G-quadruplex DNA as a molecular target for induced synthetic lethality in cancer cells

Article abstract of DOI:10.1021/ja404868t

Synthetic lethality is a genetic concept in which cell death is induced by the combination of mutations in two sensitive genes, while mutation of either gene alone is not sufficient to affect cell survival. Synthetic lethality can also be achieved "chemically" by combination of drug-like molecules targeting distinct but cooperative pathways. Previously, we reported that the small molecule pyridostatin (PDS) stabilizes G-quadruplexes (G4s) in cells and elicits a DNA damage response by causing the formation of DNA double strand breaks (DSB). Cell death mediated by ligand-induced G4 stabilization can be potentiated in cells deficient in DNA damage repair genes. Here, we demonstrate that PDS acts synergistically both with NU7441, an inhibitor of the DNA-PK kinase crucial for nonhomologous end joining repair of DNA DSBs, and BRCA2-deficient cells that are genetically impaired in homologous recombination-mediated DSB repair. G4 targeting ligands have potential as cancer therapeutic agents, acting synergistically with inhibition or mutation of the DNA damage repair machinery.

Full text of DOI:10.1021/ja404868t

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Communication
G-Quadruplex DNA as a Molecular Target for
Induced Synthetic Lethality in Cancer Cells
Keith Ian Edward McLuckie, Marco Di Antonio, Heather Zecchini, Jian Xian, Carlos Caldas,
Ben-Fillippo Krippendorff, David Tannahill, Christopher Lowe, and Shankar Balasubramanian
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja404868t • Publication Date (Web): 19 Jun 2013
Downloaded from http://pubs.acs.org on June 23, 2013
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G-Quadruplex DNA as a Molecular Target for Induced Syn-
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†
†,‡
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†,
⊥
†,
⊥
Keith I. E. McLuckie, Marco Di Antonio, Heather Zecchini, Jian Xian, Carlos Caldas, Ben-
†
†
‡
*,†,‡,§
Fillippo Krippendorff, David Tannahill, Christopher Lowe and Shankar Balasubramanian
†
Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.
‡Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Department of Oncology, University of Cambridge, Hills Road, Cambridge, CB2 0XZ.
School of Clinical Medicine, University of Cambridge, Cambridge, CB2 0SP, UK.
⊥
§
Supporting Information Placeholder
employed the small molecule G4-stabilizing ligand pyri-
dostatin (PDS) to target G4s in the DNA of human can-
ABSTRACT: Synthetic lethality is a genetic concept in
which cell death is induced by the combination of muta-
tions in two sensitive genes, while mutation of either
gene alone is not sufficient to affect cell survival. Syn-
thetic lethality can be also achieved “chemically” by
combination of drug-like molecules targeting distinct but
cooperative pathways. Previously, we reported that the
small molecule pyridostatin (PDS) stabilizes G-
quadruplexes in cells and elicits a DNA damage re-
sponse (DDR) by causing the formation of DNA double
strand breaks (DSB). We hypothesize that cell death me-
diated by ligand-induced G-quadruplex stabilization
could be potentiated in cells deficient in DNA damage
repair genes. Here, we demonstrated that PDS acts syn-
ergistically both with NU7441, an inhibitor of the DNA-
PK kinase crucial for non-homologous end joining
7
cer cells and explicitly mapped the functional response
8
to sites in the genome by deep-sequencing . Further-
more, we have visualized telomeric and non-telomeric
G4s in human cancer cells using selective antibodies and
demonstrated that PDS can trap DNA G4s in the cell
9
nucleus. We have also mapped G4 structures in ge-
1
0
nomic DNA derived from human cancer cells. It is
apparent that G4 structures exist and can serve as targets
for small molecules in cells.
Treatment of cells with some G4-stabilizing small
molecules can lead to the formation of DNA double
8,11
strand breaks (DSBs). A single DSB is sufficient to
12
kill a cell and these lesions can be generally repaired
by distinct repair systems: homologous recombination
13
(NHEJ) repair of DNA DSBs, and BRCA2-deficient
(
HR) or non-homologous end joining (NHEJ). Defi-
cells that are genetically impaired in homologous re-
combination-mediated DSB repair (HR). G-quadruplex
targeting ligands have potential as cancer therapeutic
agents, acting synergistically with inhibition or mutation
of the DNA damage repair machinery.
ciencies or chemical inhibition of these pathways can
usefully sensitize cells towards DNA damage induced by
radiation exposure or topoisomerase II inhibition (e.g.
1
4
etoposide).
Chemically-induced synthetic lethality is an attractive
therapeutic strategy that exploits a genetically deficient
disease state or its chemical inhibition combined with a
complementary drug treatment, to enhance the overall
G-quadruplexes (G4s) are structures that form from
guanine-rich nucleic acid sequences via the formation of
guanine tetrads stabilized by a central, coordinating cati-
1
5
inhibitory effect. For example, breast cancer cells car-
rying mutations in the gene BRCA2 are deficient in the
HR repair pathway and are consequently particularly
sensitive to chemical inhibitors of alternative DNA re-
1
on. These stable non-canonical structures have been
hypothesized to exist in nature and there is evidence
linking G4s to biological processes, including correla-
1
6
pair pathways. Similarly, we anticipated that DSB
formation induced by PDS treatment would be more
pronounced in cells genetically deficient in, or chemical-
2
tions from chemical biological studies. Computational
analyses using predictive algorithms have suggested
8
3
ly inhibited in, repair pathways. Thus, while DNA G4s
sites at which G4s may form in genomes, highlighting
18
4
are emerging as targets for cancer in their own right,
we foresee these structures as potential targets for a
chemically-induced synthetic lethality strategy.
an over-representation at certain regulatory regions. The
existence of G4s in living systems was first shown at the
5
telomeres of ciliates, where the formation of such DNA
structure was linked to a cell-cycle dependent phosphor-
ylation of telomere binding proteins. More recently, we
Herein, we investigate the potential of G4 ligands for
the development of chemically-induced synthetic lethali-
6
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ty. Specifically, the anti-proliferative effects of a PDS
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analogue exhibit synergy in either cells deficient in HR
-/-
repair (BRCA2 ) or in a system where NHEJ has been
chemically inhibited.
Previously, we identified three G4 ligands from the
pyridostatin family, PDS, PDSI and PDSK (Figure 1A,
Table S1), as strong G4 stabilizers and potent inhibitors
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of cell proliferation. Using an impedance-based con-
tinuous cell-monitoring approach (see Suppl. Materials
and Methods) we examined, in real time, the growth
inhibition of human HT1080 fibrosarcoma cells, previ-
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ously shown to be sensitive to the pyridostatin family ,
in the presence of each ligand for up to 72 h. Of the
three ligands, PDSI was found to exhibit the greatest
Figure 1. (A) Structures of pyridostatins; PDS, PDSI and
PDSK. (B) Growth inhibition by PDS molecules in
HT1080 cells (GI50 in ꢀM). **P=0.006, 1 way ANOVA.
(C) Structure of DNA-PKcs inhibitor NU7441.
growth inhibition (GI : PDSI 0.43 ꢀM, PDS 0.89 ꢀM,
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PDSK 3.15 ꢀM, Figure 1B) and was therefore used in
further studies.
Since PDSI and NU7441 have distinct targets (i.e.
DNA G4 structure for PDSI and the DNA-PK protein
for NU7441), we used the Bliss Independence model for
calculating additivity, Z = X + Y(1 – X), where Z repre-
sents the expected combined output, X the cell survival
of PDSI alone and Y the cell survival of NU7441
We next investigated DNA damage induced by PDSI
in the presence of a complementary inhibitor NU7441
(2-N-morpholino-8-dibenzothiophenyl-chromen-4-one)
that effectively targets the catalytic subunit of a DNA-
dependent protein kinase (DNA-PKcs), which is re-
2
0
quired for the NHEJ mode of DNA repair (Figure 1C).
22
alone. The calculated additivity is arithmetically sub-
We confirmed that PDSI induces a DNA damage re-
sponse in HT1080 cells, as judged by an increase in
tracted from the experimentally measured cell survival
and plotted as percentage synergy on a resultant 3D re-
sponse surface using the program SynergySurface (See
Suppl. Methods, Figure 2A); a positive peak indicates a
synergistic interaction and a negative peak indicates an-
tagonism. The combination of PDSI and NU7441 (Fig-
ure 2A) showed a very high level of synergy consistent
with chemically induced synthetic lethality. The opti-
mum concentrations for synergy were 2.5 ꢀM PDSI and
21
phosphorylated-53BP1 and a dose dependent increase
in apoptotic cell death (Figures S1-2). Co-treatment of
PDSI with NU7441 resulted in a greater number of
53BP1 foci, suggesting that DNA damage induced by
PDSI is exacerbated when a DNA repair inhibitor is pre-
sent (Figure S1B).
To investigate this further, we systematically exam-
ined the combination effect on cell survival of HT1080
cells treated with PDSI and NU7441. Typically, cells
were cultured in a matrix of compound concentrations
ranging from 0 to 10 times the determined GI50 values
0
.3 ꢀM NU7441 resulting in a 45% synergy. This com-
pares favorably with other synthetic lethal studies such
as the VE-821 aminopyrazine ATR inhibitor and cispla-
2
3
tin (0.3-10 ꢀM range with ~45% synergy). VE-821
also acts synergistically with other DNA damaging
agents including carboplatin, etoposide and camptothe-
(Table S1). Cell survival was measured at 72 h by an
end-point assay (Cell Titer Glo) that uses cellular ATP as
a substrate for a luciferase, where luminescence readings
are proportional to the number of viable cells. Using this
system we can investigate if the combination of the two
drugs acts synergistically; where synergism is defined as
providing a greater response than the simple addition of
single treatments. This end-point assay measures cell
viability/survival and not cell growth; the values cannot
2
3a
cin at similar dose ranges to this study. We confirmed
that a DDR was induced at 2 ꢀM PDSI and 0.3 ꢀM
NU7441 by measuring increases in levels of nuclear
53BP1, indicative of DSB formation, and of nuclear
RAD51, indicating HR induction, by laser-scanning cy-
tometry (Figure 2B/C, Table S3) after 24h treatment.
Treatment with PDSI or NU7441 alone increased levels
of nuclear 53BP1 by up to 1.4-fold, whereas synergistic
combinations led to a greater increase of 2.1-fold (Fig-
ure 2B, Table S3). Likewise, combination of PDSI and
NU7441 resulted in a 30-fold increase of nuclear
RAD51 levels compared to single treatment of NU7441
and a 1.5-fold increase relative to PDSI alone, which is
greater than a solely additive response (Figure 2C, Table
S3). NU7441 does not promote HR-mediated repair in
the absence of PDSI, in agreement with a study that re-
be directly compared to the GI presented above.
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vealed potential inhibition of HR by NU7441.
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Figure 2. Synergistic growth inhibition of cancer cells using PDSI and NU7441. (A) 3D response surface for the combination
of PDSI with NU7441 in HT1080 cells. (B) Nuclear 53BP1 and (C) RAD51 in HT1080 cells treated with PDSI (2 ꢀM) and
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00 nM NU7441 for 24 h. Error bars indicate SEM. (D) Growth inhibition by PDSI and NU7441 in isogenic HCT116 WT or
-/-
BRCA2 cancer cells. Binary treatment was 1 ꢀM NU7441 with a range of PDSI concentrations (Binary GI ). All GI data
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quoted in ꢀM. Error bars indicate SEM; significance compared to PDSI (unpaired t-test, one tail, *P<0.05, **P<0.01). (E/F)
-/-
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D response surfaces for the combination of PDSI with NU7441 in HCT116 WT cells or BRCA2 cells. (G) Nuclear 53BP1
-/-
and (H) RAD51 in HCT116 WT and BRCA2 cells treated with PDSI (2 ꢀM) and 150 nM NU7441 for 24 h. Error bars in-
dicate SEM. Dashed lines in C/H denote level of additive response. The details of statistical significance between pair wise
combinations, for panels B,C,G and H is illustrated in Table S3. Data in Panels A, E&F were measured using an end point
luminescence assay. Data in panel D were measured by continuous cell monitoring.
To complement the chemically-induced synthetic le-
thality described above, we evaluated the effect of a
G4 ligand in cells genetically lacking a vital compo-
nent of the DNA damage repair machinery. BRCA2 is
a key gene implicated in many cancers and is clinically
treated using synthetic lethal rationales with PARP
ments also gave a synergistically greater cell death
than WT cells as indicated by I-values (I<1 for syner-
gy, I=1 for additivity and I>1 for antagonism, Figure
2
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-/-
S3). This was seen in both BRCA2 and WT cells
(Binary GI : 0.28 ꢀM vs. 1.7 ꢀM, respectively). Im-
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-/-
portantly, the growth of BRCA2 cells in the presence
of combinations of PDSI and NU7441 is severely
compromised (Figure S4).
16
inhibitors. We reasoned that BRCA2-deficient cells
would be more sensitive to G4 ligands than wild type
(
WT) cells. PDSI and/or NU7441 were therefore ap-
Using the 3D growth response analysis at 72 h as de-
scribed above, we further explored this synergy using a
matrix of concentrations (Figure 2E-F). These data
plied to a pair of isogenic HCT116 colon carcinoma
cells carrying either the wild type BRCA2 gene, or
where both copies have been removed by homologous
recombination (BRCA2 ). In BRCA2 cells, PDSI
induced a greater inhibition of cell growth compared to
WT (GI : 1.8 ꢀM vs. 3.8 ꢀM, Figure 2D, Table S2)
and NU7441 had a similar response (GI : 2.2 ꢀM vs.
4
repair are more sensitive to PDSI. Combination treat-
+
/+
-/-
indicate that the absence of HR in the BRCA2 cells
-/- 25
-/-
potentiates the synergistic effects of PDSI and
NU7441. We further confirmed a substantially larger
-/-
5
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increase in nuclear RAD51 levels in BRCA2 cells
following treatment with PDSI or in combination with
NU7441 compared to untreated and WT cells (Figure
2H, Suppl. Table S3).
50
.8 ꢀM); thus confirming that cells deficient in DNA
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(
6) Paeschke, K.; Juranek, S.; Simonsson, T.; Hempel, A.; Rhodes,
In conclusion, we have demonstrated that a G4 lig-
and can induce synthetic lethality in cancer cells either
by exploiting the inherent HR DSB repair deficiency in
BRCA2 cells, or by accompanying a second inhibitor
that targets a similarly vital DSB repair mechanism
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D.; Lipps, H.J.; Nat. Struct. Mol. Biol., 2008, 15, 598.
(
J.-F.; Balasubramanian, S.; J. Am. Chem. Soc., 2008, 130, 15758.
(8) Rodriguez, R.; Miller, K.M.; Forment, J.V.; Bradshaw, C.R.;
Nikan, M.; Britton, S.; Oelschlaegel, T.; Xhemalce, B.; Balasubra-
manian, S.; Jackson, S.P.; Nat. Chem. Biol., 2012, 8, 301.
(9) Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S.;
Nat. Chem., 2013, 5, 182
7) Rodriguez, R.; Müller, S.; Yeoman, J.A.; Trentesaux, C.; Riou
-/-
(NHEJ). G4 ligands have considerable potential as
efficacious anti-proliferative agents when used as part
of a rational synergistic strategy.
(
10) Lam, E.Y.N.; Beraldi, D.; Tannahill, D.; Balasubramanian, S.,
Nat. Com., 2013, 4, 1796.
ASSOCIATED CONTENT
(11) Douarre, C.; Mergui, X.; Sidibe, A.; Gomez, D.; Alberti, P.;
Mailliet, P.; Trentesaux, C.; Riou, J.-F.; Nucleic Acids Res., 2013,
41, 3588.
(12) (a) Ward, J.F.; Prog. Nucleic Acid Res. Mol. Biol., 1988, 35,
95. (b) Iliakis, G.; Bioessays, 1991, 13, 641.
0
1
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3
4
5
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Supporting Information. All experimental details and
supplementary tables and figures cited above. This mate-
rial is available free of charge via the Internet at
http://pubs.acs.org.
(
13) (a) Kanaar, R.; Hoeijmakers, J.H.; van Ghent, D.C.; Trends
Cell Biol., 1998, 8, 483. (b) Rothkamm, K.; Kruger, I.; Thompson,
L.H.; Lobrich, M.; Mol. Cell. Biol., 2003, 23, 5706.
(14) Jeggo, P.A.; Caldecott, K.; Pidsley, S.; Banks, G.R.; Cancer
Res., 1989, 49, 7057.
AUTHOR INFORMATION
Corresponding Author
(
(
15) Kaelin, W.G., Jr.; Nat. Rev. Cancer, 2005, 5, 689.
16) Farmer, H.; McCabe, N.; Lord, C.J.; Tutt, A.N.J.; Johnson,
*
sb10031@cam.ac.uk
D.A.; Richardson, T.B.; Santarosa, M.; Dillom, K.J.; Hickson, I.;
Knights, C.; Martin, N.M.B.; Jackson, S.P.; Smith, G.C.M.; Ash-
worth, A.; Nature, 2005, 434, 917.
(17) Aggarwal, M.; Sommers, J.A.; Shoemaker, R.H.; Brosh, R.M.
Jr.; PNAS, 2011, 108, 1525.
Funding Sources
No competing financial interests have been declared.
ACKNOWLEDGMENT
(
18) Balasubramanian, S.; Hurley, L.H.; Neidle, S.; Nat. Rev. Drug
We thank the BBRSC for project funding and Cancer
Research UK for programme funding and core support.
Discov., 2011, 10, 261.
(19) Müller, S.; Sanders, D.A.; Di Antonio, M.; Matsis, S.; Riou, J.-
F.; Rodriguez, R.; Balasubramanian, S.; Org. Biomol. Chem., 2012,
10, 6537.
REFERENCES
(
20) Yhao, Y.; Thomas, H.D.; Batey, M.A.; Cowell, I.G.; Richard-
(
(
1) Davis, J.; Angew. Chem. Int. Ed., 2004, 43, 668.
2) (a) Bochman, M. L.; Paeschke, K.; Zakian, V. A.; Nat. Rev.
son, C.J.; Griffin, R.J.; Calvert, H.; Newell, D.R.; Smith, G.C.M.;
Curtin, N.J.; Cancer Res., 2006, 66, 5354.
Genet.; 2012, 13, 770. (b) Di Antonio, M.; Rodriguez, R.; Bal-
asubramanian, S.; Methods 2012, 57, 84-92; (c) Bugaut, A.;
Balsubramanian, S.; Nucleic Acids Res., 2012, 40, 4727. (d) Sid-
diqui-Jain, A.; Grand, C.L.; Bearss, D.J.; Hurley, L.H.; Proc. Natl.
Acad. Sci. USA, 2002, 99, 11593. (e) Rankin, S.; Reszka, A.P.;
Huppert, J.; Zloh, M.; Parkinson, G.N.; Todd, A.K.; Ladame, S.;
Balasubramanian, S.; Neidle, S.; J. Am. Chem. Soc., 2005, 127,
10584. (f) Cogoi, S.; Xodo, L.E.; Nucleic Acids Res., 2006, 34,
(
21) Schultz, L.B.; Chehab, N.H.; Malikzay, A.; Halazonetis, T.D.;
J. Cell. Biol., 2000, 151, 1381.
(22) (a) Bliss, C.I.; Ann. Appl. Biol., 1939, 26, 585. (b) Jonker,
D.M.; Visser, S.A.G.; van der Graaf, P.H.; Voskuyl, R.A.; Danhof,
M.; Pharmacol. Therapeut., 2005, 106, 1.
(
23) (a) Reaper, P.M.; Griffiths, M.R.; Long, J.M.; Charrier, J.-D.;
MacCormick, S.; Charlton, P.A.; Golec, J.M.C.; Pollard, J.R.; Nat.
Chem. Biol., 2011, 7, 428. (b) Charrier, J.-D.; Durrant, S.J.; Golce,
J.M.C.; Kay, D.P.; Knegtel, R.M.A.; MacCormick, S.; Mortimore,
M.; O’Donnell, M.E.; Pinder, J.L.; Reaper, P.M.; Rutherford, A.P.;
Wang, P.S.H.; Young, S.C.; Pollard, J.R.; J. Med. Chem., 2011, 54,
2
536. (g) Kumari,S.; , Bugaut, A.; Huppert, J.L.; Balasubramanian,
S.; Nat. Chem. Biol., 2007, 3, 218.
3) (a) Huppert, J.L.; Balasubramanian, S.; Nucleic Acids Res.,
005, 33, 2908. (b) Todd, A.K.; Johnston, M.; Neidle, S.; Nucleic
(
2
2
320.
Acids Res., 2005, 33, 2901.
(4) Huppert, J.L.; Balasubramanian, S.; Nucleic Acids Res., 2007,
(
24) Tavecchio, M.; Munck, J.M.; Cano, C.; Newell, D.R.; Curtin,
N.J.; Cancer Chemother. Pharmacol., 2012, 69, 155.
(25) Caldas and co-workers, 2013, Oncogene, submitted.
3
5, 406.
(
5) Schaffitzel, C.; Berger, I.; Postberg, J.; Hanes, J.; Lipps, H.J.;
(
26) (a) Amrein, L.; Loignon, M.; Goulet, A.-C.; Dunn, M.; Jean-
Claude, B.; Aloyz, R.; Panasci, L.; J. Pharmacol. Exp Ther., 2007,
21, 848. (b) Berenbaum, M.C.; Cancer Res., 1992, 52, 4558.
Pluckthun, A.; (2001). Proc. Natl. Acad. Sci. USA, 2001, 98, 8572.
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R1
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Page 6 of 7
A
1
2
3
4
5
O
PDS: R , R , R =
NH2
1
2
3
O
N
O
N
N
O
PDSI: R =Cl, R , R =
NH2
1
2
3
R
NH
HN
R3
6
2
O
7
8
9
N
PDSK: R , R , R =
1
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
B
C
4
*
*
3
2
1
0
S
O
O
O
N
*
*
*
*
NU7441
PDSI
PDS ACS P
P
a
D
ra
S
gon Plus Environment
K
DNA-PKcs Inhibitor

Page 7 of 7
Journal of the American Chemical Society
A
B
C
HT1080 Nuclear 53BP1
HT1080 Nuclear RAD51
40
HT1080
1 3
2
0
3
20
4 1
0
5
6 10
7
0
5
2
.5
5
2.5
1
.25
1.25
8
9
0
.625
.3125
.15625
0.625
0.3125
0.15625
.078125
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
0 NU7441 [uM]
1
PDSI [uM]
2
3
4
5
D
E
F
6
2
1
1
0
5
0
5
0
5
2
0
5
BRCA2-/-
HCT116 WT
1
6
7
8
9
0
1
2
3
4
5
6
7
4
2
0
10
5
*
0
−
−
5
*
*
1
0
10
5
5
5
2.5
5
2.5
2.5
2.5
1.25
0.625
.3125
0.15625
1.25
1
.25
1.25
0.625
0.3125
0.15625
0.078125
0
.625
0.3125
.15625
0.625
0.3125
.15625
.078125
0
0
0
0
0
0
0
0
NU7441 [uM]
NU7441 [uM]
PDSI [uM]
HCT116 WT BRCA2-/-
PDSI [uM]
G
H
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
ACS Paragon Plus Environment