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Biochemical development and analysis of NAD⁺-related biomolecules

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Biochemical development and analysis of NAD⁺-related biomolecules

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Content Copyright 2020 Yiling Wang
Biochemical Development and Analysis of NAD
+
-Related Biomolecules
by
Yiling Wang

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
PHARMACEUTICAL SCIENCE

May 2020

ii

Acknowledgements
I would like to express my gratitude to my principal investigator, Yong (Tiger) Zhang, who
guided me throughout this project. Also, I would like to thank Xiao-nan Zhang, Qinqin Cheng,
Zhefu Dai, Albert Lam, Jiawei Li, Xiaojing Shi, Xinping Duan and other laboratory colleagues
for their help to my research. At last, I am honored to have Dr. Yong Zhang, Dr. Ian Haworth
and Dr. Roger F Duncan to be my thesis committee members.

iii

Table of Contents
Acknowledgements .................................................................................................................... ii
List of Figures ............................................................................................................................ v
List of Schemes ......................................................................................................................... vi
Abstract .................................................................................................................................... vii
1. Introduction ....................................................................................................................... 1
1.1 Nicotinamide adenine dinucleotide: ................................................................................. 1
1.2 Poly (ADP-ribose) polymerase 1: ..................................................................................... 1
1.3 CD38: ................................................................................................................................ 2
1.4 SG3199: ............................................................................................................................ 3
1.5 Biosynthesis of NAD
+
: ..................................................................................................... 3
1.6 NRK1: ............................................................................................................................... 6

2. Experimental Methods ...................................................................................................... 7
2.1 Human PARP1 expression and purification: .................................................................... 7
2.1.1 Human PARP1 expression: ......................................................................................... 7
2.1.2 Purification of human PARP1 ..................................................................................... 8
2.2 Synthesis of SG3199 ....................................................................................................... 10
2.2.1 Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-nitrobenzoic acid)
(Scheme 1) ......................................................................................................................... 10
2.2.2 Synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy) methyl) pyrrolidin-3-ol ..... 13
(Scheme 2) ......................................................................................................................... 13
2.3 Cloning and expression of NRK1-EGFP fusion ............................................................. 16
2.3.1 Molecular cloning of NRK1-EGFP fusion ............................................................... 16
2.3.2 Expression and purification of NRK1-EGFP fusion ................................................. 16
2.3.3 Measurement of the enzymatic activity of the fusion: .............................................. 17
2.3.4 Transfection of the fusion into mammalian cells: ..................................................... 18

3. Results and discussion .................................................................................................... 20
3.1 PARP1 expression and purification: ............................................................................... 20
3.2 SG3199 synthesis: ........................................................................................................ 23
3.2.1 Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-nitrobenzoic acid).. 23
3.2.2 Synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy) methyl) pyrrolidin-3-ol ..... 23
3.2.3 Attempt of condensation of bis acid and the pyrrolidine .......................................... 24
3.3 NRK1EGFP fusion: ..................................................................................................... 33
3.3.1 Molecular cloning of NRK1-EGFP fusion. .............................................................. 33
3.3.2 Expression and purification of NRK1-EGFP fusion ................................................. 35
3.3.3 Measurement of the activity of the fusion: ............................................................... 35
3.3.4 Transfection of the fusion into mammalian cells: ..................................................... 38
iv

4. Conclusion ...................................................................................................................... 40
References. ............................................................................................................................... 41

v

List of Figures
Figure 1. The interconversion of the reduced form and oxidized form of NAD
+
……………..5
Figure 2. Chemical structure of the starting molecules of the salvage pathway...….................5
Figure 3. The de novo synthesis pathway of NAD
+
…………………………………….……..5
Figure 4. The UV spectrum over time of size exclusion chromatographic
purification………………………………………………………………………...21
Figure 5. SDS-PAGE gel image of the fractions 8 to 13 and the combination from
8 to 13 collected from size exclusion column purification…….…………………..22
Figure 6. NMR spectrum of compound 1..…………………………………………………..25
Figure 7. NMR spectrum of compound 2……………………..………………………..........26
Figure 8. NMR spectrum of compound 3…………………..………………………………..27
Figure 9. NMR spectrum of compound 4……………..…………………………………......28
Figure 10. NMR spectrum of compound 6………………………..…………...…………….29
Figure 11. NMR spectrum of compound 7…………………………...……………………....30
Figure 12. NMR spectrum of compound 8………………………………...……..………….31
Figure 13. NMR spectrum of compound 9………………………………………..…...…….32
Figure 14. DNA agarose gel of overlap PCR product with one desired band and
other unexpected bands…….………………………………………...…………..34
Figure 15. HPLC analysis for activity assay of C-terminal NRK-EGFP fusion,
N-terminal NRK-EGFP fusion and wild type NRK1 on NR compound………....36
Figure 16. HPLC analysis for activity assay of C-terminal NRK-EGFP fusion,
N-terminal NRK-EGFP fusion and wild type NRK1 on NR4 compound..…..…..37
Figure 17. Western blot of transfected NRK1-EGFP fusion protein assay…………..………39

vi

List of Schemes
Scheme 1. Reaction scheme of the synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis
(5-methoxy-2-nitrobenzoic acid)……………………………………………………………..12
Scheme 2. Reaction scheme of the synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl)
oxy) methyl) pyrrolidin-3-ol………………...……………………………….………………15

vii

Abstract
Nicotinamide adenine dinucleotide (NAD
+
) is an essential coenzyme in human body and
can be utilized by various enzymes as substrates. Ribose-functionalize NAD
+
analogues
provide opportunities to generate functionalized poly-ADP-ribose catalyzed by poly-ADP-
ribose polymerase 1 (PARP1), which may enable development of novel therapeutic delivery
systems. In addition, the ribose-functionalized NAD
+
may allow tracking and evaluation of
NAD
+
-associated pathways in cells. In the first part of this research project, human PARP1 was
expressed and purified from Escherichia coli in large quantities through three steps of
chromatography. In the second part of this project, synthesis of a pyrrolobenzodiazepine (PBD)
dimer was attempted for generating new payload candidates for drug conjugation and
subsequent targeted delivery. Finally, human nicotinamide ribose kinase 1 (NRK1) was fused
with EGFP and transfected into mammalian cells in order to elevate the rate of transformation
of cellular nicotinamide riboside into NAD
+
, which may facilitate in situ generation of
functionalized NAD
+
for cellular studies. These biochemical studies provide useful reagents
for development of functionalized poly-ADP-ribose and its conjugates as well as for
investigation of NAD
+
-dependent cellular processes.

1

1. Introduction
1.1 Nicotinamide adenine dinucleotide:
NAD
+
is an essential molecule widespread in human body. Some of the roles it plays include
redox coenzyme (Pollak et al., 2007),

substrate of ADP-ribosyl transferases and substrate of
ADP-ribosyl cyclase (Belenky et al., 2007). NAD
+
consists of two nucleotides, adenosine
mononucleotide (AMP) and nicotinamide mononucleotide (NMN), joined through a
pyrophosphate group.

NAD
+
exists in two forms: an oxidized form and a reduced form,
abbreviated as NAD
+
and NADH.

NAD
+
can accept electrons from other molecules and
NADH can be used as a reducing agent to donate electrons.
1.2 Poly (ADP-ribose) polymerase 1:
Poly (ADP-ribose) polymerases (PARPs) are a family of proteins involved mainly in DNA
repair. The PARP family has 17 different enzymes and PARP1 is the most common PARP
protein. PARP can detect single-strand DNA breaks (SSB), bind to the DNA and utilize
NAD
+
to form poly-ADP-ribose (PAR) chains (Herceg and Wang, 2001). The formation of
PAR acts as a signal for the other DNA repairing enzymes. PARP1 is composed of four
domains including two zinc fingers involved in DNA binding. There is a third zinc finger
domain essential for activation and a BRCT domain for auto modification (Ryu et al., 2015).
The rest two are the WGR domain for DNA-dependent activity and the catalytic domain,
which binds with NAD
+
and synthesize PAR. Previous study has shown that PARP1 is often
overly expressed in cancer cells (Bürkle et al., 2005). Thus, normally PARP1 acts as the
2

target for anti-cancer therapeutics. Our previous study has identified a 3′-azido NAD
+
which
has a similar activity as normal NAD
+
and a high selectivity for PARP1 protein (Zhang et al.,
2019).

This NAD
+
is named as NAD
+
4, and the corresponding 3′-azido nicotinamide riboside
(NR) is named as NR4. This azido- modified NAD
+
can react with alkyne group through
click chemistry (Jiang et al., 2010) and has the potential of linking with various function
groups (Carter‐O'Connell and Cohen, 2015). For generating NAD
+
4-derived PAR chains,
automodified PARP1 is required. The first part of this research project was focused on the
large-scale expression and purification of human PARP1 via a 3-step chromatographic
separation.

1.3 CD38:
Cluster of differentiation 38 (CD38) is a glycoprotein (Malavasi et al., 1994) expressed on the
surface of many types of immune cells, including CD4
+
and CD8
+
T lymphocytes and B
lymphocytes (Orciani et al., 2008).

CD38 mainly functions as an ADP-ribose cyclase,
transforming NAD
+
into cyclic ADP-ribose (cADPR) (Chini et al., 2002); (Aksoy et al.,
2006). Cyclic ADP-ribose is a second messenger of Ca
2+
signaling and stimulates Ca
2+

release (Malavasi et al., 2008); (Sun et al., 2002). CD38 can also hydrolyze cADPR into
ADP-ribose and catalyze the exchange of the nicotinamide group of nicotinamide adenine
dinucleotide phosphate ( NADP) with nicotinic acid to produce nicotinic acid adenine
dinucleotide phosphate (NAADP ) (Malavasi et al., 2008).

In previous study, the mechanism
of the formation of cADPR has been found (Graeff et al., 2009). The NAD
+
molecule first
undergoes a nicotinamide-ribosyl bond cleavage and forms a high energy intermediate with
3

amino residue (Cho et al., 1998) (Zhang et al., 2011). Then the intermediate is either
transformed into cADPR or ADP-ribose (Sauve et al., 1998) (Graeff et al., 2001) (Jackson et
al., 2003).
1.4 SG3199:
SG3199 is one member of pyrrolobenzodiazepine (PBD) dimers with cytotoxicity to tumor
cells (Hartley et al., 2018). It can bind selectively with 5′-purine-guanine-adenine-thymine-
cytosine-pyrimidine-3′ sequence and form interstrand DNA cross-link. Given its high
potency, SG3199 has been emerging as an attractive payload for generating antibody-drug
conjugates. In the second part of the research project, we have aimed to synthesize SG3199
from commercially available starting materials.
1.5 Biosynthesis of NAD
+
:
NAD
+
is synthesized in two pathways: a de novo pathway from amino acids and a salvage
pathway from the precursor including NR, nicotinamide and nicotinic acid (Magni et al.,
2006). The de novo pathway first oxidizes L-tryptophan into N-formyl-L-kynurenine, then
undergoes a hydrolysis reaction to transform N-formyl-L-kynurenine into L-kynurenine. L-
kynurenine is transformed into 3-hydroxy-L-kynurenine. Afterwards, 3-hydroxyl-L-
kynurenine’s alanine residue leaves and forms 3-hydroxyanthranilate. It is then oxidized into
2-amino-3carboxymuconate-6-semialdehyde and further dehydrated into quinolinate.
Quinolinate is decarboxylated and phosphoribosylated into β-nicotinate D-ribonucleotide by
quinolinate phosphoribosyl transferase. Then β-nicotinate D-ribonucleotide reacts with ATP
and forms nicotinate adenine dinucleotide through nicotinamide/nicotinic acid
4

mononucleotide adenylyltranferase (NMNAT), and it is finally synthesized into NAD
+

through amine transfer from L-glutamine. (Figure 3) The salvage pathway can transform
nicotinic acid (Na), nicotinamide (Nam) and NR into NAD
+
. Na and Nam can be transformed
into nicotinate and get recycled into NAD
+
. NR is phosphorylated into NMN by nicotinamide
ribose kinase (NRK) (Ratajczak et al., 2016) and then transformed into NAD
+
by NMNAT.

5

Figure 1. The interconversion of the reduced and oxidized form of NAD
+
.

Figure 2. Chemical structure of the starting molecules of the salvage pathway.

Figure 3. The de novo synthesis pathway for NAD
+
. Trp stands for L-trptophan, QA stands
for quinolinate, NaMN stands for nicotinic acid mononucleotide and NaAD stands for
nicotinic acid adenyl dinucleotide

6

1.6 NRK1:
NRK1 is an enzyme that can transform NR into NMN, which is a key process of the salvage
synthesis pathway of NAD
+
. Because of the high polarity of NAD
+
, it is impossible to
directly deliver NAD
+
across cell membrane. Thus, the salvage pathway is utilized for the
delivery of modified NAD
+
into mammalian cells. Previous study shows that the
phosphorylation step that transform NR into NMN is the rate-limiting step of salvage
pathway of NAD
+
synthesis (Fan et al., 2018). To improve the rate of transformation, we
planned to elevate the expression level of NRK1 through overexpressing NRK1 enzyme in
mammalian cells.

7

2. Experimental Methods
2.1 Human PARP1 expression and purification:
2.1.1 Human PARP1 expression:
Gene encoding human PARP1 was already cloned into pET28 expression vectors that
generate proteins with 6xHis tags which makes affinity chromatography purification
available (Langelier et al., 2011).
On the first day, a small amount of PARP1 glycerol stock was inoculated into 60 mL
autoclaved LB broth along with antibiotic kanamycin in a concentration of 50μg/mL. The
medium was put into an incubator at 37°C and shaken overnight.
On the second day, the medium was added into 1 liter of autoclaved LB broth in a ratio of
10mL/L along with kanamycin in a concentration of 50μg/mL. The 1 L medium was put into
an incubator at 37°C and shaken for 2-3 hours until the optical density at 600 nm (OD 600)
reached between 0.6 and 0.8 measured through Nanodrop. Afterwards, 100 mM ZnSO 4 was
added to the medium to a final concentration of 0.1 mM. Then the medium was put back into
the incubator and shaken for another 10 to 15 minutes until the OD 600 of the medium reaches
between 0.8-1.0. After that, the medium was removed from the incubator and allowed to be
chilled at 4°C for 1 hour. Finally, a 500 mM isopropyl 1-thiogalactopyranoside (IPTG)
aqueous solution as inducer was added into the medium to a final concentration of 0.5 mM
and put back into the incubator and shaken at 16°C overnight.

8

2.1.2 Purification of human PARP1
All the buffer solution was filtered before using to prevent clogging on the columns over
time.
2.1.2.1 Extraction of the protein from cells
The medium was centrifuged down at 10000×g and 4°C for 30 minutes. The supernatant
liquid was discarded, and the pellets were resuspended with equilibration buffer (25 mM
Hepes pH 8.0, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF) as protease
inhibitor (added before use) and 1 mM DTT as disulfide bond reducer (added before use)).
The resuspended pellets were then lysed with a cell disruptor. The lysed cells were
centrifuged at 14000×g for more than 50 minutes and the supernatant was collected. Later,
the collected cell lysate was filtered to remove remaining cell debris.
2.1.2.2 HiTrap chelating column purification
The filtered cell lysate was manually loaded onto the HiTrap chelating column and washed
with low salt wash buffer (25 mM Hepes pH 8.0, 500 mM NaCl, 20 mM imidazole, 1 mM
PMSF and 1 mM DTT (added before use)), followed with wash of high salt buffer (25 mM
Hepes pH 8.0, 1 M NaCl, 20 mM imidazole, 1 mM PMSF and 1 mM DTT (added before
use)) and another wash of low salt buffer. Afterwards, the column was eluted with elution
buffer (25 mM Hepes pH 8.0, 500 mM NaCl, 400 mM imidazole, 1 mM PMSF and 1 mM
DTT (added before use)) and the elute was collected.
2.1.2.3 Heparin column purification
The elute was diluted with a no salt buffer (50 mM Tris pH 7.0, 500 mM NaCl, 1 mM EDTA,
1 mM PMSF and 1 mM DTT (added before use) with equivalent volume. Then the diluted
9

solution was loaded onto a Heparin column. After loading, the column was loaded to a FPLC
system and washed with heparin column wash buffer (50 mM Tris pH 7.0, 250 mM NaCl, 1
mM EDTA, 1 mM PMSF and 1mM DTT (added before use)) and then eluted with a mixture
of heparin column wash buffer and heparin column elution buffer (50 mM Tris pH 7.0, 1 M
NaCl, 1 mM EDTA, 1 mM PMSF and 1 mM DTT (added before use)) through modifying the
concentration gradient of heparin column elution buffer from 20% to 80%. The elute was
collected and concentrated with 30kD cut-off centrifuge tube through centrifuge at 4000×g to
500 μL.
2.1.2.4 Size exclusion column separation
The elute from heparin column was injected into the FPLC system loaded with size exclusion
column and eluted by gel filtration buffer (25 mM Hepes pH 8.0, 150 mM NaCl, 1 mM
EDTA, 1 mM PMSF and 1 mM DTT (added before use)).
2.1.2.5 Characterization of the sizes of eluted proteins
The eluted fractions were separated, added with DTT to a final concentration of 1 mM, and
then boiled for 10 minutes. Then the boiled mixture was analyzed on SDS-PAGE to
determine the size of the protein contained in the fractions. Fractions with protein of correct
size were collected and concentrated with 30 kD cut-off centrifuge tube through centrifuge at
4000×g to 500 μL to 1 mL. The concentrated protein was measured for concentration by A280
and flash frozen in a liquid nitrogen bath for storage.

10

2.2 Synthesis of SG3199
2.2.1 Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-nitrobenzoic acid)
(Scheme 1)
Step 1. Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (3-methoxybenzoic acid)
(compound 1)
Vanillic acid (10.5g, 62.4mmol) was added into a 250 mL two-neck round bottom flask
connected with a reflux condenser. Then NaOH (6.25 g, 156.25 mmol) and H2O (250 ml) was
added into the flask. The mixture was heated to reflux for 1 hour and formed a homogeneous
solution. Then 1,5-dibromopentane (7.182 g, 31.22 mmol) was added into the mixture and
reflux for overnight reaction (Kuhire et al., 2017). The reaction mixture was then cooled
down and washed with ethyl acetate to remove excess dibromo pentane. The crude was
acidified by 3M HCl and forms precipitate. The precipitate was collected through filtration
and recrystallized from ethanol. The yield was 15.6 g, 62%.
Step 2. Synthesis of methyl 4-((5-(4-acetoxy-2-methoxyphenoxy) pentyl) oxy)-3-
methoxybenzoate (compound 2)
The bis acid compound 1 (15.6 g, 38.6 mmol) was dissolved in methanol (70 mL) and added
with concentrated sulfuric acid (3 mL). Then the reaction mixture was heated to reflux and
stirred for overnight reaction. The reaction mixture is extracted with 3ⅹ50 mL of ethyl acetate
and washed with 2ⅹ50 mL of saturated sodium bicarbonate solution, 3ⅹ50mL of water and 50
mL of brine consequently. Compound 2 was obtained through evaporation of solvent from
11

the organic layer. The yield was 6.3 g, 37.7%.
Step 3. Synthesis of dimethyl 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-
nitrobenzoate) (compound 3)
The benzoate 2 (6.3 g, 14.5 mmol) was added into a round bottom flask and charged with
argon. Then, acetate anhydride (60 mL) was added. Copper nitrate (9g, 37.5mmol) was added
slowly into the flask in a time period of 1 hour (Gregson et al., 2004). Then the reaction was
stirred for overnight reaction. After TLC check for completed reactions, the reaction mixture
was extracted with 3ⅹ50 mL of ethyl acetate and washed with 3ⅹ50 mL of water and 50 mL
of brine consequently. The organic layer was dried with sodium sulfate and evaporated to
remove solvent. The yield was 2.4 g, 31.7%.
Step 4. Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-nitrobenzoic acid)
(compound 4)
Compound 3 (2.4g, 4.6mmol) was dissolved in tetrahydrofuran (50 mL) and added with
sodium hydroxide (2.3 g, 45 mmol) aqueous solution. The reaction mixture was stirred for 5-
hour reaction and acidified with aqueous hydrochloric acid to pH 7.0 (Vlahov et al., 2020).
Then the crude was extracted with 3ⅹ50 mL of ethyl acetate and washed with 3ⅹ50 mL of
water and 50 mL of brine consequently. Compound 4 was obtained through evaporation of
solvent from the organic layer.

12

Scheme 1. Reaction scheme of the synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-
methoxy-2-nitrobenzoic acid).

13

2.2.2 Synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy) methyl) pyrrolidin-3-ol
(Scheme 2)
Step 1. Synthesis of 1-benzyl 2-methyl (2R,4S)-4-hydroxypyrrolidine-1,2-dicarboxylate
(compound 6)
Commercially available compound 5 (4.2 g, 29 mmol) was added into tetrahydrofuran (100
mL) along with sodium bicarbonate solid (17 g, 202 mmol). Then, aqueous solution of
sodium bicarbonate (5 mL) was added followed by addition of benzyl chloroformate (7.4 g,
43.36 mmol). The reaction mixture is stirred for overnight reaction (Gichuhi et al., 2014). The
crude was extracted with 3ⅹ50 mL of ethyl acetate and washed with 3ⅹ50 mL of water and 50
mL of brine consequently. After evaporation to remove solvent, the mixture was purified
through column chromatography. The yield was 4.1 g, 50.6%.
Step 2. Synthesis of benzyl (2R,4S)-4-hydroxy-2-(hydroxymethyl) pyrrolidine-1-carboxylate
(compound 7)
Compound 6 (4.1 g, 14.6 mmol) was dissolved in tetrahydrofuran (30 mL) and added with
methanol (5 mL). Then sodium borohydride (0.558 g, 14.68 mmol) was added slowly into the
mixture. The reaction mixture was stirred for 3 hours and then added with hydrochloric acid
to remove excess sodium borohydride salt (Gregson et al., 2004). The mixture was extracted
with 3ⅹ50 mL of ethyl acetate and washed with 3ⅹ50 mL of water and 50 mL of brine
consequently. Compound 7 was obtained after evaporation. The yield was 3.2g, 87.2%.
Step 3. Synthesis of tert-butyl-diphenyl siloxane protected pyrrolidine compound 8
Compound 7 (3.2 g, 12.7 mmol) was dissolved in pyridine (50ml) and added with TBDPSCl
14

(4.92 g, 18 mmol) under protection of argon. The reaction was stirred for overnight and
quenched by addition of water. The crude was extracted with 3ⅹ50 mL of ethyl acetate and
washed with 3ⅹ50 mL of water and 50 mL of brine consequently. After evaporation to
remove solvent, the mixture was purified through column chromatography to obtain
compound 8. The yield was 4.2 g, 67.5%.
Step 4. Synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy) methyl) pyrrolidin-3-ol
(compound 9)
Compound 8 (4.2 g, 8.5 mmol) was placed in a sealing bottle along with palladium and 10%
C (0.8g). After removal of air and charged with argon for protection, triethyl silane (40 mL)
was added as source of hydrogen along with same volume of methanol (40 mL). The bottle
was then sealed and stirred for 5 hours. The reaction mixture was then filtered to remove
carbon and palladium and extracted with 3ⅹ50 mL of ethyl acetate and washed with 3ⅹ50 mL
of water and 50 mL of brine consequently. The final compound 9 was purified through
chromatography. The yield was 1.3g, 31%.

15

Scheme 2. Reaction scheme of the synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy)
methyl) pyrrolidin-3-ol.

16

2.3 Cloning and expression of NRK1-EGFP fusion
2.3.1 Molecular cloning of NRK1-EGFP fusion
PCR reactions for the NRK1 and EGFP fragments were performed. Then the fusion gene was
obtained from overlap PCR with the two fragments. Both EGFP-NRK1 and NRK1-EGFP
genes were obtained for activity comparison.
Both fused genes were digested along with pET28a vector by XhoI and NcoI restriction
enzymes. Then the digested fused genes were ligated with the vector and transformed into
DH10B competent cells. Afterwards, the transformed cells were inoculated on LB plates with
kanamycin and incubated for overnight at 37°C. On the second day, colonies were selected
for miniprep and sequencing and the colonies with the correct sequences were used for
preparing glycerol stock.
2.3.2 Expression and purification of NRK1-EGFP fusion
On the first day, 2 μL of NRK1-EGFP fusion clone stock was inoculated into a small amount
autoclaved LB broth along with antibiotic kanamycin in a concentration of 50 μg/mL for
starter culture. The medium was put into an incubator at 37°C and shaken overnight.
On the second day, the medium was added into 1L autoclaved LB broth in a ratio of 10 mL/L
along with kanamycin in a concentration of 50 μg/mL. The 1 L medium was put into an
incubator at 37°C and shaken for 2-3 hours until the optical density at 600 nm (OD 600)
reached between 0.6 and 0.8 measured through Nanodrop. After that, the medium was
removed from the incubator and allowed to be chilled at 4°C for 1 hour. Finally, a 500 mM
17

IPTG aqueous solution as inducer was added into the medium to a final concentration of 0.1
mM and put back into the incubator and shaken at 18°C overnight.
On the next day, the medium was centrifuged down at 10000×g and 4°C for 30 minutes. The
supernatant liquid was discarded, and the pellets were resuspended with equilibration buffer
(20 mM Tris pH 7.0, 500 mM NaCl, 20 mM imidazole, 1 mM PMSF (added before use) and
1 mM DTT (added before use)). The resuspended pellets were then lysed with a cell
disruptor. The lysed cells were centrifuged at 14000×g for more than 50 minutes and the
supernatant was collected. Later, the collected cell lysate was filtered to remove remaining
cell debris.
The filtered cell lysate was manually loaded onto the HiTrap Chelating column and washed
with equilibration buffer, followed with wash of wash buffer (20 mM Tris pH 7.0, 500 mM
NaCl, 30 mM imidazole, 1 mM PMSF and 1 mM DTT (added before use)). Afterwards, the
column is eluted with elution buffer (20 mM Tris pH 7.0, 500 mM NaCl, 400 mM imidazole,
1 mM PMSF and 1 mM DTT (added before use)) and the elute was collected.
The elute was concentrated with 10 kD cut-off centrifuge tube to 500 μL. Afterwards, the
concentrated elute was diluted with 20 mL storage buffer (20 mM Tris pH 7.0, 150 mM
NaCl, 1 mM 2-mercaptolethanol, 10% glycerol) and concentrated to 500 μL for buffer
exchange. The buffer exchange is repeated twice, and the concentration of the final solution
is measured with A 280 through Nanodrop.
2.3.3 Measurement of the enzymatic activity of the fusion:
The NRK1-EGFP fusion was added into 50 μL mixture of 50 mM Tris, 100 mM NaCl, 2 mM
18

MgCl 2, 1 mM DTT, 5 mM ATP, 1 mM NR/4-azido NR and 5 μM NMNAT1. Then the
mixture was incubated for 2 hours at room temperature and then be quenched through adding
50 μL of 20% trichloroacetic acid. The mixture was then analyzed by HPLC for the
consumption of NR and formation of NAD
+
.
2.3.4 Transfection of the fusion into mammalian cells:
The fusion was amplified with PCR reactions and digested with NheI and NotI restriction
enzymes along with a pcDNA3.1 plasmid. Then the digested gene was ligated with the
digested vector and transformed into BL21 competent cells. The cells were then inoculated
on plates and incubated at 37°C for overnight. Afterwards, selected colonies were sent for
sequencing. The colonies with the correct sequences were then inoculated for maxiprep.
The plasmid obtained from maxiprep was then transfected into HEK-293T cells with
Transporter 5 using a ratio of 1:4, and the cells were collected 48 hours after transfection.
Before collected, the cells were incubated with NR4, H2O2 subsequently. The cells were
divided into 12 wells and 2 groups. Each group had three wells with transfected cells and
three with non-transfected cells. The wells with transfected cells were treated differently with
no incubation of NR4, incubation of NR4 or incubation of NR4+olaprib, a PARP1 inhibitor.
Same chemicals were added for the non-transfected cells. The cells were arranged as
described in Table 1 in the two plates.

19

Table 1. The arrangement of the cells for transfection.
2-hour incubation 6-hour incubation
Well 1 Transfected cells Transfected cells
Well 2 Non-transfected cells Non-transfected cells
Well 3 Transfected cells+NR4 Transfected cells+NR4
Well 4 Non-transfected
cells+NR4
Non-transfected
cells+NR4
Well 5 Transfected
cells+NR4+olarprib
Transfected
cells+NR4+olaprib
Well 6 Non-transfected cells
+NR4+olarprib
Non-transfected cells
+NR4+olarprib

After cell lysis, the lysates were centrifuged to remove precipitates, and the concentration
was measured through Bradford assays.
After clicking with alkyne biotin, the mixture was analyzed with by western blot to verify the
formation of PAR. Streptavidin-conjugated HRP was used for detection. Additional western
blots were performed to verify the results of transfection using an anti-His 6 tag goat antibody
as primary antibody and HRP-conjugated anti-goat antibody as secondary antibody.

20

3. Results and discussion
3.1 PARP1 expression and purification:
E. coli cells were used for expression of the protein. The cells were grown in 6L of LB
medium in each experiment. The average yield was 2.3 mg/6 L of LB medium. Factors that
may affect the yield include the extent of the cell lysis, the time of inoculation, the amount of
wash buffer used and the time of protein expression. Lysis is the most important part of the
purification. If the cells were not fully lysed, the cell lysate could be hard to filter and lead to
lower yield of final protein. Generally, not enough time of inoculation and protein expression
can significantly decrease the yield, and overextended time could also decrease the yield. If
high salt wash buffer was added too much, some of the PARP1 may be washed off.
Meanwhile, insufficient washing could lead to impurities that may not be removed in the
following purification.
SDS-PAGE (Figures 5) results demonstrated that the proteins were well separated by size
exclusion column.
The final amount of protein obtained from 126 L LB medium was 42 mg. The yield was
reasonable but can be improved through modifications of purification procedures including
increasing the cell lysis cycle number, adding lysozyme into the cell lysates, adjusting the
formulation of buffer solution and rinsing the centrifuge tubes.

21

Figure 4. The UV spectrum over time of size exclusion chromatographic purification.
Fraction 7 to fraction 12 were collected.
22

Figure 5. SDS-PAGE gel image of the fractions 8 to 13 and the combination from 8 to 13
collected from size exclusion column purification.

23

3.2 SG3199 synthesis:
3.2.1 Synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (5-methoxy-2-nitrobenzoic acid)
The synthesis of 4,4'-(pentane-1,5-diylbis(oxy)) bis (3-methoxybenzoic acid) was an easy
reaction. However, the purification was a large problem. Because each mole of 1,5-dibromo
pentane required 2 moles of vanillic acid to react, only vanillic acid can be in excess amount.
However, the reaction could not go to completion, resulting a reaction mixture with excessive
vanillic acid, 1,5-dibromo pentane and the desired bis acid. The bis acid and vanillic acid are
both soluble in basic pH aqueous solution and their solubility both drop dramatically when
acid is added. Thus, the bis acid obtained was a mixture. Also, due to the poor solubility of
the bis acid at neutral pH in either water or organic solvents, chromatography purification is
not available. Thus, we adjusted the previous plan of direct nitrification to an indirect
nitrification with transformation into methyl ester first. The esterification successfully
increased the polarity of the compound and made it soluble in organic solvent and available
for chromatography purification. The reagent used by the nitrification in literature was nitric
acid (Gregson et al., 2001). However, the quantity of starting material we used was very
large, required volume of nitric acid would be very large, so it is not practical. Thus, we
replaced nitric acid with copper nitrate. Copper nitrate turned out to be effective and the
quantity needed is not very high, although the nitrification also introduced some impurities.
3.2.2 Synthesis of (3S,5R)-5-(((tert-butyldiphenylsilyl) oxy) methyl) pyrrolidin-3-ol
The reaction for protection of Cbz group to the amine had a rather low yield. This might
24

result from the conflict between the need of preservation of the ester group in the starting
material and the requirement of basic condition.
The reaction condition for protection of TBDPS to the hydroxyl group was attempted several
times. Both DMAP and pyridine was tested as nucleophile for the reaction but only pyridine
worked for the equivalence-controlled reaction. Also, the length of time also affected the
quantity of TBDPS groups substituted. Extended reaction time would result in excessive
TBDPS protection on the secondary hydroxyl group although the excessive protection did not
affect the next reaction.
3.2.3 Attempt of condensation of bis acid and the pyrrolidine
The reagent tried for the condensation reaction included oxalyl chloride with DMF, HATU
and iodine with triphenylphosphine. However, none of these worked. The reason of the
failures may include existence of water during the transfer of reagents and the amounts used
for the attempt were too small
25

Figure 6. NMR spectrum of compound 1.
1
H NMR (200MHz, DMSO-d 6; δ, ppm): 7.52
(d,2H), 7.42 (s,2H), 7.03 (d,2H), 4.03 (t,4H), 3.76 (s,6H), 1.79 (m,4H), 1.55 (m,2H)
26

Figure 7. NMR spectrum of compound 2.
1
H NMR (200MHz, CDCl 3; δ, ppm): 7.64 (d,2H),
7.54 (s,2H), 6.91 (d,2H), 4.09 (t,4H), 3.90 (s,6H), 3.88 (s,6H), 1.75-2.01 (m,4H), 1.55-1.61
(m,2H)
27

Figure 8 NMR spectrum of compound 3.
1
H NMR (200MHz, CDCl 3; δ, ppm): 7.42 (d,2H),
7.05 (s,2H), 4.11 (t,4H), 3.94 (s,6H), 3.88 (s,6H), 1.90-2.01 (m,4H), 1.65-1.75 (m,2H)

28

Figure 9. NMR spectrum of compound 4.
1
H NMR (200MHz, CD3OD-d 4; δ, ppm): 7.81
(s,2H), 7.63 (s,2H), 7.03 (d,2H), 4.11 (t,4H), 3.92 (s,6H), 1.90 (m,4H), 1.71 (m,2H)
29

Figure 10. NMR spectrum of compound 6.
1
H NMR (200MHz, CDCl 3; δ, ppm): 7.28-7.39
(m,6H), 4.99-5.23 (m,2H), 4.46-4.57 (m,2H), 3.76 (s,3H), 3.55-3.74(m,1H), 3.53-3.60(s,2H),
2.24-2.37 (m,1H), 2.06-2.16 (m,1H)
N
O
OMe
HO
Cbz
6
30

Figure 11. NMR spectrum of compound 7.
1
H NMR (200MHz, CDCl 3; δ, ppm): 7.28-7.39
(m,6H), 4.99-5.23 (m,2H), 4.46-4.57 (m,2H), 3.76 (s,2H), 3.55-3.74(m,1H), 3.53-3.60(s,2H),
2.24-2.37 (m,1H), 2.06-2.16 (m,1H)
31

Figure 11. NMR spectrum of compound 8.
1
H NMR (200MHz, CDCl3; δ, ppm): 7.56-7.68
(m,6H), 7.18-7,46 (m,6H), 4.87-5.16 (m,2H), 4.0-4.2 (m,2H), 3.62-3.83 (m,1H), 3.53-
3.62(s,2H), 2.29-2.41 (m,1H), 2.11-1.99 (m,1H), 1.02 (s,9H)
32

Figure 12. NMR spectrum of compound 9.
1
H NMR (200MHz, CDCl3; δ, ppm): 7.56-7.68
(m,3H), 7.28-7,43 (m,3H), 3.99-4.18 (m,1H), 3.62-3.83 (m,2H), 3.58(s,2H), 2.04 (s,1H), 1.62
(m,2H), 1.02 (s,9H)

33

3.3 NRK1EGFP fusion:
3.3.1 Molecular cloning of NRK1-EGFP fusion.
The annealing temperature used for cloning of NRK1 and EGFP fragments were around
59°C. For overlap PCR, the annealing temperature used was also 59°C. The length of our
designed NRK1 fragment was 744 bp, while EGFP was 626 bp. For the overlap PCR, the gel
showed up two bands. As shown in Figure 14 a, one of the bands had a size of around 700 bp,
and the other one had a size of 1300 bp which should be the size of the desired fusion gene.
The smaller size band might be the result of nonspecific binding of primers. The size of the
pET-28a vector plasmid is 5300 bp, as shown in Figure 14 c.
When preparing vector from E. coli, the miniprep might result in a band with a very large
size over 10000 bp. This might come from too vigorous shaking during the lysis process,
which caused significant genomic DNA contamination.
The length of ligation did have effect on the numbers of colonies growing on the plates after
transformation. However, the effect was not obvious. The result of 2-hour ligation and
overnight ligation might differ in very limited amount.

34

Figure 14. DNA agarose gel analysis of PCR products. (a) DNA gel of overlap PCR with one
desired band and other unexpected bands. (b): DNA gel of one successful overlap PCR with
band of correct size. (c): DNA gel of the digested pET28a vector.

3000 bp
1000 bp
500 bp
3000 bp
1000 bp
500 bp
a
b
c
35

3.3.2 Expression and purification of NRK1-EGFP fusion
The yield of NRK1-EGFP fusion protein was very high. 1 L of LB medium can produce more
than 10 mg protein for either C-terminal NRK1-EGFP or N-terminal NRK1-EGFP. The
extinction coefficient of NRK1-EGFP is 1.145, and NRK1 has an extinction coefficient of
1.52. The purified protein looked green, suggesting the successful fusion of EGFP to NRK1.
3.3.3 Measurement of the activity of the fusion:
For the incubation time of NR and NR4 with the fusion enzymes, we tried both 2 hours and
overnight. The result suggested that no obvious difference existed between the two incubation
lengths. Through comparing the formation of NAD
+
/NAD
+
4 and the consumption of
NR/NR4, we found out that NRK1 having EGFP fused at its N-terminus has a higher activity
than wild-type NRK1, while the NRK with EGFP fused at C-terminus has a similar activity
of wild type NRK1 (Figure 15 and Figure 16).

36

Figure 15. HPLC analysis for activity assay of EGFP-NRK1 fusion, NRK1-EGFP fusion and
wild-type NRK1 on NR compound.

Tim e
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
5.0e-1
1.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
5.0e-1
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0e-1
2.0e-1
W Y L_N R K A S S A Y _N R D iode A rray
R ange: 2.097e-1
A rea
8.07
87867
6.75
4 6485
18.97
6 49
W Y L_N R K A S S A Y _N R + nterm inal N R K E G FP D iode A rray
R ange: 9.832e-1
A rea
5.00
6353 28
3.10
1646
6.89
17018 3
13.84
71137
7.87
6004 6 8.78
58 10
10.14
932
1 8.10
2 156
W Y L_N R K A S S A Y _N R + N R K W T D iode A rray
R ange: 1.064
A rea
4 .8 4
8 39838
3.10
2216
6.90
20 3582
13.87
11572 2
7.90
7024 5 8.84
704 5
10.02
1442
18.09
2637
W Y L_N R K A S S A Y _N R + cterm inal N R K E G FP D iode A rray
R ange: 1.74
A rea
6.90
397889
4.87
75633 2
4 .39
4 398
3.07
2385
13.90
274017
7.77
42314
8.84
768 3
12.49
1433
1 0.0 7
11 49
18.09
2885
NR
NR
NR
NAD
NAD
NAD
NR
37

Figure 16. HPLC analysis for activity assay of C-terminal NRK-EGFP fusion, N-terminal
NRK-EGFP fusion and wild type NRK1 on NR4 compounds.

Tim e
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
5.0e-1
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
AU
0.0
1.0e-1
W Y L_N R K A S S A Y _N R 4 D iode A rray
R ange: 1.284e-1
A rea
6.74
4415 9
5.90
49 70
13.85
7 0305
11.80
3 776
8.22
2708
10.14
1823
19.35
2651
16.85
1176
15.92
583
W Y L_N R K A S S A Y _N R 4+nterm inal N R K E G FP D iode A rray
R ange: 1.277
A rea
6 .9 0
2 90515
4.87
8027 26
3.09
155 0
9.30
32475
7.75
30040
8.80
3513
13.85
3 9776
11.77
2826
18.07
3166
16.84
8 20
15.17
1069
1 5.8 5
938
19.34
895
W Y L_N R K A S S A Y _N R 4+N R K W T D iode A rray
R ange: 1.392
A rea
6 .9 0
3 28599
4.80
853654
3.09
161 0
9.35
5394 5
7.75
30008
8.80
3200
13.89
4 0302
11.78
2870
18.09
316 9
16.85
856
15 .1 9
107 3
15.77
1 171
19.37
870
W Y L_N R K A S S A Y _N R 4+cterm inal N R K E G FP D iode A rray
R ange: 1.492
A rea
6 .9 0
3 56709
4.87
778274
3.09
146 6
9 .4 4
902 91
7.79
33288
8.84
3277
13.92
34 569
11.82
2485
15.75
3696
15.17
1215
18.09
332 2
16.85
765
19.37
698
W Y L_N R K A S S A Y _N A D 4_S TA N D A R D D iode A rray
R ange: 8.423e-1 15.74
6.77
0.37
1 5.39
NR4

NR4

NR4
NAD4

NAD4

NAD4

NR4

38

3.3.4 Transfection of the fusion into mammalian cells:
The selected C-terminal NRK1-EGFP fusion was cloned through PCR and digested with
restriction enzyme NheI and NotI. The digested gene was then ligated with the digested
EGFP-N1 vector with a size of 3923 bp.
The plasmid was transfected into HEK 293T cells with Transporter
TM
5 in a ratio of 1:4. For
one transfection, 6-wells need to be transfected. 12 μg plasmid was used along with 48 μl
Transporter
TM
5, and they were transfected separately into 6 wells with HEK 293T cells with
80% confluence.
In the first attempt of the transfection experiment, we got a positive result for the
transfecction. The transfection was successful because EGFP was detected in all the cell
lysates transfected with the plasmid. Also, the activity of NRK1 was elevated due to the
formation of large areas of smear bands along the lanes, which suggested the formation of
PAR and the succesful click chemistry between the azide group and biotin alkyne while the
non-transfected cells showed only very tainted smear bands (Figure 17A). However, we kept
having problems when trying to repeat this positive result (Figure 17B), normally resulting in
smear bands showing up in all lanes. The reason for the failure of repetation might be not
successful transfection, too much NAD
+
4 added or PARP1 inhibitor olaprib not working.

39

Figure 17. Western blot analysis of cells treated with NR4. (A). The western blot of
transfected NRK1-EGFP fusion protein. Lane 1: transfected cells. Lane 2: transfected cells
incubated with NR4 for 2 hours. Lane 3: transfected cells incubated with NR4 for 2 hours
with inhibitor added. Lane 4: non-transfected cells incubated with NR4 for 2 hours. Lane 5:
nontransfected cells incubated with NR4 for 2 hours with inhibitor added. (B). Repeated
western blot for Figure 17A.

A
B
40

4. Conclusion
A substantial amount (42 mg) of PARP1 protein was expressed purified from E. coli in a
quite good yield. However, there are still some aspects that can be modified to optimize. The
synthesis of SG3199 is a different route from previous studies. However, it still requires more
attempts on the condensation conditions and condensation reagents. The NRK1 EGFP fusion
can indeed elevate the activity of NRK1 in mammalian cells, but the extent of the elevation
needs further determination. More studies need to be performed for more accurate and
persistent results.

41

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Abstract (if available)

Abstract Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme in human body and can be utilized by various enzymes as substrates. Ribose-functionalize NAD⁺ analogues provide opportunities to generate functionalized poly-ADP-ribose catalyzed by poly-ADP-ribose polymerase 1 (PARP1), which may enable development of novel therapeutic delivery systems. In addition, the ribose-functionalized NAD⁺ may allow tracking and evaluation of NAD⁺-associated pathways in cells. In the first part of this research project, human PARP1 was expressed and purified from Escherichia coli in large quantities through three steps of chromatography. In the second part of this project, synthesis of a pyrrolobenzodiazepine (PBD) dimer was attempted for generating new payload candidates for drug conjugation and subsequent targeted delivery. Finally, human nicotinamide ribose kinase 1 (NRK1) was fused with EGFP and transfected into mammalian cells in order to elevate the rate of transformation of cellular nicotinamide riboside into NAD⁺, which may facilitate in situ generation of functionalized NAD⁺ for cellular studies. These biochemical studies provide useful reagents for development of functionalized poly-ADP-ribose and its conjugates as well as for investigation of NAD⁺-dependent cellular processes.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Wang, Yiling (author)

Core Title Biochemical development and analysis of NAD⁺-related biomolecules

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/25/2020

Defense Date 04/24/2020

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

Tag antibody-drug conjugate,biochemistry,CD38,NAD⁺,NRK1,OAI-PMH Harvest,PARP1,pyrrolobenzodiazepine dimer

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zhang, Yong (committee chair), Duncan, Roger (committee member), Haworth, Ian (committee member)

Creator Email yilingw@usc.edu,yilingwang01@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-290324

Unique identifier UC11663543

Identifier etd-WangYiling-8334.pdf (filename),usctheses-c89-290324 (legacy record id)

Legacy Identifier etd-WangYiling-8334.pdf

Dmrecord 290324

Document Type Thesis

Rights Wang, Yiling

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