Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase

Article abstract of DOI:10.1021/jacs.7b09316

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC₅₀ = 1.7 μM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC₅₀ = 0.059 μM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 μM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.

Full text of DOI:10.1021/jacs.7b09316

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Article
Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase (OGG1)
Yu-ki Tahara, Douglas Auld, Debin Ji, Andrew A. Beharry, Anna M Kietrys,
David L. Wilson, Marta Jimenez, Daniel King, Zachary Nguyen, and Eric T. Kool
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09316 • Publication Date (Web): 27 Jan 2018
Downloaded from http://pubs.acs.org on January 28, 2018
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Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase (OGG1)
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Yu-ki Tahara,[a] Douglas Auld,[b] Debin Ji,[a] Andrew A. Beharry,[a] Anna M. Kietrys,[a] David L. Wilson,[a]
Marta Jimenez,[b] Daniel King,[b] Zachary Nguyen,[b] Eric T. Kool*[a]
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[a] Department of Chemistry, Stanford University, Stanford, California 94305, United States
[b] Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, 250
Mass. Ave., Cambridge, Massachusetts
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*to whom correspondence should be addressed: kool@stanford.edu
Abstract
The activity of DNA repair enzyme OGG1, which excises oxidized base 8-oxoguanine (8-OG)
from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test
the roles of OGG1-mediated repair in these pathways, we have undertaken the
development of noncovalent small-molecule inhibitors of the enzyme. Screening of a
PubChem-annotated library using a recently developed fluorogenic 8-oxoguanine excision
assay resulted in multiple validated hit structures, including selected lead hit
tetrahydroquinoline 1 (IC50 = 1.7 µM). Optimization of the tetrahydroquinoline scaffold over
five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 =
0.059 µM). SU0268 was confirmed by surface plasmon resonance studies to bind the
enzyme both in the absence and presence of DNA. The compound SU0268 was shown to be
selective for inhibiting OGG1 over multiple repair enzymes, including other base excision
repair enzymes, and displayed no toxicity in two human cell lines at 10 µM. Finally,
experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an
increase in accumulation of 8-oxoguanine in DNA. The results suggest the compound
SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related
pathways.
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Introduction
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The consequences of DNA damage are important to many biological pathways, and are
central to mutagenesis, genotoxicity, and tumorigenesis.1-6 Among the most frequent forms
of damage in DNA is 8-oxoguanine (8-OG), which is generated by reactive oxygen species
arising from metabolism, or exposure to agents that induce oxidative stress.5-17 Because 8-
OG is mispaired frequently with adenine during DNA replication, it is highly mutagenic,18
and the frequency of guanine oxidation combined with this mispairing causes it to be the
greatest single source of mutations in the cell.15 Mutations that occur in proto-oncogenes
can initiate tumorigenesis,19 and the presence of 8-OG in DNA can also be harmful to cell
growth by leading to DNA double strand breaks.20
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The primary enzyme for repairing 8-OG in DNA is 8-oxoguanine DNA glycosylase
(OGG1), which functions via a base excision repair (BER) mechanism.21 The enzyme exists
both in the nucleus and mitochondria, repairing DNA damage in both subcellular locations.22
It recognizes 8-OG in double-stranded DNA, and cleaves the glycosidic bond, releasing the 8-
OG free base, and generating an abasic deoxyribose in the DNA.21 This abasic site is then
further processed by lyase activities of the enzyme itself or the AP lyase enzyme, ultimately
leading to strand cleavage.20,23,24 Additional enzymes in the BER pathway can then complete
the damage repair, regenerating intact DNA with correctly paired bases.21
Because of the importance of OGG1 to DNA mutations and genotoxicity, a good deal
of study has been devoted to investigating how varied levels of this activity can affect
disease pathologies. OGG1-deficient mice display elevated levels of genomic 8-oxoguanine
and increased mutations, highlighting the importance of this enzyme in maintaining genome
integrity.25,26 OGG1 mutants with abolished or decreased 8-OG repair activity have been
found in many tumor specimens.27,28 A common OGG1 polymorphism, S326C, which lowers
BER activity, is positively associated with frequency of cancers of the digestive system and
the lung.29-31 In addition to its prominent association with genetic mutations leading to
malignancies, OGG1-mediated repair of 8-oxoguanine has been associated with
inflammation-related pathologies as well.32 8-OG is known to act as a signaling molecule
that modulates activity of several GTPases.33 Downregulated OGG1 activity results in
decreased lung inflammation in murine allergy models.32 Furthermore, polymorphisms in
the OGG1 gene have been associated with rheumatoid arthritis.34
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Taken together, the data suggest strong links between OGG1 and multiple disease
states. For further studies of these relationships, it would be desirable to have small
molecule inhibitors of the enzyme, which could be useful as tools to study OGG1-related
pathways and pathologies in cellular and animal models. The only previously known small
molecule inhibitors of OGG1 were reported recently by Lloyd, who described simple
hydrazine and hydrazone derivatives that inhibited the enzyme as measured by an assay
that measures DNA strand cleavage subsequent to base excision.35 Since hydrazones can
spontaneously hydrolyze,36 and hydrazines are known to react generally with abasic sites in
DNA,37,38 such classes of compounds may raise questions of stability and specificity. In
general, the use of BER assays that measure DNA cleavage rather than the excision event
may result in identification of hit compounds that do not act by inhibiting initial base
excision.
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For these reasons, discovery and development of highly potent, selective and stable
small molecule inhibitors of OGG1 remain an important goal, and this goal would be aided
by an assay that directly measures excision. In an effort to directly measure the base
excision activity of OGG1, we recently developed fluorogenic probe (OGR1) that can detect
the removal of 8-OG in real-time (Figure 1).39 In this probe, 8-OG acts as a fluorescence
quencher of a neighboring fluorescent DNA base, and enzymatic excision of the 8-OG
renders the probe emissive. The fluorescent signal then yields a quantitative measurement
of OGG1 activity.
H
N
O
NH2
NH
oG
N
O
N
H
5’- C
G
G
C
C
G
C
C
G
C
5’- C
G
G
C
C
G
C
C
G
C
G
A
G
A
A
A
G tCo oG G
G tCo --- G
×
×
O
O
P
O-
H
N
O P O
N
NH2
NH
oG
O
O
O-
O
O
O
O
O
N
H
O
N
O
N
O
OH
abasic
N
N
O
NH2
HN
O
N
N
NH
O
HN
O
HN
tCo
oG
tCo
(emissive)
(quenched) (fluorescence quencher)
OGG1
Inhibitor
Figure 1. OGR1 probe for assay of OGG1 base excision repair activity and inhibition.39
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Herein, we describe the development of small-molecule OGG1 inhibitors using this
fluorogenic assay in high throughput, leading to the identification of a tetrahydroquinoline
scaffold with significant inhibitory activity. We report structure-activity relationships of a
broad set of derivatives as potential inhibitors of the enzyme, and we describe the
properties of the potent and selective inhibitor SU0268 that resulted from this work.
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Materials and Methods
General Information
1H NMR spectra were recorded on Varian Inova 300 (300 MHz) spectrometers. The chemical
shifts were reported in parts per million (δ) relative to internal standard TMS (0 ppm) for
CDCl3. The peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; dt, m,
multiplet; q, quartet. The coupling constants, J, are reported in Hertz (Hz). 13C NMR spectra
were obtained by Varian Inova 300. CDCl3 was used as a NMR solvent (Acros Organics). Low-
resolution mass spectra were measured on an ESI (Electro Spray Ionization) by the ACQUITY
UPLC (Waters). High-resolution mass spectra (HRMS) were measured on an ESI by
micrOTOF-Q II (Bruker). Ultraviolet spectra were measured on a Varian Cary 300.
Fluorescence intensities were measured on Fluoroskan Ascent Microplate Fluorometer.
OGR1 probe was synthesized by using an Applied Biosystems 394 DNA/RNA synthesizer.
ClogP and tPSA were calculated by ChemDraw. Analytical TLC was performed on ready-to-
use plates with silica gel 60 (Merck, F254), Flash column chromatography was performed
over Fisher Scientific silica gel (grade 60, 230-400 mesh). All reagents were weighed and
handled in air and backfilled under argon at room temperature. Unless otherwise noted, all
reactions were performed under an argon atmosphere. All chemicals were purchased from,
Acros Organics, Alfa Aesar, Ark Pharm, Asta Tech, Combi-Blocks, Enamine, Matrix Scientific,
Sigma-Aldrich and TCI and used without further purification.
Synthesis of compounds 2-44. Details of synthesis and characterization of these analogs
and their synthetic intermediates are given in the Supporting Information file.
General procedure for OGG1 inhibitor assay. BSA, hOGG1 and NEBuffer 4 (50 mM
potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH
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7.9 at 25 °C), were purchased from New England Biolabs and used. UltraPure Distilled Water
(Invitrogen) was purchased and used for the assay.
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Synthesized compounds and hOGG1 (100 nM) were incubated in NEBuffer 4 (1 X)
with BSA (1 X) at 37 °C (15 min) in 100 μL reaction volumes in a black 96-well plate. After
that, OGR1 probe39 (1.2 μM) was added to the reaction mixture. Fluorescence at 460 nm
was measured on a Thermo Fluoroskan Ascent FL fluorescence plate reader (λex = 355 nm).
The slope of initial rate (12 min) was calculated, and percent of control was used for
inhibition activity.
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For the HTS, the assay was optimized for 384-well plates (low volume, black solid
plates, Greiner cat# 784076). For the HTS assay, 5 µL of 100nM hOGG1 was dispensed using
a FlexDrop dispenser (Perkin Elmer) in NEBuffer 4. Next library plates containing compounds
at 2 mM in DMSO were used to transfer 100 nL of the DMSO solution to the assay plate
using an acoustic dispenser (EDC Biosystems, ATS). After a 15 min incubation at 37 °C, the
enzyme reaction was started by adding 5 µL of 1.6 µM OGR1 in NEBuffer 4 using the
FlexDrop. Final concentrations of OGG1 and OGR1 were then 50 nM and 0.8 µM,
respectively. The reaction was incubated at 37 °C for 30 minutes and the fluorescence was
determined on a Perkin Elmer Envision microtiter plate reader (λex 340 nm and λem 450 nm
using Lance/Delfia dichroic mirror of 412 nm). See Supporting information for further details.
Screening of enzyme selectivity. Details of assays for MTH1, dUTPase, NUDT16, AlkBH2,
AlkBH3, and SMUG1 are given in the Supporting Information.
SPR studies. Recombinant His-tagged OGG1 (Creative Biomart) was exchanged to
phosphate buffered saline (PBS). Limited biotinylation of OGG1 surface lysines was
performed at 4 oC for 20 minutes using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) and
exchanged back to PBS on a desalting column to quench the reaction. All SPR studies were
performed on a Biacore 8K instrument (GE Healthcare Life Sciences) using 50 mM potassium
acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, 2% dimethylsulfoxide
(DMSO) and 0.005% Tween, pH 7.9 as the running buffer. 3,000 resonance units (RU) of
biotinylated OGG1 was captured on streptavidin sensor chip SA (GE Healthcare Life
Sciences). Following capture, any remaining streptavidin sites on both the reference and
active channels were blocked with biocytin. A 1:3 dilution series of compound 41 (highest
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concentration 4 μM), prepared to match the running buffer, was flowed over OGG1. All
sensorgrams shown are reference subtracted with solvent correction procedures
implemented. Data were fit to both steady state and a 1:1 kinetic model using Biacore
evaluation software.
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Michaelis Menten kinetics studies. For Michaelis-Menten curves: Inhibitor 41 and hOGG1
(100 nM) were incubated in NEBuffer 4 (1 X) with BSA (1 X) at 37 °C (15 min) in 100 μL
reaction volumes in a black 96-well plate. After that, OGR1 (0.8-10 μM) was added to the
reaction mixture. Fluorescence at 460 nm was measured on a Thermo Fluoroskan Ascent FL
fluorescence plate reader (λex = 355 nm). The initial rate (10 min) was calculated.
Lineweaver-Burk plots were derived from the Michaelis-Menten curves. Km and Vmax were
calculated from the equation. kcat was calculated from kcat = Vmax/[enzyme].
DNA binding studies. Abasic DNA: NEBuffer 4 (1 X), 1 μM abasic DNA, 2 μM 41 (1% DMSO)
were included in 1600 μL reaction volumes with 1 cm path-length quartz cells. The UV
absorbance at 260 nm was measured on a Varian Cary 300 as temperature was raised 1 °C
/min. The Tm value was calculated by Meltwin.
OGR1 probe: PBS buffer (0.1 X), 1 μM OGR1 probe, 2 μM 41 (10% DMSO) were included in
800 μL reaction volumes with 1 cm path-length quartz cells. The UV absorbance at 260 nm
was measured on a Varian Cary 300 as temperature was raised 1 °C/min. The Tm value was
calculated by Meltwin.
Toxicity via MTT assay. HEK293T cells (1.6 × 104 cells per well) and HeLa cells (concentration
of 1.2 × 10^4 cells per well) were seeded to 96-well plate in supplemented DMEM culture
medium (10% FBS, 100U Penicillin/Streptomycin) and incubated for 30 h (HEK293T cells)
and 16 h (HeLa cells) at +37 °C, 95% humidity and 5% CO2. They were incubated in 100 μl of
the fresh medium at a concentration of 100 μM, 10 μM, 1 μM, 100 nM, and 10 nM of the
compound 41 for 24 h at +37°C, 95% humidity and 5% CO2, in supplemented DMEM culture
medium. After the incubation period, 10 μL of the MTT labeling reagent (final concentration
0.5 mg/mL) was added to each well, and incubated at +37°C, 95% humidity and 5% CO2. 100
μL of the solubilization solution was added into each well, and incubated for 20 h at +37 °C,
95% humidity and 5% CO2. Absorbance of the samples was measured by using a microplate
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reader (Tecan Infinite M1000) at 550, and 650 nm. Compound 41 was provided in
concentration of 2 mM in DMSO that determined final concentration of DMSO in 100 μM at
the level of 5%. In the remaining samples (10 μM, 1 μM, 100 nM, 10 nM) concentration of
DMSO was at the level of 1%. The controls were prepared: Ctrl – cells cultured only in the
supplemented DMEM; Ctrl 5% DMSO - cells cultured in 5% DMSO in the supplemented
DMEM; Ctrl 1% DMSO - cells cultured in 1% DMSO in the supplemented DMEM.
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Detection of 8-OG in cell line by LC-MS/MS. HeLa cells (2 × 106 cells per well) were seeded
in 10 cm dishes in supplemented DMEM culture medium (10% FBS, 100U
Penicillin/Streptomycin) and incubated for 12 h (HeLa cells) at +37 °C, 95% humidity and 5%
CO2. They were incubated in 10 mL of the fresh medium at a concentration of 5 μM and 0.5
μM of compound 41 for 24 h at +37°C, 95% humidity and 5% CO2, in supplemented DMEM.
Every 6 h fresh medium with the compound 41 was added. Control HeLa cells were cultured
in supplemented DMEM culture medium. For positive oxidative damage controls, we
cultured HeLa cells in the supplemented DMEM medium with 0.5 mM H2O2 and 0.3 mM
CrCl3. After incubation, all cells were washed twice with PBS and harvested for DNA isolation.
DNA isolation was performed with Quick-DNA™ Miniprep Plus Kit (Zymo Research)
according to the manufacturer’s protocol. The concentration of DNA eluted in nuclease-free
water was measured on a NanoDrop One instrument (Thermo Scientific). The DNA (5 µg)
was dried and then digested with 0.02 U spleen II nuclease (Worthington) and 0.4 U
micrococcal nuclease (Sigma Aldrich) in 20 mM succinate buffer containing 10 mM CaCl2 for
16 h at 37 °C. The hydrolysate was treated with 0.4 µg RNase P1 (Sigma Aldrich) in 78 mM
ammonium acetate buffer (pH 4.5) and 10 mM bicine-NaOH buffer (pH 9.7) containing 10
mM MgCl2, 10 mM dithiothreitol, and 1 mM spermidine for 6 h at 37 °C. The final volume of
the mixture was 25 µL. 75.5 µL internal standard was added to all samples and blank sample
to a total volume of 100 µL. One of the blank samples was prepared without internal
standard. The reaction mixtures were filtered through Amicon Ultra 0.5 mL 3 kDa cut-off
filters (Millipore). The set of 12 spiking solutions, containing dG, dC, 8oxodG, was prepared
according to the digestion procedure for DNA. The detection range covered by spiking
solution was: 0.0625 ng/mL to 100 ng/mL for 8-oxodG, and 2.5 ng/mL to 5.97 µg/mL for dG.
Additionally, in the LC-MS/MS analysis internal standards of [15N5] dG and [15N3] dC were
used. The nucleosides were separated on a Synergi 4 µm Fusion column, RP 80 Å, 150 x 2
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mm, at 30 °C with 10 µL injection volume, and flow rate of 200 µL/min. Separation was
performed using following program: 0-3 min 100% phase A (0.1% formic acid in water), 9
min 60% phase A and 40% phase B (0.1% formic acid in acetonitrile), 12 min 10% phase A,
12.1 – 15 min 100% phase A. Collected values of 8-oxodG content (ng/mL) were normalized
to the amount of dG for all replicates.
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Inhibition of OGG1 activity in cell lysates by SU0268. HeLa and MCF-7 cells were seeded in
15 cm dishes in supplemented DMEM culture medium (10% FBS, 100U
Penicillin/Streptomycin) (Gibco) and incubated for 48 h at +37 °C, 95% humidity and 5% CO2
to full confluence. Cells were scraped, washed with 1 x PBS (pH 7.4) and decanted. For each
cell culture the packed cell volume (PCV) was estimated. The 5 x PCV of lysis buffer (10 mM
HEPES pH 7.9 (Gibco), with 1.5 mM MgCl2 (Sigma Aldrich) and 10 mM KCl (Sigma Aldrich), 1
mM DTT (Invitrogen), 1 x Protease Inhibitor Cocktail (Roche)) was used to resuspend cells.
The suspension was incubated on ice for 15 min to allow cells to swell, and then centrifuged
for 5 minutes at 420 x g, at 4°C. The cell pellet was resuspended in 2 x PCV of lysis buffer
and lysed using a syringe with a narrow-gauge (No. 25) hypodermic needle. Disrupted cells
were centrifuged 20 minutes at 11,000 x g, at 4°C. The collected supernatant was the
cytoplasmic fraction. The nuclei pellet was resuspended in 2/3 x PCV of extraction buffer (20
mM HEPES pH 7.9 (Gibco), with 1.5 mM MgCl2 (Sigma Aldrich), 0.42 M NaCl (Sigma Aldrich),
0.2 mM EDTA (Invitrogen), 25% (v/v) Glycerol (Sigma Aldrich), 1 mM DTT (Invitrogen), 1 x
Protease Inhibitor Cocktail (Roche)), and shaken for 60 min at. at 4°C. The suspension was
centrifuged for 5 min at 21,000 x g, at 4°C, and the supernatant containing nucloplasmic
fraction was collected. Two volumes of cytoplasmic fraction were mixed with one volume of
nucleoplasmic fraction and this protein mixture was used in the experiment. Concentration
of proteins was measured using the Bradford Protein Assay (Biorad) according to the
manufacturer's protocol. Reactions were performed with a total volume of 80 µL containing
100 ng/µL total cell protein, 2 µM pOGR1 probe, and 10 mM MgCl2, and were incubated for
24 h at 37°C in a black 384-well plate. Fluorescence was measured at 460 nm using a
Fluoroscan Ascent FL plate reader (Thermo Scientific) and excitation wavelength 355 nm.
Procedure for CACO-2 assay. Measurements were carried out by Absorption Systems LLC.
See Supporting Information for experimental details.
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Results and Discussion
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Small molecule screening for OGG1 inhibition. The identification of OGG1 inhibitors was
initiated with a high-throughput screen of the PubChem-annotated library compounds using
hOGG1 enzyme and our originally developed OGR1 probe assay, using recombinant human
OGG1. Approximately 26,000 compounds were chosen for initial screening, and the assay
was carried out at a single 20 μM concentration (Figure 2). The high-throughput screen
showed good performance with a Z΄ = 0.68±0.1 and led to over 350 initial hit compounds
using a threshold of >35% inhibition, which was >3 s.d. of the maximum enzyme activity
controls treated with DMSO vehicle alone. The hit compounds showed varied chemical
structures and were then further validated by titration over an 8-fold concentration range
to determine IC50 values. The assay yielded several validated hit compounds, from which we
selected compound 1, a tetrahydroquinoline sulfonamide derivative, for further study.
Compound 1 did not score as active in another fluorescence enzyme assay which used the
same fluorescence detection protocol,40 thus confirming that its activity was not a false
positive resulting from inherent fluorescence. In addition, compound 1 (annotated in
PubChem as CID:7517554) has been tested in 84 assays. Of these assays, only two listed the
compound as active, with no IC50’s below 10 µM, supporting a selective compound profile.
Our general strategy for structural optimization of scaffold 1 is shown in Figure 2. The
chemical structure of 1 was divided into five fragments A-E; fragment B consists of the core
tetrahydroquinoline structure, and A is a substituent on the nitrogen atom of this core
structure. Fragment C, a sulfonamide, is a linker between core structure B and fragment D, a
benzene ring. Fragment E is a substituent of this aromatic ring. We varied these five
fragments to investigate their influences on OGG1 base excision as measured by the OGR1
assay.
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17
18
19
20
21
22
23
24
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28
29
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32
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35
36
37
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41
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HTS of 25,975 compounds at a screening concentration of 20 μM
5
6
7
8
9
8-point validation assay for 360 compounds >35% inhibition at 20 μM
22 confirmed inhibitors
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
O
O
S N
N
H
O
O
Validate hit compound 1
PubChem CID: 7517554
Primary screen: 91% inhibition
Absolute IC50 = 2 μM
B
O
O
S N
N
H
O
O
E
D
C
A
Figure 2. Discovery of lead compound 1 and strategy for the lead optimization. Following
the HTS compounds were validated using eight concentration points ranging from 100 to
0.03 µM. Absolute IC50 is the concentration for which the concentration response curve
crosses 50% inhibition.
Structural optimization. For the initial optimization studies, we synthesized analogs of hit
compound 1 with varied substituents (R1) on the nitrogen atom of the tetrahydroquinoline
skeleton (Table 1). Assays of these compounds were carried out over a three-log
concentration range to differentiate weak inhibitors from stronger ones. Lead compound 1
(with acetamide substituent as R1) showed significant inhibition of OGG1 at 20 μM and ca.
50 % inhibition at 2 μM, but yielded little inhibition activity at 200 nM. Homologation of
acetamide to ethylamide (2) had little effect on inhibition, while larger substituents (3-6)
lowered the activity. Interestingly, however, cyclopropylamide analog 7 showed significantly
more potent inhibition activity than the lead compound (Table 6), and was also more active
than the closely related isopropyl variant 5. A slightly larger ring (cyclobutyl compound 8)
showed somewhat lower potency. We proceeded to other R1 substituents, varying the
carbonyl itself. Sulfonamide analog 9, urea analog 10 and reduced analog 11 were prepared
and assayed. However, these compounds showed little or no activity at 20 μM. These
results establish that the carbonyl group on the tetrahydroquinoline nitrogen atom is highly
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important for activity, and among them, the cyclopropylamide group was the optimal
substituent for R1.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Examination of substituents R1 and their effects on OGG1 activity (Fragment A)
O
O
S N
N
R1
H
O
Enzyme activity,
% of control
at 20 μMa
Cmpd
R1
2 μMa,b
200 nMa, b
1
2
4.7 ± 3.0
47 ± 3.5
51 ± 1.4
74
O
3.7 ± 0.83
n.t.
O
3
4
79
51
≥ 100
93
n.t.
n.t.
O
O
O
5
52
96
n.t.
O
6
7
≥ 100
5.8 ± 1.9
33
n.t.
41 ± 1.7
69
n.t.
82 ± 12
n.t.
O
O
8
O
O S
O
9
92
96
n.t.
10
77
87
n.t.
O
N
H
11
83
≥ 100
n.t.
a % of control values are ratio of enzyme activity to control (no compound) based on slopes
of initial rate (first 12 min of assay). b n.t., not tested.
Next, we examined fragment B, the core structure of the initial hit compound (Table
2). Acyclic compound 12 and 5-membered ring dihydroindole 13 showed low activity. The 7-
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membered ring analog 14 was also examined, but displayed lower inhibition activity than
the 6-membered ring analog 7 shown in Table 1. To test the effects of the carbon skeleton
of the tetrahydropyridine ring, we introduced an oxygen atom (15), but this showed lower
inhibition, suggesting a relatively hydrophobic contact with the enzyme at this position. We
installed a methoxy substituent on the benzene ring of the tetrahydroquinoline skeleton
(16), but this almost completely abrogated inhibitory activity. Finally, we repositioned the
cyclopropylamide substituent by one atom in the tetrahydroisoquinoline derivative 17, but
this also greatly lowered activity. These data establish that the tetrahydroquinoline skeleton
is quite important to the inhibitory activity of active compounds 1 and 7.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Examination of core structure R2 and their effects on OGG1 activity (Fragment B)
O
R2
O
S N
H
O
Enzyme activity,
% of control
at 20 μMa
Cmpd
R2
2 μMa,b
47 ± 3.5
200 nMa, b
N
1
4.7 ± 3.0
5.8 ± 1.9
74
O
N
7
41 ± 1.7
82 ± 12
O
NH
12
13
70
96
95
n.t.
n.t.
O
N
≥ 100
O
N
14
15
35
41
76
90
n.t.
n.t.
O
O
N
O
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MeO
5
6
7
8
N
16
17
93
88
≥ 100
n.t.
n.t.
O
9
N
94
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
a % of control values are ratio of enzyme activity to control (no compound) based on slopes
of initial rate (first 12 min of assay). b n.t., not tested.
We proceeded to test variations of Fragment C, the sulfonamide linker between the
tetrahydroquinoline and the aromatic substituent, preparing diverse linkers in compounds
18-21 (Table 3). N-hydroxy and N-methyl groups combined with sulfonamide (18 and 19)
were tested, but they showed lower inhibition activity than unsubstituted sulfonamide 7
(Table 1). This suggests possible polar interactions in the enzyme with the N-H group. Amide
and urea analogs (20 and 21) were synthesized and assayed, but showed almost no activity.
Therefore, the simple “N-H” benzenesulfonamide structure in Fragment C was selected as
most appropriate for further studies.
Table 3. Examination of linker R3 and their OGG1 inhibition activity (Fragment C)
R3
N
O
Enzyme activity,
% of control
at 20 μMa
Cmpd
R3
2 μMa,b
200 nMa, b
1
7
4.7 ± 3.0
47 ± 3.5
41 ± 1.7
74
O
O
S N
5.8 ± 1.9
82 ± 12
H
O
O
O
S N
O OH
O
S N
O
18
19
21
79
79
84
≥ 100
n.t.
O
O
N
H
20
≥ 100
≥ 100
n.t.
O
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O
O
21
≥ 100
≥ 100
n.t.
N
N
H
H
a % of control values are ratio of enzyme activity to control (no compound) based on slopes
of initial rate (first 12 min of assay). b n.t., not tested.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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We next investigated the effects of variations of Fragments D and E of the core
structure (Table 4). Ethyl sulfonamide derivative 22 proved to be quite poorly active,
suggesting the importance of the aromatic framework. Subsequently, a variety of
substituents on the benzene ring were prepared and assayed with OGG1 and the OGR1
probe. Compound 23, carrying a 4-propylphenyl substituent, showed inhibition activity
nearly the same as that of compound 7 possessing 4-ethoxyphenyl group. Compound 24,
which extends the alkyl chain compared with 7 was tested, but this did not improve activity.
The analogs possessing nitrogen and sulfur atoms on the benzene ring were tested, but the
heteroatoms had slightly deleterious effects on activity (25 and 26). The disubstituted
compound 27 showed lower inhibition activity than mono-substituted compound 7.
Unexpectedly, testing of compound 28 which has a diphenyl ether group, showed almost
the same activity as compound 7 with its ethoxy group. Therefore, we designed and
synthesized the dibenzofuranyl analog 29 and biphenyl analog 30, which are rigidified
variants of the diphenyl ether compound. Interestingly, compound 29 showed low activity,
but compound 30 had more potent activity than either diphenyl ether 28 and ethoxyphenyl
compound 7. Cyclohexylphenyl derivative 31 showed almost the same activity as biphenyl
derivative 30, but a more polar morpholinophenyl substituent (32) had a poor effect on the
activity. Pyrazolylphenyl and pyridylphenyl analogs (33 and 34) containing a polar nitrogen
atom were subjected to the assay. We found that compound 34 gave slightly stronger
inhibitory activity than biphenyl compound 30, displaying ca. 60% inhibition of activity at
200 nM.
Table 4. Examination of substituents R4 and their effects on OGG1 activity (Fragments D, E)
O
R4 S N
N
H
O
O
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7
Enzyme activity,
Cmpd
R4
% of control
2 μMa,b
200 nMa, b
at 20 μMa
8
1
7
4.7 ± 3.0
5.8 ± 1.9
74
47 ± 3.5
41 ± 1.7
94
74
82 ± 12
n.t.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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59
60
O
22c
23
14
35
74
24
n.t.
19
95
O
HN
25
26
10
58
53
≥ 100
75
S
3.6
27
28
22
71
n.t.
O
8.3
41 ± 2.0
≥ 100
O
29
71
82
n.t.
O
30
31
32
33
n.t.
n.t.
29 ± 7.0
25
90 ± 6.2
87
O
N
n.t.
76
84
N
14 ± 3.2
45 ± 1.3
81
N
N
34
2.0
23 ± 1.5
61 ± 1.3
a % of control values are ratio of enzyme activity to control (no compound) based on slopes
of initial rate (first 12 min of assay). n.t., not tested. Acyl group of nitrogen atom on
tetrahydroquinoline skeleton is propionyl group.
b
c
Finally, we explored structural effects of the substituents on the terminal aromatic
ring of lead compound 30, synthesizing compounds 35-39 (Table 5). When 4-trifluoromethyl
as an electron withdrawing group and 4-methoxy as an electron donating group were
compared (35 and 36), the more polar and electron-donating group (4-methoxy) assisted
the activity more than the electron-withdrawing group. Moving this substituent’s position,
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the 3-methoxy compound 37 resulted in more potent activity than the 4-substituted
compound 36. Therefore, varied substituents at the meta-position of this benzene ring were
examined. Compound 38 (with a dimethylamino group) and compound 39 (possessing an
acetylamino group) were synthesized and assayed. The compound 38 showed similar
activity as methoxy compound 37, but the acetamide 39 showed higher activity than either
of these, with inhibition activity of about 50 % at 200 nM. When we investigated the
compounds 40 and 41 which have reverse amide structure, these compounds showed yet
higher activity, and compound 41 (SU0268), possessing a meta-carboxamide group, had the
strongest inhibitory activity of all compounds in the study.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. Examination of substituents R5 and their OGG1 inhibition activity (Fragment E)
O
R5
S N
N
H
O
O
Enzyme activity,
% of control
at 20 μMa,b
Cmpd
R5
2 μMa,b
200 nMa
1
7
4.7 ± 3.0
47 ± 3.5
41 ± 1.7
74
O
5.8 ± 1.9
82 ± 12
F3C
35
36
n.t.
n.t.
72
31
97
79
MeO
MeO
37
38
n.t.
n.t.
13
67
70
N
n.t.
O
NH
39
40
41
n.t.
n.t.
n.t.
12 ± 2.0
n.t.
53 ± 4.9
48
O
HN
O
H2N
4.8
39 ± 2.5
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a % of control values are ratio of enzyme activity to control (no compound) based on slopes
of initial rate (first 12 min of assay). b n.t., not tested.
6
7
8
9
Synthesis of SU0268 (inhibitor 41). The synthesis of tetrahydroquinoline biphenyl
sulfonamide derivative 41, the most potent OGG1 inhibitor in this study, is depicted in
Scheme 1. We commenced the preparation of 41 with acylation of the commercially
available tetrahydroquinoline 42 with cyclopropylcarbonyl chloride in the presence of N,N-
diisopropylethylamine.41 Subsequent 1-atm hydrogenation of the acylated intermediate in
the presence of palladium on carbon gave the amine 43 in 77%.41 Sulfonylation of 43 with 4-
bromobenzene sulfonylchloride afforded the sulfonamide 44 in excellent yield.42
Construction of the biphenyl structure using Suzuki-Miyaura coupling in the last step with 3-
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aminocarbonylphenyl
boronic
acid
in
the
presence
of
tetrakis(triphenylphosphine)palladium(0) gave the desired 41 in 82%.43 The intermediates
43 and 44 were obtained by crystallization, and this protocol required only one silica column
chromatography purification step to isolate 41.
O
1)
O
S Cl
O
Cl
(1.1 eq.)
Br
(1.2 eq.)
O
DIPEA (1.2 eq.) / DCM, rt, ON
H2N
N
Br
S N
N
H
O
O2N
N
10% Pd/C, H2
pyridine, 0 °C - rt, ON
2)
H
O
43
O
MeOH/DCM, rt, ON
42
44
77%
95%
O
H2N
B(OH)2
(4.0 eq.)
O
H2N
Pd(PPh3)4 (20 mol%)
Cs2CO3 (4.0 eq.)
O
S N
O
N
H
toluene/MeOH, 80 °C, 3 h
O
41
83%
Scheme 1. Synthesis of the OGG1 inhibitor SU0268 (41).
Properties of selected inhibitors. Properties of selected inhibitors were further examined.
Titration curves and IC50 values of the hit compound 1, selected key intermediate inhibitors
(7, 28, 34 and 39) and optimized compound 41 were determined using the initial rates
method (Figure 3 and Table 6). The IC50 of the hit compound 1 was 1.7 µM. The IC50 values
of compounds 7 and 28, with modified substituents on tetrahydroquinoline and
benzensulfonamide, were slightly more potent than that of 1. The compounds 34 (with
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pyridine ring) and 39 (with acetylamino group) had considerably higher activity than the
lead compound 1, with IC50 values below 0.3 µM. The IC50 value of the optimized compound
41 was 0.059 µM. We calculated cLogP (hydrophobicity) and tPSA (topological polar surface
area) values for the compounds (Table 6) as useful measures of potentially bioactive
compounds.44,45 The values of clogP of the compound 7 and 28 were higher than the hit
compound 1, but their values of tPSA were almost the same. Our potent inhibitors 34, 39
and 41 showed moderate cLogP values (3.4-3.8), and their tPSA values were 79-110, which
also fall in the generally accepted range for potential bioactivity.44,45
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19
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21
22
23
24
25
26
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
120
1
7
100
ꢀ
28
34
39
80
41
60
40
20
0
1
10
100
1000
10000
100000
-20
Compound concentration (nM)
Figure 3. Titration curves of selected OGG1 inhibitors in this study
Table 6. IC50 of OGG1 inhibitors, and their cLogP and tPSA
Cmpd IC50 (μM)
cLogPa
2.5
3.6
tPSAa
76
76
1
1.7
1.1
7
28
34
39
41
1.5
4.9
3.4
3.8
3.3
76
79
96
110
0.27
0.25
0.059
a see SI for calculation methods.
Surface plasmon resonance (SPR) was used to validate the series and confirm the
compounds acted through OGG1. Compound 41 was titrated against biotinylated OGG1
previously captured on a SA streptavidin sensor chip (Figure 4). Sensorgram plots showed a
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saturable dose response consistent with specific binding to OGG1. The data, fit against 1: 1
kinetic and steady state models, gave Kd’s consistent with IC50 of the biochemical assay.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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30
31
32
33
34
35
36
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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60
Figure 4. SU0268 (41) specifically binds OGG1. 3000 RU (Resonance Units) of recombinant
biotinylated OGG1 was immobilized onto streptavidin sensor chip SA and experiments were
performed on a Biacore 8K with a 1:3 dilution series of inhibitor 41 (highest concentration 4
µM) (a) Sensorgram plots of inhibitor 41. (b) Steady state Kd determination of compound 41.
(C) Kinetic and binding constants.
To investigate the effects of SU0268 on inhibition of OGG1, we measured Michaelis-
Menten parameters in the absence and presence of this compound at 200 nM and 2 μM. A
Lineweaver-Burk plot was obtained from Michaelis-Menten curves (Figure 5), and Km and
kcat values were calculated (Table 7). Measuring the effect of the inhibitor showed an
increase in Km and little or no effect on kcat. These results indicate that inhibitor 41 acts as a
competitive inhibitor against OGG1.46
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Lineweaver-Burk plots of OGG1 activity without inhibitor and with inhibitor 41
Table 7. Km and kcat without inhibitor and with inhibitor 41
No Inhibitor
1.2
200 nM 41
1.8
2 μM 41
8.2
0.057
0.0070
Km (μM)
kcat (s-1)
0.046
0.039
0.040
0.022
kcat/Km (s-1μM-1)
We further investigated interaction of abasic DNA (without 8-OG) and OGR1 probe
(with 8-OG) with inhibitor 41 (Table 8). Abasic DNA has C instead of tCo and was introduced
abasic site mimic (X) instead of 8-OG. If the interaction is observed, melting temperature
(Tm) would be changed significantly.47 In both cases of abasic DNA and OGR1 probe, no
measurable changes of Tm were observed, providing evidence that inhibitor SU0268 does
not bind DNA, and thus interacts with OGG1 specifically rather than its substrate.
Table 8. Thermal denaturation studies of OGR1 probe and Abasic DNA in the absence (Tm(-)
and presence (Tm(+)) of OGG1 inhibitor 41
)
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5’- C
G
G
C
C
G
C
C
G
C
5’- C
G
G
C
C
G
C
C
X
C
G
C
G
A
G
A
A
A
G tCo oG G
G
G
OGR1 Probe
Abasic DNA
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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DNA
Tm(-) / ˚C
Tm(+) / ˚C
ΔTm / ˚C
0.0
Abasic DNAa 71.3 ± 0.63 71.3 ± 0.29
OGR1 Probeb 82.2 ± 0.77 82.5 ± 0.25
0.3
a
b
[DNA] = 1 μM, [41] = 2 μM (1% DMSO), NEBuffer. [DNA] = 1 μM,
[41] = 2 μM (10% DMSO), PBS buffer and betaine (10 mM).48
The selectivity of inhibitor SU0268 (41) was investigated by testing it against other
repair enzymes that process either 8-oxoguanine nucleotides, bind nucleotides or DNA, or
employ base excision mechanisms. MTH1 (human MutT Homolog 1),49 dUTPase
(Deoxyuridine 5’-Triphosphate Nucleotidohydrolase),50 NUDT16 (Nucleoside Diphosphate
linked moiety X-type motif 16),51 hABH2 (Human AlkB Homolog 2),52 hABH3 (Human AlkB
Homolog 3),53 and SMUG1 (Single-strand Selective Monofunctional Uracil DNA
Glycosylase)54 were assayed with the compound at 20 µM (Figure 6). The activities against
all enzymes of compound 41 were low or negligible, thus establishing high selectivity of
compound 41 to OGG1.
120
100
80
60
40
20
0
MTH1
DUT
NUDT16
ABH2
ABH3
SMUG1
Figure 6. Selectivity of compound 41, tested by assaying against varied repair enzymes at 20
µM.
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The toxicity of compound 41 was tested in two human cell lines (HEK293T and HeLa)
by the MTT assay (Figure 7). The compound showed little or no cytotoxicity in both cell lines
at concentrations 10 nM to 10 μM, and exhibited moderate apparent toxicity only at the
highest 100 μM concentration (30-50% toxicity). Thus, the compound is not significantly
toxic at concentrations well above its IC50.
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HEK293T (5% DMSO)
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HeLa (5% DMSO)
100
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80
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0
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100 μM
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10 μM
1 μM
100 nM
10 nM
(5% DMSO) (5% DMSO) (1% DMSO) (1% DMSO) (1% DMSO) (1% DMSO) (1% DMSO)
Figure 7. Toxicity of 41 (SU0268) by MTT assay with HEK293T and HeLa cell lines
Next, we examined the membrane permeability of SU0268 (41) using the Caco-2
(clone C2BBel) assay, measuring permeability in both the apical-to-basolateral and
basolateral-to-apical directions (Table 9). Recoveries based on the apical side (A-to-B) and
basolateral side (B-to-A) were 65% and 79%, showing that the permeability of compound 41
is satisfactory.
Table 9. CACO-2 assay with the OGG1 inhibitor 41
Papp (10-6 cm/s)
Direction
Recovery (%)
Efflux Ratio
6.2
R1
14.2
63.5
R2
AVG
12.5
77.4
65
79
10.8
91.3
A-to-B
B-to-A
Finally, we carried out tests of the ability of SU0268 to engage OGG1 and inhibit its
activity in a human cell line. Since the cellular role of the enzyme is to remove 8-oxoG from
DNA, inhibition of the enzyme is expected to measurably increase levels of the damaged
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base in DNA. To evaluate this possibility, we incubated cells for 24 h with or without SU0268
at 0.5 µM, and used LC/MS-MS methods to measure levels of this damaged base in DNA
isolated from the cells. The results (see Figure 8a) confirm that the drug increases levels of
8-oxodG in DNA by 25% after only 24 h relative to the mock-treated controls. The amount of
increase is similar to or greater than that provided by a positive control of oxidatively
damaging conditions. We also employed a fluorescence probe of OGG-1 activity39 to directly
test the ability of SU0268 to inhibit OGG1 activity in HeLa and MCF-7 tumor cell lines, which
have relatively low and high OGG1 activities, respectively. Results of the assay are shown in
Fig. 8b; the data confirm the inhibition of OGG1 activity at 0.5 μM of the compound,
amounting to 64% and 95% inhibition of signal in the two lysates as measured at 20 h. Thus,
the collective data provide evidence that SU0268 (41) can be active at the intended cellular
target, both in lysates and in living cells.
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Figure 8. Inhibition of cellular OGG1 activity by 41 (SU0268). (a) Incubation with 0.5 μM
SU0268 increases the number of oxo8dG lesions in HeLa cell DNA. (b) SU0268 (0.5 μM)
inhibits OGG1 activity as measured by fluorescence probe (pOGR1)39 in HeLa and MCF-7 cell
lysates.
A previous report described the discovery of hydrazine and hydrazone inhibitors of
OGG1 using a DNA strand cleavage assay.35 We independently confirmed that two of the
most potent of those compounds are indeed inhibitors of the enzyme in vitro (SI Figure S1)
using our base excision (OGR1) assay. The results show that the new compound SU0268 (41)
(IC50 = 0.059 µM) is more potent than those prior compounds as measured by the initial
rates method. Notably, the prior compounds exhibit apparently delayed kinetics of
inhibition, which suggests the possibility that the enzyme may first excise the 8-OG base
before the compounds can be active (see SI Figure S1). In contrast, as measured by the
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OGR1 assay, the current inhibitor SU0268 binds directly to OGG1 enzyme even in the
absence of DNA, and directly inhibits base excision, the first step of repair of this lesion by
this enzyme.
In summary, we have developed highly potent and selective OGG1 inhibitor SU0268
which has an acyl tetrahydroquinoline sulfonamide skeleton. The structure-activity
relationships of the compounds were outlined by synthesizing a broad range of analogs.
Synthesis of the optimized OGG1 inhibitor is quite straightforward from commercially
available stating materials. The compound shows good membrane permeability and no
cytotoxicity in HEK293T and HeLa cells at active concentrations, and demonstrates activity in
inhibiting the enzyme in human cell lines. Further studies are being directed to applications
of this inhibitor in varied cellular and animal models of multiple diseases.
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Acknowledgement. We thank the U.S. National Institutes of Health (CA217809, GM067201,
GM110050) for support, and the Ajinomoto Corporation for providing postdoctoral support
to Y.T. A.A.B was supported by a postdoctoral fellowship from the Human Frontiers Science
Program. We thank the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford
University Mass Spectrometry for assistance with mass spectrometry measurements. We
thank Kirk Wright (NIBR) for providing SPR resources. We also acknowledge Ann Schlesinger
at NIBR and Stanford ChEM-H for supporting the research collaboration.
References
(1) Helleday, T.; Petermann, E.; Lundin, C.; Hodgson, Ben; Sharma, R. A. Nat. Rev. Cancer
2008, 8, 193-204.
(2) Phillips, D. H.; Arlt, V. M. Molecular, Clinical and Environmental Toxicology, Springer, Basel,
2009, 87-110.
(3) Dietlei, F.; Thelen, L.; Reinhardt, H. C. Cell Press 2014, 326-339.
(4) Kelley, M. R.; Logsdon, D.; Fishel, M. L. Future Oncol. 2014, 10, 1215-1237.
(5) Gavande, N. S.; VanderVere-Carozza, P. S.; Hinshaw, H. D.; Jalal, S. I.; Sears, C. R.;
Pawelczak, K. S.; Turchi, J. J. Pharmacology & Therapeutics 2016, 160, 65-83.
(6) Chatterjee, N.; Walker, G. C. Environ. Mol. Mutagen. 2017, 58, 235-263.
(7) Kasai, Hiroshi; Nishimura, S. Nucleic Acids Res. 1984, 12, 2137-2145.
(8) Dizdaroglu, M. Biochem J. 1986, 238, 247-254.
(9) Kasai, H.; Nishimura, S. Environ. Health Perspect. 1986, 67, 111-116.
ACS Paragon Plus Environment

Page 25 of 27
Journal of the American Chemical Society
1
2
3
4
5
(10) Kasai, H; Crain, P. F.; Kuchino, Y.; Nishimura, S.; Ootsuyama, A.; Tanooka, H.
Carcinogenesis 1986, 11, 1849-1851.
6
7
8
(11) Kasai, H.; Nishimura, S.; Kurokawa, Y.; Hayashi, Y. Carcinogenesis 1987, 8, 1959-1961.
(12) Ames, B. N.; Gold, L. S. Mutation Research 1991, 250, 3-16.
9
(13) Abu-Shakra, A.; Gold, B. Nucleic Acids Res. 2011, 39, 6789-6801.
(17) Nakabeppu, Y. Int. J. Mol. Sci. 2014, 15, 12543-12557.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(18) Shibutani, S.; Takeshita, M.; Grollman, A. P. Nature 1991, 349, 431-434.
(19) Anderson, M. W.; Reynolds, S. H.; You M.; Maronpot, R. M. Environ. Health Pespect. 1992,
98, 13-24.
(20) Kozmin, S. G.; Sedletska, Y.; Reynaud-Angelin, A.; Gasparutto, D.; Sage, E. Nucleic Acids
Res. 2009, 37, 1767-1777.
(21) Lu, R.; Nash, H. M.; Verdine, G. L. Curr. Biol. 1997, 7, 397-407.
(22) Nakabeppu, Y. Prog. Nucleic Acid Res. Mol. Biol. 2001, 68, 75-94.
(23) Bauer, N. C.; Corbett, A. H.; Doetsch, P. W. Nucleic Acids Res. 2015, 43, 10083-10101.
(24) Zhang, Y.; Rohde, Larry, H.; Wu, H. Current Genomics 2009, 10, 250-258.
(25) Klungland, A.; Rosewell, I.; Hollenbach, S.; Larsen, E.; Daly, G.; Epe, B.; Seeberg, E.;
Lindahl, T.; Barnes, D. E. Proc. Natl. Acad. Sci. USA 1999, 96, 13300-13305.
(26) Osterod, M.; Hollenbach, S.; Hengstler, J. G.; Barnes, D. E.; Lindahl, T.; Epe, B.
Carcinogenesis 2001, 22, 1459-1463.
(27) Mahjabeen, I.; Ali, K.; Zhou X.; Kayani, M. A. Tumor Biol. 2014, 35, 5971-5983.
(28) Ali, K.; Mahjabeen, I.; Sabir, M.; Mehmood, H.; Kayani, M. A. Dis. Markers, 2015, 690878.
(29) Sugimura, H.; Kohno, T.; Wakai, K.; Nagura, K.; Genka, K.; Igarashi, H.; Morris, B. J.; Baba,
S.; Ohno, Y.; Gao, C.; Li, Z.; Wang, J.; Takezaki, T.; Tajima, K.; Varga, T.; Sawaguchi, T.; Lum, J.
K.; Martinson, J. J.; Tsugane, S.; Iwamasa, T.; Shinmura, K.; Yokota, J. Cancer Epidemiol.
Biomarkers Prev. 1999, 8, 669-674.
(30) Zhong, D.; Li, G.; Long, J.; Wu, J.; Hu, Y. Sci. Rep. 2012, 2, 548.
(31) Das, S.; Nath, S.; Bhowmik, A.; Ghosh, S. K.; Choudhury, Y. Springerplus. 2016, 29, 227
(32) Bacsi, A.; Aguilera-Aguirre, L.; Szczesny, B.; Radak, Z.; Hazra, T. K.; Sur, S.; Ba, X.;
Boldogh, I. DNA Pepair 2013, 12, 18-26.
(33) Aguilera-Aguirre, L.; Bacsi, A.; Radak, Z.; Hazra, T. K.; Mitra, S.; Sur, S.; Brasier, A. R.; Ba,
X.; Boldogh, I. J. Immunol. 2014, 193, 4643-4653.
(34) Chen, S. Y.; Wan, L.; Huang, C. M.; Huang, Y. C.; Sheu, J. J. C.; Lin, Y. J.; Liu, S. P.; Lan,
Y. C.; Lai, C. H.; Lin, C. W.; Tsai, C. H.; Tsai, F. J. Rheumatol. Int. 2012, 32, 1165-1169.
(35) Donley, N.; Jaruga, P.; Coskun, E.; Dizdaroglu, M.; McCullough, A.; K.; Lloyd, R. S. ACS
Chem. Biol. 2015, 10, 2334-2343.
(36) Testa, B.; Mayer, J. M. Hydrolysis in Drug and Prodrug Metabolism Chemistry, biochemistry
and enzymology, Wiley-VCH, Weinheim, 2003, Chapter 4, 148-162.
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Page 26 of 27
1
2
3
4
5
(37) Sugiyama, H.; Kawabata, H.; Fujiwara, T.; Dannoue, Y.; Saito, I. J. Am. Chem. Soc. 1990,
112, 5252-5257.
6
7
8
9
(38) Chowdhury, G.; Guengerich, F. P. Chem. Res. Toxicol. 2009, 22, 1310-1319.
(39) Edwards, S. K.; Ono, T.; Wang, S.; Jiang, W.; Franzini, R. M.; Jung, J. W.; Chan, K. M.;
Kool, E. T. ChemBioChem 2015, 16, 1637-1646.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(40) Beharry, A. A.; Lacoste, S.; O’Connor, T. R.; Kool, E. T. J. Am. Chem. Soc. 2016, 138,
3647-3650.
(41) Sloss, M. K.; Mckenna, J.; Yoon, W. H.; Norris, S.; Robinson, D.; Parnes, J. Shevlin, G.
WO2009089042.
(42) Rashad, A. A.; Jones, A. J.; Avery, V. M.; Baell, J.; Keller, P. A. ACS. Med. Chem. Lett.
2014, 5, 496-500.
(43) Katagiri, K.; Sakai, T.; Hishikawa, M.; Masu, H.; Tominaga, M.; Yamaguchi, K.; Azumaya, I.
Cryst. Growth Des. 2014, 14, 199-206.
(44) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Del. Rev. 1997, 23, 3-
25.
(45) Cox, P. B.; Gregg, R. J.; Vasudevan, A. Bioorg. Med. Chem. 2012, 20, 4564-4573.
(46) Mohan, C.; Long, K. D.; Mutneja, M. An Introduction to Inhibitors and Their Biological
Applications, EMD MILLIPORE, Billerica, 2013, 3-22.
(47) Yoshimoto, K.; Nishizawa, S.; Minagawa, M.; Teramae, N. J. Am. Chem. Soc. 2003, 125,
8982-8983.
(48) Rees, W. A.; Yager, T. D.; Korte, J.; von Hoppel, P. H. Biochemistry 1993, 32, 137-144.
(49) Carter, M.; Jemth, A.-S.; Hagenkort, A.; Page, B. D. G.; Gustafsson, R.; Griese, J. J.; Gad,
H.; Valerie, N. C.K.; Desroses, M.; Boström, J.; Berglund, U. W.; Helleday, T.; Stenmark, P. Nat.
Commun. 2015, 6, 7871.
(50) Cedergren-Zeppezauer, E. S.; Larsson, G.; Nyman, P. O.; Dauter, Z.; Wilson, K. S. Nature
1992, 355, 740-743.
(51) Iyama, T.; Abolhassani, N.; Tsuchimoto, D.; Nonaka, M.; Nakabeppu, Y. Nucleic Acids Res.
2010, 38, 4834-4843.
(52) Yang, C.-G.; Yi, C.; Duguid, E. M.; Sullivan, C. T.; Jian, X.; Rice, P. A.; He, C. Nature 2008,
ꢀ
452, 961-965.
(53) Sundheim, O.; Vågbø, C. B.; Bjørås, M.; Sousa, M. ML.; Talstad, V.; Aas, P. A.; Drabløs, F.;
Krokan, H. E.; Tainer, J. A.; Slupphaug, G. EMBO J. 2006, 25, 3389-3397.
(54) Zhang, Z.; Shen, J.; Yang, Y.; Li, J.; Cao, W.; Xie, W. ACS Chem. Biol. 2016, 11, 1729-1736.
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O
H2N
O
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42
S N
N
H
O
O
SU0268
OGG1
O
N
H
N
NH
NH2
O
N
OH
O
H
O
O
O
N
NH2
NH
N
O
O
N
H
O
O
8-oG
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