic advances
culminating in the FDA approval of raltegravir and elvitegravir in the marketing stage of
approval.

1.8.1 AIDS Vaccine
The innate biological response to protect against pathogens and foreign antigens
begins in the blood stream and systemic mucosal lining. Once HIV enters the blood
stream, the virus gains access to the lymphatic drainage system triggering a chain of viral
infections and severely hampering the immune system's innate and secondary response.
41,
45, 64, 105
T-helper lymphocytes and leukocytes have CD4 receptors that HIV recognizes
for attachment, and the ensuing infection hampers the ability of these T-cells to recruit
and select progenitor cytotoxic T-cells (CD8+ thymus cells) and antibody-producing B-
cells (bone marrow-derived memory cells) for differentiation. Further exacerbating the
problem, initial HIV infection in CD4+ lymphocytes and macrophages releases
inflammatory cytokines and chemokines, such as human monocyte chemo-attractant
protein-1, which triggers a domino effect of cellular recruitment for further infection.
1, 70

An AIDS vaccine is the ideal therapeutic strategy to prepare the immune system
T-helper and B cells for readily recognition of HIV particles at the inception of viral

15
infection. Several attempts to produce an effective vaccine have failed to protect a
significant number of people in Africa and Southeast Asia. The arduous task of creating
a comprehensive vaccine stems from high viral mutation rates evident with the nine
phylogenic diverse major (M) strain subtypes (A-D, F-H, J and K) and numerous
circulating recombinant forms (including CRF01_AE, CRF02_AG, and CRF03_AB).
54

Prior to HIV infection, an ideal AIDS vaccine will induce antigen presenting cells'
major histocompatibility complexes (MHC) II to elicit systemic cell-mediated and
humoral immune responses, producing immunogenic memory for select viral
components.
76, 90, 97
Ensuing HIV infection, B memory plasma cells will secrete
antibodies capable of recognizing these vaccine induced viral antigen epitopes. Next,
neutralizing antibodies

that recognize the foreign HIV particles will trigger an
inflammatory complement fixation (C1 antibody recognition and complement cascade
activation) and directing cytotoxic T lymphocytes (CTL) to the site of infection.
13

Recruited CTL cytolysis and/or macrophage phagocytosis of infected cells and foreign
viral particles conclude the efficacy of a vaccination.
More than 200 studies examining preventive and therapeutic vaccines are listed as
completed or currently ongoing according to aidsinfo.nih.gov. Many of the failed
vaccines were composed of prevalent glycoprotein receptors and env encoded structural
proteins to provide the immune system with various antigen epitopes to help restore
immune response.
103
Vaccines composed of HIV protein fragments are presented by the
MHCII antigen presenting complex to T helper cells to signal macrophage phagocytosis
and the production of B cell neutralizing antibodies.

16
After HIV infection, successful vaccine-induced immune systems have a higher
likelihood to resist the infection and combat the virus. Ethical issues arise when
neutralizing antibodies specific for one viral strain do not have a high affinity for
different viral strain substrates. Although this stage presents the body with a
predetermined antiviral protective strategy where inducing an antibody response
increases immune response upon initial contact, a vaccine specific for one strain can
leave an immune system susceptible and compromised to other globally circulating
strains. Currently, there are no FDA approved AIDS vaccines available.
1.8.2 Stem Cell Therapeutics
In 2007, a leukemia patient, also infected with HIV, was given a bone marrow
stem cell transplant from a donor that had a delta-32 CCR5 mutation, a genetic mutation
that prevented HIV from infecting the cell. The patient was cured of HIV, where no viral
levels were undetectable and the patient exhibited a systemic recovery of helper T cells.
Stem cell therapeutics is an avenue under investigation in many labs across the world.
1.8.3 HIV HAART
HAART is a multi-drug regimen that typically includes two nucleoside reverse
transcription inhibitors (NRTI) combined with a non-nucleoside RT inhibitor (NNRTI),
PR inhibitors, or raltegravir, an integrase inhibitor. In treatment-experienced patients,
new NRTIs combined with raltegravir or an HIV fusion inhibitor, enfurvirtide, is used
instead of NNRTIs or PR inhibitors.
3
Ideally, treatment-experience patients need new
drugs to overcome the relapse of HIV infection. However, viral suppression to
undetectable levels will not immediately replenish CD4+ T cell levels nor completely
restore the ability of the thymus gland to produce enough diverse T cells to combat new

17
infections. Immune system boosting or stem cell therapies need to be developed to
restore the immune system during HAART treatment. Furthermore, several immune
system-boosting drugs, including interleukin-2, significantly improve HAART efficacy
while improving CD4+ T cell count.
117
Also, maturation and CCR5 targeting inhibitors
are under investigation in clinical trials.
The error-prone HIV system of reverse transcription, RNA alternative splicing,
and gag-pol frame-shifts give the HIV life cycle an inherent method to evade therapeutic
pressure with every viral progeny. Drug resistant mutations within RT and viral PR often
confer cross-resistance to other HAART components targeting these proteins.
55, 120
This
pattern of resistance will also be prevalent in new IN metal-chelating inhibitors and other
parallel inhibitors that target the same viral proteins. HAART suppression of viral
replication in HIV infected patients reduces viral loads below detection limits. However
the sustained reduction in viral load is accompanied by a wide range of adverse effects
including metabolic, cardiovascular, renal, immunologic, hematologic, neurologic, and
gastrointestinal toxicities.
115
Technology now exists to computationally filter lead
molecules that have unfavorable absorption, distribution, metabolic, excretion, and
toxicology properties.
100
Also, considering limitations of therapeutic options once viral
strains emerge, new antiretroviral drugs targeting different stages of the viral life cycle
with unique mechanics is essential.
60

18
1.8.4 Raltegravir, Elvitegravir, and "Metoogravir" IN Inhibitors
HIV-1 IN is a validated and important target for the development of novel
antiviral drugs. First, IN is essential for retroviral genomic incorporation and thus
replication.
32
Second, the lentiviral IN has no cellular homologue. Third, several
sensitive assays have been developed for testing IN enzymatic activity and protein-
protein binding affinity in vitro.
11, 18, 58
Finally, the combination of IN inhibitors with PR
and RT inhibitors has shown to be synergistic in several models and is now used in first-
line HAART treatment.
131
These features and the availability of IN co-crystal structures
in complex with viral DNA substrate, and cellular cofactors, such as LEDGF/p75, make
IN a well-suited target for rational drug design.
16, 66

Despite extensive efforts, only two IN inhibitors have been successful in clinical
trials: raltegravir and elvitegravir (on FDA fast-track for treatment of HIV). Raltegravir
has a fluorobenzene, indicative of IN ST specific inhibition, and a diketo-enol in a central
pyrimidine to evade metabolic reduction, a negative physiochemical property associated
with the failure of a previous clinical candidate, S1360 (Figure 12).
21, 24, 89
Mechanistic
studies show raltegravir, elvitegravir, and other strand transfer inhibitors are susceptible
to emergent drug-resistant viral strains. Successful viral progeny after monotherapy with
the metal-chelating IN inhibitors have residues in the CCD active site different from the
wild type parent viruses. These new viral strains are no longer subject to the same
monotherapy and often show cross-resistance to other IN inhibitors that have the same
mechanism of inhibition.
26, 95, 133

19

Figure 12. Structures of IN inhibitors that entered into clinical trials past and present.
Metal-chelating IN inhibitors bind to residues adjacent to D64, D116, and E152 in
the CCD active site. Thus, the two primary mutations, N155H and Q148K/R/H, lead to
subsequent secondary mutations and completely abolish raltegravir-induced inhibition of
IN catalysis and antiviral activity.
25, 82, 86
elvitegravir, a quinolone carboxylic acid,

20
induces several primary mutations including T66I, E92Q, Q146P, and S147G (Figure
13). Elvitegravir-resistant T66I and secondary mutant viral strains show significant
resistance to other diketo-acid derivatives such as S1360 and L731,988. Furthermore, the
elvitegravir-induced E92Q mutant shows greater than a ten-fold resistance to IN inhibitor
L870,810, a diketo-enol derivative. Subsequent or combined secondary mutations
(E92Q/H51Y/S147G) reveal greater than a 30-fold resistance to L870,810 and
L731,988.
71, 119

Figure 13. Structures of metal chelating IN inhibitors raltegravir, elvitegravir, and
S1360. Strand transfer specific moieties in red and metal chelating moieties in blue.

Failing to fully suppress HIV leads to new viral strains resistant to antiretroviral
drugs that have a similar mechanism of action. This emergence of drug-resistant strains
led to changing the therapeutic regime from a monotherapy, like AZT, to a rational-based
multi-drug regimen. Although a combination of three - four antiviral drugs suppress

21
most viral strains (1% of the viral systemic population), drug-resistance to specific
HAART regimens have also developed.
77, 128
Novel therapeutic options are needed to
remain several steps ahead of viral evolution by squelching replication at different stages
of the viral life cycle so that when mutations arise, they also will have a significant effect
on the viral fitness.
1.8.5 Allosteric IN Inhibitors
Raltegravir, elvitegravir, and metoogravir-resistant viral strains are observed
across the spectrum of lentiviruses.
118
Inhibition of HIV-1 IN and viral replication
through an alternate mechanism is the next step to eliminate the AIDS pandemic.
Suppression of HIV to undetectable levels before the immune system is compromised
will slow the progression of AIDS. With the inclusion of raltegravir in HAART, all HIV
enzymes have a counter therapeutic component specific to their enzymatic function. New
allosteric IN inhibitors has been the focus of current research although there are no leads
in clinical trials.
Clinical IN inhibitors traditionally bind within the IN active site by disrupting the
coordination of magnesium and the DD(35)E motif, however, DNA intercalation
inhibitors can also inhibit catalytic function by targeting the 3' end of the viral substrate.
The next step of inhibition is targeting HIV-1 IN at a region topologically different for
the DD(35)E catalytic active site entails targeting the nonspecific DNA binding site, the
dimer-dimer interface, or inhibition of the interaction between IN and another essential
viral or cellular protein. Allosteric inhibition of HIV-1 IN targeting the nonspecific DNA
binding region is ambitious because of the length of the CTD lysine chain. However,

22
disruption of the dimer-dimer interface using coumarins, for example, has shown success
inhibiting HIV-1 IN and viral replication in synergy with S1360.
5

Inhibition of the interaction between HIV-1 IN and RT is another strategy to
inhibit viral replication.
141
Inhibition of this interaction destabilizes the integrity of the
RTC. The disruption prevents IN from providing the RT nucleoprotein structural support
and preventing the transition from RTC into the PIC. In addition to inhibiting the
interaction between IN with other viral proteins, disrupting the interaction between HIV-
1 IN and a cellular protein essential to viral replication is the best strategy to overcoming
the high mutation rate of HIV.
HIV evolved to incorporate specific cellular proteins to aid in replication; thus,
inhibitors designed to prevent these key interactions are less likely to encounter resistant
strains that still maintain strong viral fitness properties. The crystallization of the
LEDGF/p75-IN protein-protein complex are a monumental success because it defines
exact 3D spatial coordinates of key residues responsible for the interaction at a position in
time. The allosteric pocket defined at the IN dimer-dimer interface between the CCD and
NTD domains of separate dimers provides further evidence a small molecule could bind
within this important pocket. Furthermore, polypeptides derived of the LEDGF/p75 IBD
inhibit the formation of the LEDGF/p75-IN complex and shift the oligomerization of IN
to an inactive tetramer form.
4, 51
Recently, small molecules reported to inhibit
LEDGF/p75-IN inhibition also have antiviral activity. The first reported LEDGF/p75-IN
inhibitor, D77, showed 41% inhibition at 100 µM in a yeast hybrid system (Figure 14).
28

23

Figure 14. Structures of LEDGF/p75-IN inhibitors recently published from other labs.
1.8.6 Design of Inhibitors Disrupting LEDGF/p75-IN Interaction
IN is a viral protein vital to various important stages of the HIV life cycle prior to
the the formation of a provirus. Design of small molecules to inhibit every important
phase of IN function counters the benefit IN affords the HIV virus, from formation of the
PIC to the two stages of viral DNA integration. The trail of potential IN targets is still
continuing to explode while new crystal structures and identification of new host factors
are found. Several cellular proteins aid in protecting, chaperoning, and guiding the IN-
led PIC to the nucleus and site of viral genomic integration. Inhibition of the interaction
between IN and a small group of essential cellular cofactors will significantly improve
the efficacy of HAART and diminish HIV infection to undetectable levels.
The discovery of LEDGF/p75 as a key constituent for viral DNA integration, the
availability of a crystal structure of LEDGF/p75-IN in a complex, and the small molecule
capacity of the LEDGF/p75 IBD pocket on IN suggests inhibitors with an affinity to
disrupt the LEDGF/p75-IN interaction will also effectively inhibit viral replication; if the

24
compound can reach its target. Recently, a handful of LEDGF/p75-IN inhibitors have
been published in various journals; however, diversity and drug-ability is lacking. The
first LEDGF/p75-IN inhibitor reported, D77, had a central rhodanine moiety and had an
antiviral EC
50
value of 24 µg/ml in MT-4 cells and 5 µg/ml in C8166 cells.
28

The first LEDGF/p75-IN inhibitor from this study was identified employing
pharmacophore models with an internal laboratory database built from catalytic IN
inhibitors over the years. The identification of an active LEDGF/p75-IN inhibitor using
these models helped validate pursuing the discovery and design of LEDGF/p75-IN
inhibitors using two novel pharmacophore models in two funded grants.

Figure 15. Lowest energy docking pose of the first LEDGF/p75-IN inhibitor identified
from an internal database before investing in commercial compounds.

Two additional papers have been published, exploring compounds with a central
indole attached to a 2-hydroxy-4-oxobut-2-enoic acid chain that inhibited LEDGF/p75-
IN interaction. These compounds have a terminal diketo-enol acid chain, a moiety
previously described to be metabolically active in early IN inhibitors, indicating these
compounds will not serve as leads. The most active inhibitor from the second
LEDGF/p75-IN inhibition study had an IC
50
value of 35 µM.
22
Subsequent structure
similarity searches yielded three compounds that inhibited LEDGF/p75-IN interaction

25
with IC
50
values around 10 µM in a follow-up study.
23
Compound 6c is the only
compound that protected the metabolic diketo-enol acid within a ring and still had an
inhibitory profile. None of the compounds in these two studies exhibited antiviral
activity. Also, two crystal structures of LEDGF/p75-IN inhibitors with a common 2-
(quinoline-3-yl) acetic acid were solved and reported in a separate study, showing the
binding interaction at the IN dimer-dimer interface (PDB entry code 3LPT and 3LPU).
The most active compound VI inhibited LEDGF/p75-IN interaction with an IC
50
value of
1.4 µM and an antiviral EC
50
value of 2.4 µM, providing a proof of concept that targeting
the LEDGF/p75-IN interaction is valid for antiretroviral therapeutic intervention.
17

LEDGF/p75 IBD recombinant mutants I365A, D366A, and D366N abolish the
LEDGF/p75-IN complex formation, highlighting these residues as an ideal template for
the rational design of pharmacophore models to identify a training set of small molecules
to screen against the important interaction. Pharmacophore model development using a
receptor or ligand as a template can successfully lead to training sets of molecules has
several classes of inhibitors as occupants of its successful history. Utilizing the crystal
structure to build receptor-based pharmacophore models of key LEDGF/p75 KID
residues provides a rationally-based starting point to the identify LEDGF/p75-IN
inhibitors.
Chapter 2. Acylhydrazone-based Small Molecules as LEDGF/p75-IN
Inhibitors

The role of cellular protein LEDGF/p75 in IN-led PIC chromosome targeting
provides a functional advantage for lentiviradae. Disrupting the interaction between IN
and LEDGF/p75 offers an unexplored path to inhibit HIV replication. New HIV
infections are likely to have a multitude of different viral strains. New resistant-viral

26
strains evading LEDGF/p75-IN inhibitors are also unlikely to have significant viral
survival fitness because of the important role of the cellular LEDGF/p75 cofactor. Key
LEDGF/p75 residues K364, I365, and D366 are the basis for pharmacophore models
developed to identify novel LEDGF/p75-IN inhibitors. Analysis of LEDGF/p75-IN
crystal structures and mutagenesis studies reveals LEDGF/p75 residues K364, I365, and
D366 form hydrogen bonds with HIV-1 IN residues E170 and H171 and van der waals
interactions within the A128-W132 and A169 hydrophobic dimer-dimer pocket.
Here forth this dissertation reports more than 100 novel acylhydrazone, pyrazole,
rhodanine, and other leads as inhibitors of the LEDGF/p75-IN protein-protein interaction
with IC
50
values below 50 µM.
2.1 Methodology
2.1.1 Selection and Preparation of Compounds
Interaction-based 3D pharmacophore models with 4 features, developed using
Discovery Studio, was imported to Catalyst for search queries to retrieve compounds for
biological screening. The database search query retrieved a wide variety of compounds
with novel chemical scaffolds composed of the desired chemical features from the multi-
conformer Catalyst-formatted database. The Fast Flexible Search database/spread sheet
methods were employed to search a commercial database consisting of 365,000
compounds (ASINEX Corp, North Carolina, USA).

27
2.1.2 Molecular Docking
A. GOLD
346 compounds retrieved by the pharmacophore search were exported and fully
minimized before performing docking analysis. After building all the required ligand
structures of a specified configuration, molecular docking was conducted. The generic
optimization ligand design (GOLD version 3.2) molecular docking program, which uses
a genetic algorithm method for conformational search and docking, is widely regarded as
one of the best docking programs.
99
For the protein target, a 20 Å radius active site was
defined considering the carboxylate oxygen atom of residue D168 as the center of the
active site. We used an X-ray determined structure of the HIV-1 IN catalytic core
domain in complex with the LEDGF/p75 IBD (PDB entry code 2B4J). The structural file
consists of four chains: chains A and B form the IN catalytic core domain dimer; chains
C and D are LEDGF/p75 monomers binding to IN in opposite directions. All water
molecules and the LEDGF/p75 monomer chain D were removed and proper protonation
states assigned for acidic and basic residues before docking.
On the basis of the GOLD fitness score, each molecule's bound conformation that
matches the high fitness score was considered as the best bound-conformation (Figure
15). In the initial virtual screening the best 100 compounds were selected solely on
GOLD fitness scoring function. Next, we selected 50 non-identical compounds for
experimental analysis on the basis of GOLD score, pharmacophore fitness, and structural
diversity. All docking runs were carried out using standard default settings with a
population size of 100, a maximum number of 100,000 operations, and a mutation and
crossover rate of 95. The GoldScore and ChemScore were calculated for comparison. The

28
GOLD score fitness function was set to the default setting, optimized for the prediction of
ligand binding positions and taking into account H-bonding energy, van der Waals
energy, and ligand torsion strain; whereas, the ChemScore fitness function estimates the
total free energy change that occurs upon ligand binding and was trained by regression
against binding affinity data.
B. CDOCKER
A radius of 12 Å was defined as the active site for the target IN, considering the
carboxylate oxygen atom of residue D168 as the center. The X-ray crystallographic
determined structure of HIV-I IN catalytic core domain in complex with LEDGF/p75
(PDB entry code 2B4J) was used as in the GOLD study. CDOCKER grid-based
molecular docking method employs CHARMm and the following steps were included in
the CDOCKER protocol.
135
Initially, a set of ligand conformations were generated using
high-temperature molecular dynamics with different random seeds. Random orientations
of the conformations were then produced by translating the center of the ligand to a
specified location within the receptor active site and performing a series of random
rotations. A softened energy is calculated and the orientation is kept if the energy is less
than a specified threshold. This process continues until either the desired number of low-
energy orientations is found, or the maximum number of bad orientations has been
attempted. Each orientation is subjected to simulated annealing molecular dynamics. A
final minimization of the ligand in the rigid receptor using non-softened potential was
performed. For each final pose, the CHARMm energy (interaction energy plus ligand
strain) and the interaction energy alone were calculated. The poses were then sorted by

29
CHARMm energy and the top scoring (most negative Gibbs free energy value) poses
were retained.
2.1.3. Preparation of Recombinant Proteins
Protein expression plasmids pGEX-6P-3 and pET-15b-IN encoding the
LEDGF/p75 fusion protein gene and HIV-1 IN gene respectively, were transformed into
competent E. coli pLysS BL21 (DE3) bacterial cells through heat shock and plated on
100 μg/ml ampicillin LB-agar plates. The plasmid pGEX-6P-3 encoded for a FLAG
gene (Figure 16), while pET-15b included a hexa-histidine tag on the IN C-terminus
(Figure 17). The plasmids contain a T7 promoter region to promote the overexpression
of transformed genes only and an ampicillin-resistant gene to positively select for
correctly transformed colonies. The bacteria were grown in large culture vats at 37 ˚C,
250 rpm, and protein expression was induced with 1 mM IPTG. The bacteria was
centrifuged using a Beckman Coulter 6KR centrifuge for 20 minutes at 3000 rpm and
lysed using French Pressure Cell Press (ThermoSpectronic, Inc.). Recombinant
LEDGF/p75 was isolated using a general non-tagged purification protocol using Heparin
HP and Sepharose SP columns according to the manufacture (Thermoelectronic, Inc.).
Recombinant IN was isolated using a nickel-chelating column and extracted with
increasing imidazole concentrations at 4 ˚C. Overnight dialysis at 4 ˚C concentrated
purified proteins in 50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM DTT,
10% glycerol buffer stock solutions, and the purified protein solutions were kept at -80
˚C for storage.

30

Figure 16. Cellular LEDGF/p75 pGEX-
6P-3 plasmid map with a FLAG peptide
gene encoded.
Figure 17. HIV-1 IN pET-15b plasmid
map includes a hexa-histidine tag on the C-
terminus.

2.1.4. LEDGF/p75-IN AlphaScreen Proximity Luminescent Assay
The amplified luminescent proximity hybridization assay (AlphaScreen) was
performed according to the manufacturer's protocol (Perkin Elmer, Waltham, MA).
58

Reactions were performed in a 25 μl final volume in 384-well Optiwell™ microtiter
plates (Figure 18). The reaction buffer contained 25 mM Tris–HCl (pH 7.4), 150 mM
NaCl, 1 mM MgCl
2
, 0.01% (v/v) Tween-20 and 0.1% (w/v) bovine serum albumin. Wild
type IN had a His
6
-tag (300 nM final concentration) and was pre-incubated with each
inhibitor for 30min at 4°C. Next, 100 nM FLAG peptide-tagged LEDGF/p75 was added
to the reaction and incubated for an additional hour at 4°C. Subsequently, equal volumes
of Ni-chelate –coated donor beads and anti-FLAG acceptor beads were added to a final
concentration of 20µg/ml for each beads. Next, the proteins and beads were incubated
for 1h at 30°C to allow association to occur. Exposure of the reaction to direct light was
omitted as much as possible and the emission of light from the acceptor beads was
measured in the EnVision plate reader (Perkin Elmer, Benelux) and analyzed using the
EnVision manager software.

31

Figure 18. Cartoon of LEDGF/p75-IN protein-protein interaction with AlphaScreen
bead technology. Excitation at 580 nM triggers the nickel-chelating donor beads to
release singlet oxygen which is picked up by adjacent anti-FLAG acceptor beads that
emit light for the EnVision plate reader.

2.1.5 AlphaScreen Counterscreen
A counterscreen assay was conducted to observe a compound's ability to
"quench" the AlphaScreen bead signal and inhibit the non-specific interaction between
the protein tags and their affinity to the beads (Figure 19). Some compounds absorbed
energy at the emission frequency, significantly diminishing the output signal for accurate
reading. As another example, molecules can also interfere with the interaction of the
FLAG peptide with the anti-FLAG acceptor bead reporting as a false positive. An
internal control substrate, provided by the company, was used to determine the
nonspecific inhibition of the active compounds with the beads and tags as well as the
disruption of the emitting signal. None of the compounds reported in this study exhibited
quenching effects.

32

Figure 19. Cartoon of AlphaScreen counterscreen in the absence of LEDGF/p75 and IN
recombinant proteins. The assay uses a hexa-histidine tagged peptide conjugated to a
FLAG peptide to identify false positive LEDGF/p75-IN inhibitors.

2.1.6. Integrase Catalytic Assay
To determine the extent of 3'-processing and strand transfer, wild-type IN was
pre-incubated at a final concentration of 200 nM with the inhibitor in reaction buffer (50
mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10% glycerol
(w/v), 7.5 mM MnCl
2
, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol,
10% dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30
º
C for 30 min. Then, 20 nM
of the 5'-end
32
P-labeled linear oligonucleotide substrate was added, and the incubation
was continued for one hour. Reactions were then quenched by the addition of an equal
volume of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol
and 0.025% bromophenol blue). An aliquot (5 - 10 µl) was electrophoresed on
denaturing 20% polyacrylamide gels (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20%
acrylamide, 8M urea). Next, the gels were dried, exposed in a PhosphorImager cassette,
and analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences) and
quantitated using ImageQuant 5.2.
Percent inhibition (%I) was calculated using equation: % I = 100 X [1 - (D - C)/(N - C)],
where C, N, and D are fractions of 21-mer substrate converted to 19-mer (3' processing
product) or strand transfer products for DNA alone, DNA plus IN, and IN plus drug,

33
respectively. The IC
50
values were determined by plotting the logarithm of drug
concentration versus percent inhibition to obtain the concentration that produced 50%
inhibition.
2.1.7 Cytotoxicity Assays
The inhibitory effect of compounds on the HIV-induced CPE in CEM and MT-4
cell culture and compound cytotoxicity in HCT116 cells were determined by flow
cytometry and MTT-assay.
106
The MTT assay measures the reduction of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mitochondrial dehydrogenase
of metabolically active cells to a blue formazan derivative, which can be measured with a
spectrophotometer. The 50% cell culture infective dose (CCID
50
) of the HIV strains was
determined by titration of the virus stock using CEM or MT-4 T-lymphocytic cells. MT-
4 cells were grown in RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 2
mM L-glutamine, 0.1% sodium bicarbonate and 20 µg/mL of gentamycin. The cells
were infected with HIV strains in the presence of select LEDGF/p75-IN inhibitors.
None of the compounds reported in this chapter exhibited significant cytotoxic
effects in a representative cell line. Only compounds 15, 24, 26, and 31 showed a
cytotoxic profile with IC
50
values of 10 µM. Compounds 1 and 2 were screened for
antiviral activity and were not active at 10 µM.
2.2. Results and Discussion
2.2.1. LEDGF/p75-IN Interaction-based 3D-Pharmacophore Model
The design of LEDGF/p75 receptor-based pharmacophore models to filter large
commercial databases to identify novel small molecules that disrupt the crucial
LEDGF/p75-IN protein-protein interaction have led to the discovery of several lead

34
acylhydrazone, rhodanine, and pyrazole-based inhibitors. Key contact residues between
the cellular protein LEDGF/p75 and HIV-1 IN were used as a template to develop
interaction-based three dimensional (3D) pharmacophore models. This computer
generated representation was then used as a query to identify small-molecule inhibitors
that could disrupt the LEDGF/p75-IN protein-protein interaction. Experimental evidence
and molecular dynamic simulations show that two regions of LEDGF/p75 protein
(residues 364-368 and residues 402-408) are involved in binding to IN.
16
Analysis of the
binding domains between HIV-I IN and LEDGF/p75 reveals that the LEDGF/p75 tri-
peptide K364, I365, and D366 makes effective hydrogen bonding interactions with IN
residues Q168, E170, and H171 (Figure 20). These interactions were the basis of
generating two 3D pharmacophore models using an X-ray crystallographic structure of
the LEDGF/p75-IN complex (PDB entry code: 2B4J).

Figure 20. K364, I365 and D366 LEDGF/p75 residues make effective hydrogen-bonding
interactions with IN residues Q168, E170, and H171 and are the basis for interaction-
based 3D-pharmacophore models.

35
The two pharmacophore models have three common descriptors and either a
negative ionizable (NI) or a hydrogen bond acceptor (HBA) for the carboxylate group of
LEDGF/p75 residue D366 which makes strong hydrogen bond (H-bond) interactions
with the IN residues E170 and H171 NH amide. Next, the model was merged with two
hydrogen-bond donor (HBD) groups representing LEDGF/p75 residue K364 and the I365
NH amide backbone which makes H-bond connections with IN residues Q168 and E170.
Lastly, a hydrophobic (HYD) representative was used for the LEDGF/p75 residue
I365 side chain. The co-crystal structure of the LEDGF/p75-IN complex depicts I365 to
be in a hydrophobic environment within the IN dimer-dimer pocket and comes in close
contact with residues M178, W131, W132, A128 and A129. The proposed interaction-
based 3D pharmacophore model shown in Figure 21 utilizes key LEDGF/p75-IN
interactions and was employed as a query to search small molecule databases. The initial
search yielded 346 hits.

Figure 21. Interaction-based 3D-pharmacophore and an active compound mapped on the
interaction based 3D pharmacophore. The alignment of chemical features with
pharmacophore features of the compound shown are negatively ionizable group (NI),
hydrophobic (HYD) and two H-bond donors (HBD).

36
2.2.2 Filtering Drug-like Compounds for Experimental Analysis
The LEDGF/p75 lysine, isoleucine, aspartic acid (KID) pharmacophore model
retrieved 346 compounds with a wide variety of chemical scaffolds from a database of
365,000 compounds. Before experimental analysis was pursued several criteria were
considered to further filter and focus the initial 346 hits. Consideration for further
analysis was given to compounds that adopted the required binding orientation and a
pharmacological potential of the compound's chemical scaffold. High docking scores
were also evaluated using GOLD docking program.
99
The last parameter satisfied was
Lipinski’s rule-of-five which applies drug-like limitations derived from FDA-approved
drugs.
74, 75
The filtering process represented in Figure 22 resulted in a pilot training set of
50 compounds for biological evaluation. The chemical structure and purity of each
compound was confirmed by nuclear magnetic resonance and mass spectrometry. None
of the reported inhibitors show significant cytotoxicity in a representative cancer cell line.

37

Figure 22. Representation of in silico and experimental screening protocol implemented
in the discovery of novel LEDGF/p75-IN disrupting compounds. After filtering, a
training set of diverse compounds were selected for screening against recombinant
LEDGF/p75-IN protein-protein interaction in vitro.
2.2.3. Pharmacophore Search and Acylhydrazone-based Inhibition

The initial search yielded 346 compounds, where the top pharmacophore model-
based fitness values determined the first pool for analysis. A total of 150 selected
compounds fit the pharmacophore model with a score above 2.0. Based on structural
information of the LEDGF/p75 binding pocket of IN, molecular docking analysis was
performed to select small molecules that favorably bind to IN within this region.
Irrespective of fitness value strengths, all 346 compounds retrieved were docked into the
LEDGF/p75 binding site of IN. AlphaScreen proximity luminescent-based interaction
assays were performed to identify molecules that disrupt the LEDGF/p75-IN interaction

38
in vitro. Inhibitors that showed greater than 45 percent inhibition at 100 μM were
considered for dose response and optimization studies.
Promising molecules were tested to determine the IC
50
value of each for the
LEDGF/p75-IN protein-protein interaction. The training set had several diverse
compounds that inhibited the LEDGF/p75-IN complex formation with IC
50
values of 100
μM and below (Table 1). Compound 1 was among the most potent compounds identified
with an IC
50
value of 9 μM, however no antiviral activity was observed. On the other
hand, compound 2 showed moderate/weak antiviral activity with an effective
concentration (EC
50
) of 40 μM in HIV infected MT-4 cells. GOLD docking scores and
CDOCKER interaction energy calculations were also correspondingly high for both.
Table 1. LEDGF/p75-IN inhibitors retrieved from the first pharmacophore search.

Compound
LEDGF/p75-IN IC
50
IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
(μM) 3’ Processing Integration
1.

9 ± 6 >100 >100
2.

14 ± 7 >100 >100
3.

68 ± 17 >100 <100
4.

100 >100 >100

39
Table 1 continued.
5.

100 >100 >100
6.

100 9 6
a
LEDGF/p75-IN 100 μM IC
50
value represents 45 - 55% inhibition of at 100 μM.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
Both derivatives 1 and 2 had pharmacophore fitness values of 3.05 and 2.65,
respectively, and a GOLD docking score above 45, suggesting bromine had an important
role in antiviral activity and target presentation. Compounds 3 - 6 were selected from the
initial model search and exhibited weak activity (Table 1). Further investigation into
acylhydrazone derivatives coupled with substructure database searches yielded an
additional 35 compounds (not shown), where eight had IC
50
values of 100 μM or below
(Table 2). Compound 7 was the most active acylhydrazone derivative discovered in this
fashion, inhibiting LEDGF/p75-IN interaction with an IC
50
value of 7 μM. Potency
decreased in compounds with a methyl group that shifted to meta (8) and para (9)
positions, revealing an inhibitory profile ranging from 7 to 26 and 100 μM, respectively.

40
Table 2. LEDGF/p75-IN inhibitors retrieved from optimization studies of initial hits.

Compound
LEDGF/p75-IN IC
50
IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
(μM) 3’ Processing Integration
7.

7 ± 3 >100 >100
8.

26 ± 5 >100 >100
9.

100 >100 >100
10.

100 >100 >100
11.

14 ± 5 >100 >100
12.

100 >100 >100
13.

71 >100 >100
14.

22 ± 6 >100 >100
a
LEDGF/p75-IN 100 μM IC
50
value represents 45 - 55% inhibition of at 100 μM.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

41
A close analogue to compound 1, compound 13 contained a nitrosylated
phenylacetamide and showed a significant decrease in activity with an IC
50
value of 71
μM. Reducing the acylhydrazone to a hydrazine, as observed in 14, also exhibited
inhibition with an IC
50
of 22 μM. The activity observed for many acylhydrazones tested
in this study provided a foundation for further investigations. Furthermore,
acylhydrazones have a wide range of applicability against different therapeutic targets
including HIV-1 capsid,
61, 127
MurC and MurD ligases,
123
and even serve as a
cyclooxygenase COX-2 inhibitor.
129

Comparing compounds 1 and 2 with other acylhydrazones revealed the HBA or
NI representative on the aromatic ring improved LEDGF/p75-IN complex disruption.
Another active acylhydrazone, compound 3 had a central indole attached to a long
aliphatic acetamide. With an IC
50
value of 68 μM, compound 3, containing a long alkyl
chain, appeared to be advantageous for LEDGF/p75-IN complex disruption, while GOLD
docking calculations predicted an excellent docking score at the LEDGF/p75-IN site.
Compounds 3 - 14 did not show antiviral activity.
2.2.4 Protein-ligand Interactions
One of the more active LEDGF/p75-IN complex inhibitors identified in this study
was compound 1 with an IC
50
value of 9 µM, a close analogue to antiviral compound 2.
Compound 1 also had the lowest energy docking poses at the LEDGF/p75 binding region
along the IN dimer-dimer interface as shown in Figure 23. A closer view of the proposed
binding mode showed the inhibitor fit well in the LEDGF/p75 binding site and is in
involved in several H-bond and van der Waals interactions with IN residues. Docking of
compound 1 with its lowest energy conformations revealed the molecule comes in close

42
contact with IN residues Q95, T125, A128, W131, W132, Q168, A169, E170, H171,
T174, and M178. These interactions were comparable with the LEDGF/p75 interactions
shown in the X-ray co-crystallized structure, PDB 2B4J. The two oxygen atoms of
compound 1 carboxylate makes an H-bond interaction with the side chain amide proton
of Q95 and the backbone amide proton of E170. The acylhydrazone amide proton of
compound 1 can also make a strong H-bond interaction with the side chain oxygen atom
of Q168.

Figure. 23. Binding mode of compound 1 core-contact residues (Q95, T125, A128,
W131, W132, Q168, A169, E170, H171, T174, and M178 ) which make effective H-
bond or hydrophobic interactions with the inhibitor.
The binding mode of compound lead 1 showed an edge-to-face interaction with
the aromatic ring of IN residue W131 and a face-face π-interaction with W132 aromatic
ring. Earlier molecular dynamics simulation studies and binding free energy
decomposition analysis found the free energy contribution of each LEDGF/p75 residue
upon binding to IN was ΔG
b
res
≤ -1.0 kcal, a value comparable with the small molecule
inhibitors described in our study.
43
Docking results of the most active LEDGF/p75-IN

43
inhibitors of each class revealed similar docking results as compound 1 with low-energy
conformations docked within the IN dimer-dimer interface and LEDGF/p75 binding
pocket.

2.2.5 Inhibition of IN Enzymatic Function
Only compound 6 from the initial training sets of acylhydrazone-based
LEDGF/p75-IN inhibitors inhibited IN catalytic function.
2.2.6 Implications for Drug Design
Cellular transcriptional cofactor LEDGF/p75 enhances HIV-1 IN enzymatic
function, solubility, and DNA binding ability. Conformational changes key to the IN
intermediate states presents the opportunity for LEDGF/p75 to bind at a dimer interface,
an evolutionary advantage afforded the virus to utilize the transcriptional chaperone's
chromosomal docking properties. Knockdown of LEDGF/p75 significantly impairs HIV
replication, thus designing compounds that would interrupt IN and LEDGF/p75 binding
defines an alternative therapeutic strategy that would be effective in raltegravir and
elvitegravir drug-resistant viral profiles and enhance antiretroviral regimens.
110
To date,
there are no IN inhibitors in clinical trials that target allosteric sites such as the cofactor
binding site of the LEDGF/p75 binding region.
Disruption of the LEDGF/p75-IN interaction represents a new mechanistic class
of HIV-1 IN targeted therapeutics, where molecules identified bind to a region
topologically different from the IN catalytic active site. Compound 2 showed antiviral
activity validating studies into other acylhydrazones and hydrazine derivatives to improve
antiviral profile. In addition to synergistic effects, antiviral compounds specific for
LEDGF/p75-IN complex disruption would be active in viral strains resistant to known

44
classes of antiretroviral agents including IN inhibitor resistant strains. Also,
LEDGF/p75-IN inhibitors targeting the binding site of the necessary cellular protein has a
much higher genetic barrier compared to a viral protein like HIV-1 IN; thus greatly
decreasing the rate at which new drug resistant viral strains will emerge.
Chapter 3. Second Generation Acylhydrazone-based LEDGF/p75-IN
Inhibitors

Two lead compounds 1 and 2, identified using LEDGF/p75 KID pharmacophore
models, had a central N-acylhydrazone in common. Substructure and similarity
investigations revealed additional acylhydrazone-containing compounds inhibited
LEDGF/p75-IN complex formation. These observations coupled with selective screening
of an in-house database of 10,000 compounds led to the discovery of N-acylhydrazones
as a novel highly active chemical class of LEDGF/p75-IN inhibitors.
As a chemical drug class, N-acylhydrazones have been used to treat adentitis and
tuberculosis (TB). Figure 24 shows the chemical structures of acylhydrazone-based
prodrugs used to treat different forms of TB. Isoniazid and other acylhydrazone anti-
tuberculosis drugs exhibit a mechanism of action that involves targeting the biosynthesis
of mycolic acids found in the pathogenic bacteria cell wall.
84, 102
These antibacterial
compounds also led to the discovery that acylhydrazones could be used as hydrolytically-
cleavable prodrugs.
53

45

Figure 24. Clinical acylhydrazone and hydrazine-containing drugs have been widely
used in antituberculosis, rheumatoid arthritis, psoriasis, and Crohn's disease.
7, 56, 68

Herein, we report acylhydrazone, hydrazine, and diazene-containing LEDGF/p75-
IN inhibitors and the structure-activity relationship (SAR) of active and inactive
analogues.
3.1 Methodology
3.1.1 Selection and Preparation of Compounds
Substructure and similarity investigations revealed acylhydrazone-containing
compounds successfully inhibited LEDGF/p75-IN complex formation. Selective
screening of compounds with an acylhydrazone or hydrazine were conducted in an in-
house database of 10,000 compounds. All compounds were purchased from Enamine,
dissolved in DMSO, and stock solutions stored at -20 ⁰C.

46
3.1.2 AlphaScreen Assays
Recombinant human LEDGF/p75 and HIV-1 IN proteins were produced from
protein expression plasmids pGEX-6P-3 and pET-15b-IN, respectively. The plasmids
were transformed into competent E. coli pLysS BL21 (DE3) bacterial cells through heat
shock and plated on 100 μg/ml ampicillin LB-agar plates. The plasmid pGEX-6P-3 had a
gene that encodes for a FLAG peptide, while pET-15b included a hexa-histidine tag on
the IN C-terminus. The plasmids contain a T7 promoter region to promote the
overexpression of transformed genes only and an ampicillin-resistant gene to positively
select for correctly transformed colonies. Bacteria were grown in large bacterial culture
vats at 37 ˚C, 250 rpm, and protein expression was induced with 1 mM IPTG. The
AlphaScreen assay was performed according to the manufacturer's protocol. Wild type
integrase with a His
6
-tag (300 nM final concentration) was pre-incubated with each
inhibitor and 100 nM Flag peptide tagged-LEDGF/p75 was then added to the reaction.
Finally, Ni-chelate–coated donor beads and anti-Flag acceptor beads were added to a
final concentration of 20µg/ml and incubated at 30 °C in order to allow association.
Emission signals were read by an EnVision plate reader and analyzed in EnVision
manager software.
The concentrations were screened at a maximum concentration of 50 µM to
reduce dmso content, improve accuracy of the assay, and avoid weakly active
LEDGF/p75-IN inhibitors. The counterscreen assay was conducted as described to
identify quenching compounds with non-specific activity.

47
3.1.3 Integrase Catalytic Assay
To determine the extent of 3'-processing and strand transfer, wild-type IN was
pre-incubated at a final concentration of 200 nM with the inhibitor in reaction buffer.
Then, 20 nM of the 5'-end
32
P-labeled linear oligonucleotide substrate was added and
reactions were quenched with an equal volume of loading dye containing formamide. An
aliquot of each sample was electrophoresed on denaturing 20% polyacrylamide gels and
the gels dried, exposed in a PhosphorImager cassette, analyzed using a Typhoon 8610
Variable Mode Imager, and quantitated using ImageQuant 5.2. The IC
50
values were
determined by plotting the logarithm of drug concentration versus percent inhibition to
obtain the concentration that produced 50% inhibition.
3.1.4 Cytotoxicity Assays
The inhibitory effect of compounds on the HIV-induced CPE in CEM cell culture
and compound cytotoxicity in HCT116 cells were determined by flow cytometry and
MTT-assay, respectively.
106
Select compounds were also screened in infected MT-4 cells.
3.1.5 GOLD Molecular Docking
LEDGF/p75-IN complex inhibitors with IC
50
values below 20 µM were imported
into GOLD and the lowest energy conformation was used to select residues for
mutagenesis. For the LEDGF/p75 binding domain at the IN dimer-dimer interface, a 20
Å radius active site was defined considering the carboxylate oxygen atom of residue
D168 as the center of the active site. The X-ray crystal structure of LEDGF/p75 IBD in
complex with an IN dimer, PDB entry code 2B4J, was used to map the ligand inhibitors.
All water molecules and the LEDGF/p75 monomer chain D were removed and proper
protonation states assigned for acidic and basic residues before docking. The GOLD

48
score fitness function was the default setting. On the basis of the GOLD fitness score,
each molecule's bound conformation that matches a fitness score above 30 in fast-flexible
docking was re-evaluated with more iterations.
3.2 Results and Discussion
3.2.1 HIV-1 IN and LEDGF/p75 Interaction Acylhydrazone Inhibitors
The most active LEDGF/p75-IN inhibitor reported (21) was identified through a
series of SAR investigations from a commercial database of a half million compounds.
The first inhibitor identified in these searches had a central acylhydrazone (15).
Substructure similarity searches yielded additional inactive compounds including 16 and
17. Pyridine-containing compound 18 inhibited LEDGF/p75-IN interaction with an IC
50

of 4 µM. Changing the position of the pyridine nitrogen from meta to an ortho position
improved the inhibition three-fold with an IC
50
of 1.3 µM (compound 19). Compound 21
pyrazine-2-carbohydrazine had the best IC
50
value of 0.4 µM, a significant improvement
compared to compound 22, inactive at 50 µM with a pyrazine opposite a
dichlorobenzylidene in place of the phenol.
Table 3. Pyridine, pyrazine, and pyrazole LEDGF/p75-IN lead inhibitors.
Compounds
LEDGF/p75-IN
IC
50
(µM)
3’ Proc.
IC
50
(µM)
Integration
IC
50
(µM)
Antiviral
15.

93 ± 14 --

--

>20
16.

>50
a
-- -- --

49
Table 3 continued.
17.

>50 >100 >100 --
18.

4 ± 2 >100 >100 --
19.

4 ± 1 >100 >100 >3
20.

1.3 ± 0.3 17 ± 5 13 ± 3 >22
21.

0.4 ± 0.1 >100 >100 >40
22.

>50 >100 >100 --
23.

6 ± 3

>100 >100 --
24.

>50 -- -- --
25.

>50 >100 >100 >40

50
Table 3 continued.
26.

>50 -- --
>20
27.

2 ± 1 >100 >100 >4
28.

4 ± 1

29 ± 12 15 ± 3 --
29.

4 ± 2

>100 >100 >17
30.

7 ± 3 >100 >100 >14
31.

>50 -- -- --
32.

>50 -- -- --
33.

6 ± 2 <100 <100 >20
b

51
Table 3 continued.
34.

21 ± 2 -- -- --
35.

>50 -- --

--

36.

38 ± 6 >100 >100 --
37.

50 100 100 --
38.

50 >100 >100 --
39.

50 -- -- --
40.

8 ± 3

90 ± 14 75 ± 35 --
41.

13 ± 5 90 80 >40

52
Table 3 continued.
42.

>50 -- -- --

43.

>50 -- -- --
44.

5 ± 0.2 >100 >100 >50
b

a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
b
Antiviral results from virally infected CEM and MT-4 cells.

Examples of pyrazole-based acylhydrazones that inhibited LEDGF/p75-IN
interaction followed a pattern similar to pyridine and pyrazine inhibitors. Compounds 24
- 26 were inactive at 50 µM, while 27 had an IC
50
of 2 µM, at least a 25-fold
improvement. 2-hydroxyl-naphthalene compounds 23 and 29 had similar LEDGF/p75-
IN inhibitory profiles and contained a pyridine and tetrahydro-1H-indazole, respectively.
Compound 30, containing a pyrazole in place of a tetrahydro-1H-indazole, also yielded
similar activity with an IC
50
of 7 µM. Primary and secondary nitrogen positions in 29
tetrahydro-1H-indazole and 30 pyrazole were reversed, indicating the position switch or
removal of the non-aromatic ring attached to a pyrazole was not important for inhibition.
The presence of a phenol opposite of nitrogen-containing aromatic rings was
necessary to exhibit inhibitory activity of the reported acylhydrazones. However, other
examples of acylhydrazone-based inhibitors were also identified (Table 3). Although
isoniazid did not inhibit the LEDGF/p75-IN interaction at 1000 µM, examples of "pre-

53
processed" acylhydrazones with a good inhibitory profile were discovered. For instance,
compound 33 had an IC
50
of 6 µM and with a carboxamide replacing the acetyl
hydrazine, as observed in compound 34, the scaffold still exhibited moderate activity
with an IC
50
of 20 µM.
Visualizing the molecular shape through ChemBioDraw 3D software, the active
2-phenol-containing inhibitors all conform to a bent shape with a hydrogen bond between
the acylhydrazone carbonyl and hydroxyl group of the 2-phenol, or 2-
hydroxylnapthalene, as the most energetically favorable conformation. These
observations were confirmed during molecular docking of the most active LEDGF/p75-
IN inhibitors, although the docking pose showed a spread molecule spanning the dimer-
dimer pocket (Figure 25). Active N-acylhydrazones and their analogues' central carbonyl
group acts to form hydrogen bonds with the main chain amide of E170 and H171.

Figure 25. GOLD molecular docking poses of the most active acylhydrazone-containing
LEDGF/p75-IN inhibitor, compound 21. The pyrimidine comes in close contact with
H171 where a possible stacking interaction can form and the carbohydrazide carbonyl
oxygen is close enough to E170 and H171 main chain amide to also form hydrogen
bonds.

Most of the isoniazid-like hydrazides tested do not have a 2-phenol to cause the
bent fold including inactive compounds 31 and 32. Also, replacing the pyrazole of 30

54
with a thiofuran shown in 35 did not maintain LEDGF/p75-IN inhibitory activity.
Several other acylhydrazone-containing LEDGF/p75-IN inhibitors absent of a nitrogen-
containing aromatic ring were also identified (Table 3). Compound 36 had a 2-phenol
but also showed weak inhibitory activity. While the 1,3-dioxo-quinoline in 40 and 4,6-
dioxo-pyrimidine in 41 had an IC
50
of 8 and 13 µM, respectively. The presence of the 2-
phenol improved inhibition in many cases, however this functional group was clearly not
the only factor contributing to activity. Moreover, removing an acetophenone and
leaving a terminal hydrazine (44) yielded a 20-fold improvement in inhibition activity.
LEDGF/p75-IN N-acylhydrazone derivatives were also screened against HIV-1
IN catalytic activity in the presence of MnCl
2
in vitro (Table 3). The most active
LEDGF/p75-IN inhibitor 21 did not inhibit IN catalysis at 100 µM. A structurally similar
acylhydrazone 20 however, inhibited IN enzymatic function with IC
50
values of 20 and
15 µM for 3' processing (3'P) and strand transfer (ST), respectively. The remaining
LEDGF/p75-IN inhibitors containing pyridines or pyrazines were all inactive at 100 µM
for IN catalysis. Bromylated compound 28 was the only other acylhydrazone to exhibit
moderate IN enzymatic inhibition. Further analogues need to be tested to determine a
more comprehensive SAR for an IN inhibitory profile for catalysis.
Several acylhydrazones were chosen for antiviral screening in CD4 T-
lymphocytes (CEM cells), and MT-4 cells for compounds 33and 44. All acylhydrazone-
based compounds tested were ineffective at inhibiting viral replication. Further
optimization is needed to improve acylhydrazone activity profiles in cells.

55
3.2.2 Hydrazines, Diazenes, and Their Derivatives
A total of 40 novel acylhydrazone, hydrazine, and diazene-based compounds
inhibited LEDGF/p75-IN interaction with IC
50
values below 50 µM. In addition to
acylhydrazone compounds, hydrazines were also identified to be potent inhibitors of the
LEDGF/p75-IN interaction (Table 4). Hydrazine 45 had a pyrimidine opposite a meta-
phenol but did not inhibit LEDGF/p75-IN interaction. More SAR information will be
required to analyze the basis of 45 inactivity. Interestingly, compound 46, containing a
2-hydroxyl-naphthalene, displayed an IC
50
of 6 µM, while compound 47 containing the
2-phenol did not show inhibition.
Table 4. LEDGF/p75-IN inhibitors with a central hydrazine and diazene linker.
Compounds
LEDGF/p75-IN
IC
50
(µM)
3’Proc.
IC
50
(µM)
Integration
IC
50
(µM)
Antiviral
45.

>50 -- -- --
46.

6 ± 1 77 ± 12 52 ± 3 --
47.

>50

>100 >100 --
48.

13 ± 3 >100 >100 >28
b

49.

6 ± 4 >100 >100

12
b

56
Table 4 continued.
50.

25 ± 12 >100 >100 >10
51.

6 ± 3 -- -- --
52.

3 ± 2

>100 >100 >20
53.

40 ± 12 -- -- --
54.

46 ± 8 -- -- --
55.

20 ± 8

>100 >100 >5
56.
2 ± 1 >100 >100 >100
b

57
Table 4 continued.
57.

12 ± 2 >100 >100 --
58.

36 ± 9 -- -- --
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
b
Antiviral results from virally infected MT-4 cells.
Compound 49 was the most active LEDGF/p75-IN inhibitor that exhibited
antiviral activity an EC
50
value of 12 µM, more than a two-fold improvement compared
to a di-hydroxylated compound 48. Compound 52 had a hydrazine linker and inhibited
LEDGF/p75-IN with an IC
50
of 3 µM, a substantial improvement in inhibition compared
to analogues 53 and 54. Interestingly, apilimod, a hydrazine compound in clinical trials
for rheumatoid arthritis bears some resemblance to hydrazine 52. Compound 55,
containing a diazene linker in addition to the hydrazine, inhibited the LEDGF/p75-IN
interaction with an IC
50
of 20 µM. The most active diazene tested (52) had an IC
50
value
of 2 µM in vitro but displayed no antiviral activity.
We reasoned that cyclization and/or conjugation of the hydrazine to cyclic
moieties may improve the metabolic profile and reduce non-specific interaction of this
chemical class of LEDGF/p75-IN inhibitors. Compound 59 had a pyrazine and an
oxygen in the ortho position of the flanking ring, however the inhibitory activity was
moderate at best, while 60 containing a 2-phenol was inactive at 50 µM. The change in

58
distance between opposite aromatic rings or the lack of acylhydrazone flexibility could be
responsible for the lack of activity, although compounds 61 and 62 were active with IC
50

values below 10 µM (Table 5).
Table 5. LEDGF/p75-IN inhibitors with an acylhydrazone or hydrazine in a cyclic ring.
Compounds LEDGF/p75-IN
IC
50
(µM)
3’Proc.
IC
50
(µM)
Integration
IC
50
(µM)
Antiviral
59.

39 ± 6 -- -- --
60.

>50 -- -- --
61.

3 ± 1 >100 100 >40
62.

6 ± 2 >100 >100 >100
b

63.

35 ± 13 >100 >100 --
64.

24 ± 6 -- -- --
65.

19 ± 1 >100 >100 >100
b

66.

50 -- -- --

59
Table 5 continued.
67.

32 ± 7 >100 >100 --
68.

16 ± 5 69 ± 30 31 ± 19
3
(slight
toxicity)
69.

21 ± 5 >100 >100 >100
b

70.

34 ± 11 -- -- 24
b

71.

15 ± 5

-- -- >10
72.

12 ± 4
>100 >100 >10
73.

14 ± 6
>100 >100
>5
>15
b

74.

8 ± 1
>100 >100
30% at 5
>60
b

60
Table 5 continued.
75.

23 ± 3
>100 >100 --
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
b
Antiviral results from virally infected CEM and MT-4 cells.
Compounds 63 - 69 had a hydrazine linked to a piperazine moiety, where the
more active inhibitors had electron-withdrawing groups. Compounds 73 - 75 were
selected for screening because they had a protected hydrazine conjugated to a rhodanine,
a moiety from another class of LEDGF/p75-IN inhibitors that is discussed in chapter 5.
Importantly, LEDGF/p75-IN inhibitor 74 had an IC
50
value of 8 µM and 30% antiviral
inhibition at 5 µM.
Hydrazine-containing compounds 44, 46, and 68 were the only examples that
inhibited both LEDGF/p75-IN protein-protein interaction and IN catalytic activities.
Compounds 49 and 70 shared a sulfonamide linker and showed good antiviral activity in
MT-4 cells with EC
50
values of 12 and 24, a selectivity index of >8 and >1, respectively;
MT-4 cytotoxicity not provided. Compounds 68 and 74 also had antiviral activity in
CEM cells, although 68 cytotoxicity altered its inhibitory profile and 74 did not show
antiviral activity in MT-4 cells warranting further investigation in CEM cells (Table 5).
3.2.3 GOLD Docking and Molecular Modeling
Select compounds from this study were docked in the LEDGF/p75 binding cavity
of the IN dimer-dimer interface using the 2B4J crystal structure. For comparison of the
docking interactions, we merged the co-crystal LEDGF/p75-IN inhibitor (976) imported

61
from the IN crystal structure 3LPU,
17
which lacks the complete binding site, onto 2B4J
16

by superimposing both structures. Figure 26 shows that inhibitor 976 forms hydrogen
bond with the main chain nitrogen atoms of E170 and H171 as well as side chain oxygen
atom of T174. Docking of selected active compounds from this chapter formed similar
strong hydrogen bonds with these residues.

Figure 26. Published ligand structure of LEDGF/p75-IN inhibitor 976 that was adopted
from the 3LPU crystal structure and merged into the 2B4J crystal structure to build
comprehensive pocket.
The oxygen atoms from the carbohydrazide moieties of LEDGF/p75-IN inhibitors
21 and 33, and the sulfur from the thiazole moiety of 74, form hydrogen bonds with main
chain nitrogen atoms of E170 and H171. The pyrazino nitrogen of compound 21 (Figure
25), and the terminal nitrogen from the carbohydrazide moiety of 33 (Figure 27), both
form a second hydrogen bond with T174. The phenyl ring of compound 21, the ethyl
amino moiety of 33, the dimethyl-amino-phenyl of 56, and the dimethoxyphenyl of 74,
all pack in a hydrophobic cavity near A128. LEDGF/p75-IN inhibitor 56 chlorophenyl
moiety forms a pi-pi stacking interaction with IN residue His171.

62

Figure 27. Examples of the molecular docking of three active LEDGF/p75-IN inhibitors
representative of the classes presented in this study. A) Compound 33 has a
carbohydrazide oxygen atom docking close to E170 and H171 main chain nitrogen
atoms. B) The active diazene compound 56 chlorophenyl moiety docks near H171 for a
possible pi-pi stacking interaction. C) Compound 74 dimethoxylphenyl moiety docks in
the hydrophobic region while the thiol group docks near the E170 and H171 main chain
amides for hydrogen bonding.
The inactive anti-tuberculosis drug, isoniazid, which contains a carbohydrazide
moiety, did not dock near the A169 - T174 region (Figure 28) and thus cannot form any
hydrogen bonds with these residues to disrupt LEDGF/p75 interaction, as is observed
with the inactivity at 1 mM.

Figure 28. Molecular docking pose of an inactive compound, the anti-tuberculosis drug
isoniazid.

63
3.2.4 Cytotoxicity profile

Several acylhydrazone, hydrazine, and diazene LEDGF/p75-IN interaction
inhibitors had cytotoxic properties in human colon cancer HCT116 p53 +/+ and -/- cell
lines. Most of the pyridine and pyrazine-2-carbohydrazides (15 - 23) and pyrazoles (24 -
30) had cytotoxicity IC
50
values around 10 µM revealing a non-existent therapeutic index
for LEDGF/p75-IN inhibitors (Table 6). The most active LEDGF/p75-IN inhibitor 21
had a cytotoxic IC
50
value of 9 µM in HCT116 cells, a potential therapeutic index greater
than 100-fold. However, compound 21 did not inhibit viral replication in HIV infected
CEM cells at 40 µM, the same result observed in the second most active LEDGF/p75-IN
inhibitor reported (20) here, their activity and promotion of evading metabolic properties
warrant more evaluation into anticancer studies.
19, 92
As a class, acylhydrazone-containing compounds showed minimal cytotoxicity.
Seven compounds had cytotoxic IC
50
values below 5 µM. Active LEDGF/p75-IN
inhibitors 18, 27, and 28 and in the inactive acylhydrazones 16 and 25 successfully
inhibited cellular proliferation with an IC
50
around 5 µM. Compounds 17 and 26,
containing a methoxyl group at the second position, were inactive against the
LEDGF/p75-IN interaction in vitro and displayed no effect on cellular viability in MTT
assays; however, a chlorine in the second position had a mixed cytotoxic profile where
compound 22 did not inhibit cellular growth at 10 µM while 25 had a IC
50
< 2 µM in
cells. Hydrazines 45 and 46 revealed cytotoxicity with IC
50
values below 5 µM, while
diazenes 57 and 58 showed an active cytotoxic profile with IC
50
values below 1 µM. In
order to evade a toxic and metabolic response, central acylhydrazones and cyclic/cyclic-

64
conjugated hydrazines were explored. Overall, compounds 60 - 74 had a more favorable
toxicity profile than flexible hydrazine-based LEDGF/p75-IN inhibitors reported.
Table 6. Cytoxicity profile against HCT116 p53 +/+ and -/-.
Compounds HCT116 IC
50
(µM) Compounds HCT116 IC
50
(µM)
15 >10 45 4
16 2.5 46 <1
17 -- 47 --
18 4 48 --
19 -- 49
20 -- 50
21 9 51 >10
22 >10 52 >10
23 -- 53 >10
24 <1 54 3.3
25 >10 55 >10
26 5 56 >10
27 6 57 <1,0.5 (-)
28 5 58 <1,0.4 (-)
29 <1 59 --
30 1.5 60 10
31 -- 61 --
32 10 62 10
33 -- 63 >10
34 -- 64 <10
35 <1.1, 0.3 (-) 65 >10
36 8 66 >10
37 3.3 67 >10
38 >10 68 >10
39 6.5 69 >10
40 6.5 70 --
41 10 71 >10
42 -- 72 >10
43 -- 73 >10
44 -- 74 >10
Cytoxicity profile of acylhydrazone, hydrazine, and diazene-containing compounds.

65
3.2.5 Implications for Drug Design
Molecular docking of LEDGF/p75-IN inhibitors presented in this study mimicked
the binding site and close approximation to residues identified in the 976 ligand crystal
structure imported from 3LPU. The crystal structure representation showed ligand 976
contorted indicating better molecules can be designed to fit the dimer-dimer pocket with
lower energy, higher affinity, and span the same IN residues A128, W131, E170, and
H171. LEDGF/p75-IN inhibitors identified from this study provide a beginning template
to further design new small molecules.
The energy gained from breaking an intramolecular hydrogen bond of the bent
shaped acylhydrazones increases the affinity and interaction with IN residues E170 and
H171 amide backbone. Next, a stacking interaction with H171 and the ligand's aromatic
ring can stabilize the interaction. This phenomena helps explain the elevated activity
found in 18 - 30, where compounds with a nitrogen in the ortho position had improved
inhibition, suggesting increased electron density improved the internal hydrogen bonding
and thus better interaction with IN.
The prodrug products presented in this study did not show antiviral activity.
Notably, salinizide, aconiazide, and other acylhydrazone prodrugs are hydrolyzed to their
hydrazide component such as isoniazid (Figure 29). Further hydrolysis of isoniazid
mediated by amidase produces the cytotoxic hydrazine which triggers a hydrogen
peroxide response in the cell. Initial aconiazid derivatives are 2-formylphenoxyacetic
acid and isoniazid, 2-formylphenoxyacetic acid binds to the metabolic products hydrazine
and acetylhydrazine to minimize cytotoxicity in the liver.
104, 108, 125

66
Figure 29. Metabolism of acylhydrazones. Aconiazide undergoes hydrolysis to form
isoniazid which in turn is inactivated to an acetylated derivative or further hydrolyzed by
amidohydrolase/amidase to hydrazine.

The most active LEDGF/p75-IN inhibitor identified had an IC
50
value of 400 nM.
Exploration into acylhydrazone prodrugs including the anti-tuberculosis drug isoniazid
did not produce any immediate candidates. Although compounds 33 and 44 had a
terminal hydrazine and effectively disrupted the LEDGF/p75-IN interaction, they did not
show antiviral activity. It is hypothesized that future investigations into other prodrug
candidates will lead to improved activity and SAR analysis will yield new antiviral leads.
Additional compounds containing hydrazine linkers were also identified to have
antiviral properties. Compound 49 has a similar structure to the antikeratolytic
compound linarotene, a dermatological drug that has a sulfone and a hydrazine connected
to an ethylidene. Compound 49 had an antiviral EC
50
value of 12 µM and a CC
50
>100, a
therapeutic index of >8. Furthermore, the LEDGF/p75-IN inhibitor 70 that showed
antiviral activity also has a sulfonamide with a diazapane replacing the hydrazine
suggesting the hydrazine can be replaced while maintaining most of the antiviral activity.
Further investigations into these antiviral inhibitors and their close analogues are
currently underway.

67
Chapter 4. Pyrazole Small Molecules as LEDGF/p75-IN Inhibitors

4.1 Methodology
4.1.1 Selection and Preparation of Compounds
Pyrazoles presented in this chapter were selected to investigate lead compounds
identified from the LEDGF/p75 KID receptor-based pharmacophore models. The
database search queries retrieved a wide variety of pyrazole derivatives. The Fast
Flexible Search database was employed to select potential LEDGF/p75-IN inhibitors
from a database consisting of 365,000 commercially available compounds (ASINEX
Corp, North Carolina, USA). Second generation pyrazoles were synthesized by Dr.
Jung's laboratory in the USC Chemistry Department. Although these compounds were
originally synthesized to study their effects on IN catalytic function, they were also
screened in LEDGF/p75-IN interaction assays because of their close structural
relationship to the pyrazoles identified from the KID pharmacophore models.
4.1.2 Biological Assays
Protein expression plasmids pGEX-6P-3 and pET-15b-IN encoding the
LEDGF/p75 fusion protein gene and HIV-1 IN gene, respectively, were transformed into
competent E. coli pLysS BL21 (DE3) bacterial cells through heat shock and plated on
100 μg/ml ampicillin LB-agar plates. The plasmid pGEX-6P-3 had a gene that encodes
for a FLAG peptide, while pET-15b included a hexa-histidine tag on the IN C-terminus.
Purified recombinant human LEDGF/p75 and HIV-1 IN were used in proximity
luminescent assays to screen for potential inhibitors to disrupt the protein-protein
interaction. An AlphaScreen assay was performed according to the manufacturer's
protocol. A counterscreen assay was conducted to observe compounds' ability to

68
"quench" the AlphaScreen bead signal or inhibit the non-specific interaction between the
protein tags and their affinity to the beads.
To determine the extent of 3'-processing and strand transfer, wild-type IN was
pre-incubated at a final concentration of 200 nM with the inhibitor in reaction; then, 20
nM of the 5'-end
32
P-labeled linear oligonucleotide substrate was added, and the
incubation was continued. Reactions were ended using quenching dye containing
formamide and an aliquot was electrophoresed on denaturing 20% polyacrylamide gels.
The inhibitory effect of compounds on the HIV-induced CPE in CEM and MT-4
cell culture and compound cytotoxicity in HCT116 cells were determined by flow
cytometry and MTT-assay, respectively.
57, 106
The 50% cell culture infective dose
(CCID
50
) of the HIV strains was determined by titration of the virus stock using MT-4
cells. For the drug-susceptibility assays, MT-4 cells were infected with 100-300 CCID
50

of the HIV strains in the presence of five-fold serial dilutions of the antiviral drugs. The
concentration of the compound achieving 50% protection against the CPE of HIV, which
is defined as the EC
50
, was determined. Cytotoxicity of the compounds was determined
by measuring the viability of mock-infected MT-4 cells after 5 days of incubation.
4.2 Results and Discussion
4.2.1 5-oxo-pyrazoles
Investigation into lead molecules other than acylhydrazones yielded three more
unique classes of LEDGF/p75-IN inhibitors. Substituted compounds with 5-oxo-
pyrazoles, 2-iminothiazolins, and thioxothiazolidin-based acids yielded more
LEDGF/p75-IN inhibitors with IC
50
values below 50 μM. The 5-oxo-pyrazole scaffold
in Table 7 has a furan benzoic acid attached to pyrazole position 4 and an aromatic ring

69
with HBA functionality important for activity. Active pyrazole derivatives 76, 77, and 78
inhibited LEDGF/p75-IN protein-protein interaction with IC
50
values of 23, 9, and 19 μM
respectively. The activity stemmed from strong hydrogen bond acceptors on the benzene
attached to pyrazole position 1. Compound 77 had a carboxylic acid representing the
HBA and showed slightly better activity than chlorinated 76. When either the chlorine or
acid was substituted with a tri-fluoromethyl (79), inhibitory activity was abolished. The
pyrazole derivative 80 had an unsubstituted benzene and showed no inhibition at 50 μM,
reinforcing the importance of HBA influence on activity.
Table 7. Compounds with a 5-oxo-pyrazole moiety inhibit LEDGF/p75-IN interaction.
Compound
LEDGF/p75-IN IC
50
IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
(μM) 3’ Processing Integration
76.

23 ± 6

>100

>100
77.

9 ± 3

>100

>100
78.

19 ± 1

73 ± 23

20 ± 9
79.

>50

>100

>100

70
Table 7 continued.
80.

>50

>100

>100
81.

38 ± 4

24 ± 6

7 ± 0.1
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
Qualitatively analyzing the structure activity relationship of the pyrazoles
revealed compounds with improved HBA chemical moieties had a significant effect on
inhibiting LEDGF/p75-IN complex formation. The lead scaffold also offers potential for
optimization at the central pyrazole ring positions 2 and 3. For example, the methyl
group at the 3 position highlights the potential for improved activity, solubility, or
physiochemical properties. Second generation pyrazoles were explored with various
groups substituting the 3-methyl.
Furthermore, pyrazole derivative 81 showed some activity despite having a bulky
phenylthiazole replacing the 3-methyl group and a hydrazine instead of a furan linker.
Also, 81 inhibited IN catalytic activity with IC
50
values of 24 and 7 μM for 3' processing
and strand transfer, respectively. Both 79 and 80 showed no ability to inhibit IN catalytic
activity at 100 μM, while 78 and 81 effectively inhibited both catalytic activities with
strand transfer IC
50
values of 20 and 7 µM, respectively. Pyrazole derivative 78,

71
containing a sulfonamide group, likely provided metal-chelating abilities within the IN
DD(35)E catalytic triad.
Combining active LEDGF/p75-IN inhibitors with catalytic IN inhibitors from the
same class of molecules may potentially show synergistic effects inhibiting viral
infectivity.
4.2.2 Second Generation 5-oxo-pyrazole-based IN inhibitors
Six of the reported second generation pyrazole-based LEDGF/p75-IN inhibitors
had IC
50
values below 50 μM (Table 8). Compounds 82 - 85 did not inhibit LEDGF/p75-
IN interaction at 50 μM, while 86, with a hydroxyl group instead of a methoxy, had a
LEDGF/p75-IN IC
50
value of 40 μM. Although compounds 82 and 83 did not inhibit
LEDGF/p75-IN interaction at 50 μM, compound 83 inhibited IN strand transfer activity
comparably to 86. The IN catalytic inhibitory profile of 83 and 86 likely improved
because the 3-methyl group in 82 was replaced with a trifluoromethyl. Compound 87
also inhibited IN catalytic function with a carboxylic acid replacing the trifluoromethyl,
however the 4-fluorobenzyloxy and hydroxyl group positions are reversed from 86.
Interestingly, compound 88 had a nitro group replacing the bromine in 87 and had a
LEDGF/p75-IN inhibitory IC
50
of 22 μM. Compound 88 also exhibited a 15-fold
improvement in IN strand transfer inhibition compared to 87.

72
Table 8. Compounds with a 5-oxo-pyrazole moiety inhibit LEDGF/p75-IN interaction.
Compound
LEDGF/p75-IN
IC
50
(μM)
3’ Proc.
IC
50
(μM)
Integration
IC
50
(μM)
Antiviral
82.

>50 >100 >100 >10
83.

>50 50 ± 8 30 ± 7 >10
84.

>50 24 ± 1 18 ± 2
13
(IC
90
20)
85.

>50 180 ± 14 170 ± 14 >20
86.

40 ± 10 >100 22 ± 5 >25
87.

>50 >100 30 ± 0.1 >25
88.

22 ± 12 >10, 2.3
2 ± 1

>25
89.

34 ± 6 >100 95 ± 7
12 ± 3

73
Table 8 continued.
90.

35 ± 0.1 8 ± 4 2 ± 1 --
91.

>50 60 ± 14 14 ± 4 >25
92.

50
>33

8 ± 3

>25
93.

>50 15 11 >25
94.

>50 80 ± 29 27 ± 11 >25
95.

30 ± 1 >100 50 --
96.

>50 >100 >100 >25
97.

>50 >100 100 >25

74
Table 8 continued.
98.

50 70 56 >25
99.

38 ± 7 >100 >100 >20
100.

>50 >100 >100 >25
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
Compound 89 was the only pyrazole presented in this chapter that both inhibited
LEDGF/p75-IN protein-protein interaction and exhibited antiviral activity with an IC
50
of
12 μM. Importantly, antiviral compound 89 did not effectively inhibit IN catalytic
function in vitro suggesting the mechanism of action is distant from the catalytic active
site and possibly at the dimer-dimer interface. Compound 90, a salt analogue of 89,
comparatively inhibited LEDGF/p75-IN but also had a strand transfer IC
50
value of 2
μM. The antiviral activity was not determined. Similar to compounds 83 and 86, a
difference in activity is observed between 92 and 93 where only the substitution of a
hydroxyl group with a methoxy abolishes LEDGF/p75-IN inhibitory activity at 50 μM.

75
Chapter 5. Rhodanine Small Molecules as LEDGF/p75-IN Inhibitors
5.1 Methodology
5.1.1 Selection and Preparation of Compounds
Rhodanines presented here were selected to investigate lead compounds identified
from the LEDGF/p75 KID receptor-based pharmacophore models. The database search
queries retrieved a wide variety of rhodanine derivatives. The Fast Flexible Search
database tool was employed to select potential LEDGF/p75-IN inhibitors from a database
consisting of 365,000 commercially available compounds (ASINEX Corp, North
Carolina, USA). Further active analogues were identified from an in-house database of
10,00 compounds purchased from Enamine. All compounds were dissolved in DMSO,
and stock solutions were stored at -20 ⁰C.
5.1.2 Biological Assays
Protein expression plasmids pGEX-6P-3 and pET-15b-IN encoding the
LEDGF/p75 fusion protein gene and HIV-1 IN gene respectively, were transformed into
competent E. coli pLysS BL21 (DE3) bacterial cells through heat shock and plated on
100 μg/ml ampicillin LB-agar plates. The plasmid pGEX-6P-3 encoded for a GST gene,
while pET-15b included a hexa-histidine tag on the IN C-terminus. Purified recombinant
human LEDGF/p75 and HIV-1 IN were used in proximity luminescent assays to screen
potential inhibitors to disrupt the protein-protein interaction. The AlphaScreen assay was
performed according to the manufacturer's protocol. A counterscreen assay was
conducted to observe compounds' ability to "quench" the bead signal or inhibit the non-
specific interaction between the protein tags and their affinity to the AlphaScreen beads.

76
To determine the extent of 3'-processing and strand transfer, wild-type IN was
pre-incubated at a final concentration of 200 nM with the inhibitor in reaction; then, 20
nM of the 5'-end
32
P-labeled linear oligonucleotide substrate was added, and the
incubation was continued. Reactions were ended using quenching dye containing
formamide and an aliquot was electrophoresed on denaturing 20% polyacrylamide gels.
The inhibitory effect of compounds on the HIV-induced CPE in CEM cell culture,
MT-4 cell culture, and compound cytotoxicity in HCT116 cells were determined by flow
cytometry and MTT-assay.
106

5.2 Results and Discussion
5.2.1 2-imino-thiazolidin-4-ones
Exploration into compounds with a central 2-iminothiazolidin-4-one ring led to
the discovery of a new class of LEDGF/p75-IN inhibitors. Six 2-iminothiazolidins
moderately inhibited LEDGF/p75-IN complex formation below 40 μM (Table 9).
Compounds 101, 102, and 103 had a common (E)-3-((3-methyl-4-oxothiazolidin-2-
ylidene)amino)benzoic acid attached to a benzylidene and had IC
50
values of 38, 34, and
39 μM, respectively. LEDGF/p75-IN inhibitor SAR analysis suggests the activity was
maintained because of weak HBA or even a hydrophobic functionality.

77
Table 9. 2-iminothiazolidin-4-one compounds inhibit LEDGF/p75-IN interaction.
Compound
LEDGF/p75-IN IC
50
IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
(μM) 3’ Processing Integration
101.

38 ± 17 >100 >100
102.

34 ± 8 >100 100
103.

39 ± 8 >100 100
104.

19 ± 5 >100 >100
105.

>50 78 43
106.

20 ± 7 -- --
107.

29 ± 11 -- --
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

78
Some similarity is observed between the first published LEDGF/p75-IN inhibitor
(Figure 14) and the thiazolidins presented in Table 9. The most active 2-iminothiazolidin
derivative tested was 104 with a phenol replacing the benzoic acid and an additional
phenol extending from position 3 on the central thiazolidin. Compound 104 had an IC
50

value of 19 μM and a nitro group on the benzylidene. Thiazolidin derivative 105 showed
no activity at 50 μM. Although 105 had three HBA moieties attached to the benzylidene,
one was hindered by an aromatic and another had weak H-bond functionality. Derivatives
of the 2-iminothiazolidin-containing compounds identified from the LEDGF/p75
pharmacophore model were screened to identify further LEDGF/p75-IN inhibitors.
Compounds 106 and 107 are examples of these analogues that exhibited LEDGF/p75-IN
inhibition with IC
50
values of 20 and 29 uM, respectively. In addition, thiazolidin 105
inhibited both IN catalytic function with IC
50
values of 78 µM for 3' processing and 43
µM for integration.
Analysis into the most energetically favorable conformations of each compound
revealed an overall bent geometry of all active compounds. Superimposed at the
thiazolidin ring the resulting geometry revealed activity related to the HBA functionality
and spatial orientation. Superimposing the different classes of LEDGF/p75-IN complex
inhibitors at the 5-oxo-pyrazole and 2-iminothiazolidin-4-one ring revealed a similar
geometry and distance from the aromatic HBA, suggestive that more variations of the
central ring can be tolerated. It is highly likely the overall shape of the molecules played
a vital role in their inhibitory profile. Active LEDGF/p75-IN inhibitor 104 uniquely did
not conform to the active bent geometry as compared to the other active thiazolidin
derivatives. Furthermore, the aromatic ring attached to the tertiary amine at position 5 of

79
compound 104 lends to a three-prong scaffold similar to a trigonal pyramidal shape. This
orientation may lead to a different 3D scaffold all together.
The 2-iminothiazolidin-4-one scaffold allowed for the 5 position to be substituted
with both an alkyl group and an additional ring as long as a polar group was present.
Further evidence of this pattern is observed with compounds 106 and 107 where an acid
replaces the rhodanine methylamine. This presents an opportunity for optimization and a
site to improve physiochemical properties.
5.2.2 (Z)-5-benzylidene-4-oxo-2-thioxothiazolidin-3-carboxylic acids
The thiazolidin ring is also known as a rhodanine, a retinoid derivative of vitamin
A, that has been shown to reduce viral infection and has been extensively explored in IN
catalytic inhibitory studies.
20, 63, 112, 113
Analogous to 2-iminothiazolidins, an additional
class of rhodanines was crafted from the LEDGF/p75 KID tri-peptide pharmacophore
model and optimization studies. Replacing the 2-imino benzoic acid with a thioxo group,
and the methyl group with an acid, revealed a new 4-oxo-2-thioxothiazolidin-3-
carboxylic acid class of inhibitors. Eight of ten compounds selected for experimental
analysis from this class successfully inhibited LEDGF/p75-IN protein-protein interaction
around or below 50 µM (Table 10).

80
Table 10. Compounds with a thioxothiazolidin group inhibit LEDGF/p75-IN interaction.
Compound
LEDGF/p75-IN μM IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
IC
50
3’ Processing Integration
108.

52 ± 17 83 ± 25 49 ± 9
109.

46 ± 17 >100 100
110.

44 ± 3 >100 99 ± 1
111.

>50 39 ± 13 13 ± 3
112.

>50 84 ± 15 62 ± 22
113.

8 ± 1 <100 <100
114.

9 ± 3 <100 <100
115.

32 ± 2 >100 >100

81
Table 10 continued.
116.

25 ± 7 22 ± 10 17 ± 4
117.

31 >100 >100
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
Bearing some structural similarity to 105, thiazolidin derivative 108 showed some
LEDGF/p75-IN complex inhibition with an IC
50
value of 52 µM. Compound 108 had a
carboxylic acid on the central thiazolidin tertiary amine and a methoxybenzylidene in
addition to a more restrained para-ether representing the HBA functionality. Modifying
the 108 ether linker to acetyl groups did not alter the moderate inhibitory activity between
109 and 110. However, the di-chlorinated compounds 111 and 112 did not inhibit the
LEDGF/p75-IN interaction at 50 µM.
The most active rhodanines reported were compounds 113 and 114 with IC
50

values of 8 and 9 µM, respectively (Table 10). Compound 113 had a bromine
representing the HBA on the benzylidene and a propanamido benzoic acid attached at the
3 position of the central thioxothiazolidin ring. An analogue of 113, compound 115 had a
methoxybenzylidene in the para position representing the HBA and a longer amido chain
separating the benzoic acid and central thiazolidin. The four-fold decrease in activity of
115 versus 113 likely related to the H-bond capabilities of the para-methoxyl group
versus that of the meta-bromine.

82
The modified molecular scaffold of the propanamido benzoic acid permitted
changing the benzylidene to different aromatic rings while still maintaining activity. For
instance, compound 116 had an IC
50
value of 25 µM and substituted a methylene
thiophene for the benzylidene. Similarly, 114 replaced the benzylidene with a furan
benzoic acid, plus a nitrobenzene substitute for the propanamido benzoic acid or
thioxothiazolidin carboxylic acids. There were no analogues close enough to 114 to
observe proper SAR patterns, however, there was symmetry when viewing the molecule
through the central alkene bond. This superimposition can help explain the activity
observed and possibly a similar binding mechanism of 113 and 114. The emerging
pattern also highlighted improved activity related to a minimal presence of HBA on the
5-benzylidene. Furthermore, the bent geometry observed in active pyrazoles was also
observed in active 2-iminothiazolidin-4-ones, and the 3-(4-oxo-2-thioxothiazolidin-3-
yl)propanamido benzoic acids, revealing candidates for activity-based models.
Although 111 and 112 had failed to inhibit LEDGF/p75-IN complex formation,
both compounds inhibited IN catalytic activity. Thioxothiazolidin 111 inhibited IN
strand transfer activity with an IC
50
of 13 µM, while 108 and 112 had IC
50
values of 49
and 62 µM, respectively. The additional chlorine on the terminal aromatic increased IN
catalytic inhibition greater than four-fold when comparing 112 and 111, and three-fold
when comparing 108 with 111. None of the rhodanine-containing compounds in chapter
5 sent for testing against HIV showed antiviral activity.

83
5.2.3 Implications for Drug Design
SAR fragment hopping led to the discovery of LEDGF/p75-IN inhibitors 73 - 75
with a hydrazine protected in a central rhodanine ring and a potential for antiviral activity
with compound 74 (Table 5). The antiviral compounds reported in chapters 3 and 4
justifies exploring each lead's fragments to identify the importance of each feature to
improve activity. Like the hydrazine-containing LEDGF/p75-IN inhibitors 49 and 70,
compound 89 was identified because it had a central 5-oxo-pyrazole similar to the 5-oxo-
pyrazoles identified from the pharmacophore model hits. Thus, new compounds
combining different moieties of active LEDGF/p75-IN inhibitors such as acylhydrazone
conjugated or protected within central 5-member cyclic rings such as rhodanines,
pyrazoles, imidazoles, triazoles, pyrroles, furans, and thiofurans, warrants further
investigations to identify more novel lead candidates.
Chapter 6. New Potential Lead LEDGF/p75-IN Inhibitors
6.1 Methodology
6.1.1 Selection of Compounds
Indoline and indole-based compounds that showed good fitness values from the
pharmacophore models described in chapter 2, were selected for screening in
LEDGF/p75-IN assays. 2,3-dihydroxybenzamide were synthesized by collaborator Dr.
Long to study their effects on IN catalytic function. They were screened in the
LEDGF/p75-IN complex formation assay to further study their binding affinity to IN and
a cellular cofactor, LEDGF/p75. The synthetic scheme for the 2,3-dihydroxybenzamides
and their IN catalytic and LEDGF/p75-IN inhibitory profile was published in JBMC.
34

84
The remaining inhibitors in section 6.2.2. were identified through random
screening of an in-house database of 10,000 compounds (Enamine, Inc.). Analogues and
structure similarity searches in the internal laboratory database of 50,000 small molecules
were conducted to investigate the SAR of active inhibitors. Some lead inhibitors did not
have any analogues available and have not been further pursued. All commercial
compounds were purchased from Enamine and ASINEX. All compounds were dissolved
in DMSO, and stock solutions stored at -20 ⁰C.
6.2 Results and Discussion
6.2.1 Inhibitors Identified from SAR, Fragment Hopping, and
Structure Similarity

A. 1,3-dioxoisoindolines and Indoles
Chapter 2 examined lead LEDGF/p75-IN inhibitors identified from the
LEDGF/p75 KID pharmacophore models. The acylhydrazones first discovered also had
a central indoline fragment (compounds 1 and 2). Additional LEDGF/p75-IN inhibitors
were identified from LEDGF/p75 pharmacophore model hits 118 (isoindolines) and 3
(indoles) derivative searches. The indolines presented in Table 11 all fit the
pharmacophore models, however, not all the compounds selected as hits form the models
showed LEDGF/p75-IN inhibition. Compounds 118 and 120 inhibited LEDGF/p75-IN
interaction with IC
50
values below 25 μM, and had two isoindoline units, while 119 had
only one isoindoline unit and was inactive at 50 μM. The distance between the two
isoindoline units in compound 118 varied from inactive compound 121 with different
linkers, while active compound 122 had a different linker spacing the isoindoline units at

85
a similar atomic distance as 118. Compound 123 was inactive at 50 μM and was missing
the 5-carboxylic acid on the isoindoline units of active compound 122.
Table 11. LEDGF/p75-IN inhibitors with 1,3-dioxoisoindolines or indole moieties.
Compound
LEDGF/p75-IN μM IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen
a
IC
50
3’ Processing Integration
118.

23 ± 5 --

--

119.

>50 --

--

120.

17 ± 3 --

--

121.

>50 --

--

122.

20 ± 4 --

--

123.

>50 --

--

86
Table 11 continued.

124.

28 ± 4
--

--

125.

19 ± 12 -- --
126.

>50 -- --
127.

43 ± 12 -- --
128.

12 ± 5

--

--

129.

>50 -- --

87
Table 11 continued.
130.

22 ± 8 47 ± 5 7 ± 3
131.

45 ± 6 --

--

132.

>50 --

--

a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.
LEDGF/p75-IN inhibitors 124 and 125 were identified through substructure
searches of the 1,3-dioxoisoindoline fragment. Similar substructure training sets were
built for indole fragments observed in the LEDGF/p75 pharmacophore hit compound 3.
Compounds 126 - 132 had two indole units attached to a third aromatic ring with a
tertiary carbon in a three-prong structure. The third aromatic ring of compound 126 was
a pyridine and it did not inhibit LEDGF/p75-IN interaction at 50 μM. Compound 127
had a dimethylaniline and showed some activity, while compound 128 replaced the
tertiary amine with a nitro group and improved inhibitory activity more than three-fold
with an IC
50
of 12 μM. The compound 130 dihydroxybenzene moiety replaced by
methoxy groups reduced activity in 131 and abolished activity in 132. Although 130 was
missing a 2-methyl group on both indole units and exact comparisons cannot be

88
confirmed, the results do suggest functional groups with better HBA ability will have
better LEDGF/p75-IN inhibitory profiles.
B. 2,3-dihydroxybenzamides
2,3-dihydroxybenzamide containing LEDGF/p75-IN inhibitors were synthesized
by Dr. Long's group to study their effects on IN catalytic function.
34
They were also
screened against LEDGF/p75-IN complex formation to study their binding affinity and
ability to disrupt IN and LEDGF/p75 interaction. Compound 133 did not significantly
improve inhibition compared to compound 134 indole, while shortening the distance
between the carboxamide and indole abolished activity at 50 μM. Altering the
naphthalene (133) or indole (134) with a furan (136 and 137) did not exhibit inhibitory
activity, while replacing the furan ring in 137 with a cyclohexane (138) showed greater
than a six-fold improvement in activity with an IC
50
value of 8 μM. Interestingly,
replacing the hydroxyl group in the meta position of the benzene with a methoxyl group
in compound 140 also showed inhibition with an IC
50
of 13 μM (Table 12).
Table 12. LEDGF/p75-IN inhibitors containing 2,3-dihydroxybenzamide groups.
LEDGF/p75-IN μM IN catalysis IC
50
(μM) MnCl
2

Compound Alpha-Screen
a
IC
50
3’ Processing Integration
133.

34 ± 9 >100 26 ± 4
134.

45 ± 9 >100 24 ± 2

89
Table 12 continued.
135.

>50 >100 58 ± 12
136.

>50 90 ± 14 15 ± 5
137.

>50 >100 13
138.

8 ± 1 53 ± 4 19 ± 3
139.

>50 >100 39 ± 8
140.

13 ± 4 >100 >100
141.

50 >100 >100
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

90
6.2.2 LEDGF/p75-IN Inhibitors Identified from Random Screening
A. Indene-1,3-diones
Compounds 142 and 143 were identified from random screening of an internal
commercial database of 10,000 compounds. Available analogues were searched,
screened, and added to Table 13 to reveal SAR tendencies. LEDGF/p75-IN inhibitor 142
with a 4-cyclopentane had an IC
50
value of 8 μM, at least a six-fold improvement from a
morpholine moiety (144) for example. Compound 142 did not show antiviral activity.
Table 13. LEDGF/p75-IN inhibitors with an indene-1,3-dione.
Compounds
LEDGF/p75-IN
IC
50
(μM)
3’ Proc.
IC
50
(μM)
Integration
IC
50
(μM)
142.

8 ± 1 -- --
143.

23 ± 10 -- --
144.

>50 -- --
145.

50 -- --

91
Table 13 continued.
146.

>50 -- --
a
Compounds tested at a maximum concentration of 50 µM to reduce the DMSO content.
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

B. Pyrrole Propanoic acids
Compounds 147 - 153 had a common central pyrrole propanoic acid and were
also identified from random screening of an internal commercial database of 10,000
compounds (Enamine). Altering functional groups of the aromatic ring attached to the
pyrrole tertiary amine did show much variation in activity from the analogues available.
Compounds 151 - 153 exhibited a better inhibitory profile with a thiofuran replacing one
benzene ring (Table 14). More analogues are needed to complete SAR investigations of
pyrrole propanoic acids.
Table 14. LEDGF/p75-IN inhibitors containing pyrrole propanoic acids.
Compound
LEDGF/p75-IN μM IN catalysis IC
50
(μM) MnCl
2

Alpha-Screen IC
50
3’ Processing Integration
147.

38 ± 9 -- --
148.

38 ± 6 -- --

92
Table 14 continued.
149.

35 ± 4 -- --
150.

33 ± 5 -- --
151.

11 ± 4 -- --
152.

14 ± 4 -- --
153.

22 ± 4 -- --
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

93
C. Orphan LEDGF/p75-IN Inhibitors with Unavailable Analogues
LEDGF/p75-IN inhibitors 154 - 167 presented in Table 15 were identified
through random screening of an internal laboratory database of 10,000 compounds
(Enamine). SAR analysis could not be conducted because analogues of these compounds
are not available. Compound 160 was the most active with an IC
50
value of 1.3 μM and
had a moderate antiviral activity profile with an EC
50
value of 35 μM, more than a 12-
fold improvement from 161 that replaced the benzene attached to the 1,3-diothiole with
another carboxylate. Also, compound 161 was missing a 7-methoxy group and had a
trifluoroacetyl group attached to the quinolin tertiary amine which could also explain the
diminished inhibitory activity. Compound 161 did not show antiviral activity.

94
Table 15. Uninvestigated potential lead LEDGF/p75-IN inhibitors.
Compounds
LEDGF-IN
IC
50
(μM)

Compounds
LEDGF-IN
IC
50
(μM)
154

17 ± 9
155

7 ± 1
156

20 ± 8
157

33 ± 9
158

<25
159

10 ± 1
160

1.3 ± 0.5
161

16 ± 3
162

28 ± 9
163

4 ± 1
164

19 ± 4
165

10 ± 2
166

21 ± 11
167

21 ± 10
IC
50
was determined using wild type IN and LEDGF/p75 in the presence of Mg
2+
.
IC
50
for IN catalysis was determined using wild type IN in the presence of Mn
2+
.

95
6.2.3 Implications for Drug Design
Many LEDGF/p75-IN inhibitors presented in this chapter have a similar bent or a
three-prong structure to inhibitors described in previous chapters. More analogues and
optimization studies are necessary to understand the SAR of each compound and their
potential to be lead candidates of new classes of LEDGF/p75-IN inhibitors. The dithiole
moiety in compound 160 presents a new central 5-member ring scaffold to develop more
potent LEDGF/p75-IN inhibitors that can also improve on the moderate antiviral activity.
Conclusion
The essential role of cellular LEDGF/p75 in HIV infection makes it a rational
target for therapeutic intervention. Discovery of LEDGF/p75-IN inhibitors described in
this study provides an unexplored therapeutic tool to inhibit HIV replication and
overcome the emergence of multi-drug resistant strains. LEDGF/p75 KID tri-peptide
residues key to viral replication served as a valid pharmacophore template and starting
point to identify novel potent LEDGF/p75-IN inhibitors. The identification of
LEDGF/p75-IN inhibitors that also show antiviral activity suggests a starting scaffold to
further develop second generation inhibitors. All compounds presented in chapters 2 - 5
satisfy ADMET parameters and are presented as lead compounds for the inhibition of this
essential viral-cellular cofactor interaction.

96
All LEDGF/p75-IN inhibitors that had an IC
50
value below 25 µM were screened
against HIV-1. Seven compounds were identified as antiviral. Antiviral LEDGF/p75-IN
inhibitors 49, 70, and 160 represent the best templates to improve the current batch of
lead inhibitors. Hybrid compounds like LEDGF/p75-IN inhibitors 73 and 74 also show
promise for further development of lead antiviral inhibitors. Several approaches were
employed to optimize and improve activity of the lead LEDGF/p75-IN inhibitors
presented in this study, including altering linkers, exploiting metabolically active groups
to identify prodrugs, and masking metabolically active moieties within cyclic rings.
Future
Pharmacophore model development mimicking the LEDGF/p75 IBD KID
residues led to the discovery of more than 100 active LEDGF/p75-IN inhibitors with IC
50

values below 50 µM. Analogues and optimization studies are necessary to explore the
properties of identified antiviral inhibitors and understand the SAR of the new
LEDGF/p75-IN inhibitors identified in chapter 6. Chemo-informatics, clustering, and
importing small molecules identified from this study into the HypoGen and HipHop
functions of Catalyst will lead to new activity-based pharmacophore models and identify
more novel lead candidates to test against HIV infection. Although random screening
generated the new list of potential leads presented in chapter 6 including the antiviral
compound 160, many of the inhibitors reported in chapters 2 - 5 were pooled from
various sources and would not have been identified without the aid of a rational tool like
the KID receptor-based pharmacophore models.

97
New receptor-based pharmacophore models can also be developed using another
localized location within the dimer-dimer pocket mimicking IBD 2nd chain hydrophobic
residues F406 and V408. Also, a ligand-based pharmacophore model can be generated
from the crystallography coordinates of the 976 LEDGF/p75-IN inhibitor ligand. In all,
there are still at least three methods to build new pharmacophore models, receptor-based
models mimicking IBD hydrophobic residues and ligand-based models built from
LEDGF/p75-IN inhibitors. It is easy to visualize in the near future the use of small
molecules that inhibit the interaction between cellular LEDGF/p75 and HIV IN as being
pivotal in combination cocktails for the initial treatment of HIV infection, treatment-
experienced patients, and new HIV infections that consist of multi-drug resistant strains.
Mutant viral progeny that prevent the interaction between IN and LEDGF/p75 to evade
inhibitor binding are also likely to have low viral fitness and an inability to successfully
deliver the IN-led PIC to the site of integration, thus impeding viral replication.

98
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Abstract (if available)

Abstract Cellular transcription co-activator p75, also known as lens epithelial-derived growth factor (LEDGF/p75), plays an essential role in HIV-1 IN-led integration of viral DNA into the human genome. Designing compounds to disrupt LEDGF/p75-IN complexes serves as a novel mechanistic approach different from current antiviral therapies. The evolution of HIV has afforded the viral species to adapt to external therapeutic pressure and raltegravir-resistant viral strains have been well documented, increasing the need for new IN inhibitors with a different mechanism of action. Mechanistic studies show the majority of reported IN inhibitors effectively chelate with a magnesium ion in the catalytic active site, a region topologically different from the LEDGF/p75 binding site. Thus, key LEDGF/p75 K364 I365 D366 amino acid residues from the IN binding domain laid a foundation for ligand-based pharmacophore models. Herein we report funding from the National Institute of Allergies and Infectious Diseases and the California HIV/AIDS Research Program led to the discovery of more than 100 novel small molecules that successfully inhibited LEDGF/p75-IN complex formation with IC50 values below 50 µM. A small molecule database of 365,000 was searched and compounds that had fitness values above 2.0 were selected for prescreening in an in vitro luminescent proximity assay. Lead inhibitors expanded into training sets using substructure similarity tools and ADMET filters led to the discovery of four novel classes of LEDGF/p75-IN inhibitors and additional potential lead inhibitors. Seven antiviral LEDGF/p75-IN inhibitors were identified from this study.

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Asset Metadata

Creator Sanchez, Tino Wilson (author)

Core Title Design and discovery of small molecules inhibiting the interaction of cellular LEDGF/p75 and HIV-1 integrase

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 04/17/2012

Defense Date 12/08/2011

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

Tag AIDS,antiviral,chaperone,drug design,HIV,host factor,integrase,LEDGF,OAI-PMH Harvest,Pharmaceutical Sciences,pharmacophore,small molecules,therapeutics,virus

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Haworth, Ian S. (committee chair), Neamati, Nouri (committee chair), Jung, Kyung Woon (committee member), Louie, Stan G. (committee member)

Creator Email nightsuave@yahoo.com,twsanche@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-8559

Unique identifier UC1111524

Identifier usctheses-c3-8559 (legacy record id)

Legacy Identifier etd-SanchezTin-617.pdf

Dmrecord 8559

Document Type Dissertation

Rights Sanchez, Tino Wilson

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA