Irreversible inhibition of DNA polymerase β by small-molecule mimics of a DNA lesion

Article abstract of DOI:10.1021/ja411733s

Abasic sites are ubiquitous DNA lesions that are mutagenic and cytotoxic but are removed by the base excision repair pathway. DNA polymerase β carries out two of the four steps during base excision repair, including a lyase reaction that removes the abasic site from DNA following incision of its 5′-phosphate. DNA polymerase β is overexpressed in cancer cells and is a potential anticancer target. Recently, DNA oxidized abasic sites that are produced by potent antitumor agents were shown to inactivate DNA polymerase β. A library of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to irreversibly inhibit DNA polymerase β. One candidate (3a) was examined more thoroughly, and modification of its phosphate backbone led to a molecule that irreversibly inactivates DNA polymerase β in solution (IC₅₀ ≈ 21 μM), and inhibits the enzyme's lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates, more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase β that works synergistically with a DNA damaging agent.

Full text of DOI:10.1021/ja411733s

Article
pubs.acs.org/JACS
Irreversible Inhibition of DNA Polymerase β by Small-Molecule
Mimics of a DNA Lesion
Dumitru Arian,† Mohammad Hedayati,‡ Haoming Zhou,‡ Zoe Bilis,‡ Karen Chen,‡
Theodore L. DeWeese,‡,§ and Marc M. Greenberg*,†
†Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
‡Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 401 North
Broadway, Baltimore, Maryland 21231, United States
§Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United
States
S
* Supporting Information
ABSTRACT: Abasic sites are ubiquitous DNA lesions that are
mutagenic and cytotoxic but are removed by the base excision repair
pathway. DNA polymerase β carries out two of the four steps during
base excision repair, including a lyase reaction that removes the abasic
site from DNA following incision of its 5′-phosphate. DNA polymerase
β is overexpressed in cancer cells and is a potential anticancer target.
Recently, DNA oxidized abasic sites that are produced by potent
antitumor agents were shown to inactivate DNA polymerase β. A library
of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to
irreversibly inhibit DNA polymerase β. One candidate (3a) was examined more thoroughly, and modification of its phosphate
backbone led to a molecule that irreversibly inactivates DNA polymerase β in solution (IC50 ≈ 21 μM), and inhibits the enzyme’s
lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates,
more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-
fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase β that works synergistically with
a DNA damaging agent.
alleles of the gene for this enzyme are embryonically lethal, and
knocking down Pol β activity sensitizes cells to DNA damaging
agents.3 Consequently, Pol β has attracted interest as a target
for antitumor therapy. Inhibiting Pol β potentiates the cytotoxic
effects of DNA damaging agents and can be cytotoxic in its own
right. We report on a series of Pol β inhibitors whose design
was inspired by DNA lesions that irreversibly inactivate the
enzyme by targeting its lyase active site.4−7
Pol β is a bifunctional enzyme that contains an 8 kDa lyase
active site separate from its polymerase active site.8−10 The
enzyme excises the 5′-phosphorylated 2-deoxyribose (dRP)
produced upon Ape1 incision of DNA containing an AP site
(Scheme 2). Lys72 is the primary amine responsible for Schiff
base formation, although the enzyme retains some lyase activity
when this amino acid is mutated.11−14 Lys84, which is also
present in the lyase active site is postulated to substitute for
Lys72 in the mutated enzyme, albeit with much lower
efficiency. Following Schiff base formation, dRP elimination
leaves a single nucleotide gap that contains the appropriate end
groups for DNA synthesis (by Pol β) and ligation to complete
repair (Scheme 1). Part of the attraction of Pol β as a potential
INTRODUCTION
■
Base excision repair (BER) is a primary mechanism for
maintaining genome integrity. A large variety of modified
nucleotides resulting from DNA oxidation and alkylation are
removed by glycosylases.1 Some BER glycosylases are bifunc-
tional and cleave DNA at a transiently formed abasic site (AP)
via a lyase process.2 In other instances AP sites are produced as
metastable intermediates. AP sites are also generated via
spontaneous hydrolysis of native and damaged nucleotides.
DNA polymerase β (Pol β) plays an integral role in BER by
excising the remnant of an AP site following 5′-incision by
apurinic endonuclease I (Ape1), and subsequently filling in the
single nucleotide gap (Scheme 1). Pol β’s vitality to genome
integrity is manifested by the observation that cells lacking both
Scheme 1
Received: November 18, 2013
© XXXX American Chemical Society
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Scheme 2
Scheme 4
therapeutic target is that it is overexpressed in a variety of
cancer cells.15−17 In addition, Pol β variants are found in a large
percentage of tumors.18−20 Some of the variants exhibit
reduced activity and may contribute to tumorigenesis by
decreasing genomic stability.
Natural and unnatural products have been tested as
inhibitors of Pol β and the related enzyme, Pol λ, which is
believed to act as a backup for Pol β in BER.21−26 Some of
these molecules are believed to target the lyase domain. The
inhibitors described below were designed to mimic the
interaction between Pol β and a DNA lesion, 2-phosphato-
1,4-dioxobutane (DOB), which is produced by a family of
potent cytotoxic antitumor antibiotics following C5′-hydrogen
atom abstraction.27,28 DOB efficiently inactivates Pol β (and
Pol λ).4−6 Radiolabeling experiments, liquid chromatography,
and mass spectral analyses of protease digests indicate that the
1,4-dicarbonyl inactivates Pol β in two ways (Scheme 3). DOB
no exocyclic amines that require protection. Initially, the
phosphate diester in DNA was replaced by a more lipophilic
methyl phosphonate (1). The molecules contained a carbon
between the ring containing the 1,4-dicarbonyl and the methyl
phosphonate to improve their chemical stability in aqueous
solution by preventing elimination. Finally, structural diversity
was introduced in the form of an oxime linkage at the 3′-
terminus. The oxime group is easy to incorporate and has
proven to be a useful means for introducing structural diversity
(represented by “R”) into chemical libraries of enzyme
inhibitors.29,30
We anticipated unmasking the 1,4-dialdehyde of 1 in the final
step after the library was prepared containing single compounds
in individual wells of microtiter plates (Scheme 5). The choice
of acetal protecting group was important, as it needed to be
cleaved rapidly under mild conditions. We chose to use the
pentenyl group that has been very useful in carbohydrate
synthesis for glycosidic bond formation and is cleaved rapidly
under mild oxidizing conditions.31 The alkoxyamine (7) was
the last common intermediate in the library synthesis and was
apportioned into the wells of the microtiter plates. The methyl
phosphonate coupling to produce 6 was carried out using the
phosphonamidite of the thymidine component (5) and the
primary alcohol of the protected 1,4-dicarbonyl (4). The
coupling yields were higher using this approach due to poor
solubility of the corresponding 5′-hydroxy-3′-phthalimide
substituted thymidine (9). Following removal of the
phthalimide protecting group using hydrazine, chemical
diversity was introduced by reacting 7 separately with one of
232 aldehydes overnight in DMSO and acetic acid. DMSO was
removed from the crude mixture of 8 under vacuum prior to
deprotecting the bis-acetal with N-bromosuccinimide.
Scheme 3
forms a stable lactam following condensation with Lys72 or
Lys84, elimination, and dehydration. The lesion also forms a
stable adduct without undergoing DNA cleavage. pC4-AP that
is produced upon Ape1 incision of C4-AP also contains a 1,4-
dicarbonyl and inactivates Pol β and Pol λ.6,7 We hypothesized
that small, DNA-like molecules containing such a 1,4-
dicarbonyl motif would inactivate Pol β upon binding.
RESULTS AND DISCUSSION
The methyl phosphonamidite (5) was prepared from
previously reported 9 under standard phosphitylation con-
ditions in 60% yield.32 The requisite hydroxymethyl compound
(4) was prepared from 10 using a strategy employed previously
for the synthesis of photolabile DOB precursors (Scheme
6).33,34 The hydroxymethyl group was introduced via an aldol
condensation with formaldehyde that was ultimately trapped as
the silyl ether of the formacetal (11) using a chiral catalyst
derived from proline.35 Substituting Selectfluor for N-
■
Design and Synthesis of Small-Molecule DOB Mimics
As Potential Irreversible Inhibitors of Pol β. A library of
nucleotide inhibitors containing the 1,4-dicarbonyl group that is
present in the DOB and pC4-AP lesions that irreversibly inhibit
DNA polymerase β was conceived (Scheme 4). A thymidine
nucleotide was incorporated to make the molecule DNA-like.
Since Pol β exhibits no selectivity for one nucleotide over
another, thymidine was chosen for synthetic simplicity, as it has
B
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a
Scheme 5
a
Key: a) i. tetrazol, CH3CN, 25 °C; ii. t-BuOOH. b) N2H4, THF, 25 °C. c) RCHO, AcOH, DMSO, 37 °C. d) NBS, CH3CN, −5 °C.
a
Scheme 6
Scheme 7
a
Key: a) i. CH2O, prolinol catalyst, toluene, 25 °C; ii. TBDMSCL. b)
Selectfluor, pent-4-en-1ol, CH3CN, 25 °C. c) TBAF, THF, 0 °C.
bromosuccinimide (which was used in the synthesis of the
DOB precursor) to induce cyclization of 11 by oxidatively
cleaving the 1,3-dithiane resulted in a significant improvement
in yield, albeit as an inseparable mixture of diastereomers of
12.36 Two of the four diastereomers were separable upon
desilylation to 4. Although the stereochemistry of the acetals
was unimportant with respect to the properties of the inhibitor
candidates, working with less complex mixtures facilitated
characterizing subsequent intermediates in the synthetic
sequence. A library containing 232 members was synthesized
from 4 (Scheme 5) and screened for Pol β inhibition.
Screening Inhibitor Candidates Using a Strand
Displacement Assay. Inactivation of Pol β’s lyase activity
by DOB or C4-AP also shuts down the enzyme’s ability to
extend a primer via strand displacement synthesis.5,7 Con-
sequently, we speculated that successful small molecules that
inactivate the lyase activity would also shut down polymerase
activity. This enabled us to use a previously reported
fluorescence assay (Scheme 7) in which a ternary substrate
(13, 50 nM) containing TAMRA at the 3′-terminus of the
displaced strand and quencher (BHQ-2) at the 5′-terminus of
the template strand was subjected to Pol β (10 nM) and dTTP
(100 μM).37,38 Strand displacement synthesis results in
fluorescence by TAMRA, and Pol β inhibition is reflected by
decreased fluorescence relative to control lacking inhibitor.
The 232 candidates38 (50 μM) were screened using this
method. Although the underivatized oxime (50 μM) had no
effect on polymerase activity (data not shown), several
candidates exhibited significant inhibition in the strand
displacement assay. Of these, the molecule derived from 2,6-
dichloro-3-hydroxy-4-methoxy benzaldehyde (1a) was most
effective at reducing fluorescence. Purified 1a significantly
inhibited Pol β in the strand displacement assay at as low as 10
μM (Figure 1A). The reduction product (14) had no effect on
enzyme activity (data not shown), indicating that the 1,4-
dicarbonyl is required for inhibition. Furthermore, the extent of
inhibition was dependent upon the preincubation time (Figure
1B), a property that is consistent with irreversible inactivation.
Direct Examination of Pol β Lyase Inactivation by 1a.
The ability of 1a to inhibit Pol β’s dRPase activity was
examined using 3′-32P-15 in which the oligonucleotide
containing dRP was labeled. The DNA substrate was added
to the reaction following preincubation of 1a with Pol β and
subsequent 100-fold dilution. The lyase reaction was monitored
by gel electrophoresis. A logarithmic plot of activity in the
presence of 1a (up to 30 μM) relative to when no inhibitor is
present versus preincubation time decays linearly at each
concentration over the range tested (Figure 2A).39 In addition,
Pol β activity was not restored following dialysis of the
C
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Figure 2. Irreversible inhibition of Pol β by 1a. (A) Relative Pol β
lyase activity on 32P-15 as a function of [1a] and preincubation time of
inhibitor with enzyme. (B) Normalized lyase activity of Pol β on 32P-
15 upon dialysis following incubation with or without 1a ([1a] (μM):
0, filled square; 50, gray square).
Figure 1. Inhibition of strand displacement synthesis in 13 by 1a. (A)
Dependence on [1a] (μM): 0, filled circle; 1, gray circle; 5, open
circle; 10, open square; 25, filled square; 50, gray square. (B)
Dependence on preincubation time of 1a (10 μM) with Pol β. No
inhibitor, filled circle; preincubation time:42 5, gray square; 20, open
circle; 40, filled square; 60, gray square.
enyzme−inhibitor solution for up to 3 days (Figure 2B).40,41
Both observations are consistent with irreversible inhibition of
Pol β. However, we were unable to characterize the modified
Pol β by mass spectrometry.
Effects of Phosphate Backbone Modification on Pol β
Inactivation. Using 1a as a lead we explored the effect of
modifying the phosphorus backbone on inhibitor activity by
synthesizing the phosphate triester (2a) and phosphate diester
(3a) analogues. These candidates were prepared from 4 and 9
via a manner similar to that for 1a.38 Phosphate triester 2a
exhibited inhibition activity comparable to that of 1a using the
strand displacement assay (Scheme 7).38 This backbone motif
was also useful for establishing the necessity of the 1,4-
dicarbonyl for irreversible inactivation, as monoaldehdye 16
had no effect on Pol β lyase activity.38
In contrast, introducing the negative charge present in DNA
(3a) produced a more potent inhibitor. Direct measurement of
Pol β lyase activity showed that 3a was at least 2-fold more
potent than 1a and exhibited an IC50 of ∼21 1 μM (Figure
3). The IC50 for 1a was 42 5 μM.38 Furthermore, as for the
methyl phosphonate, dialysis of Pol β incubated with 3a
confirmed that inhibition was irreversible, and reduction to the
diol (17) provided additional affirmation that the 1,4-
dialdehyde was required for inactivation.38
Figure 3. Irreversible inhibition of Pol β by 3a. (A) Relative Pol β
lyase activity on 32P-15 as a function of [3a] and preincubation time of
inhibitor with enzyme. (B) IC50 of Pol β inactivation following 30 min
preincubation with 3a.
D
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was synthesized from 3-hydroxymethylfuran (19) and 9
(Scheme 8). Following Pb(OAc)4 oxidation and hydro-
a
Scheme 8
Selectivity and Robustness of Pol β Inactivation by
Small-Molecule DOB Analogues. There are more than one
dozen polymerases in a human cell. Because we do not have
access to each of these, we used the Klenow fragment of DNA
polymerase I from E. coli, which is often used as a model
polymerase to test the inhibitor’s (3a) selectivity. While 3a (10
μM) almost completely eliminated Pol β’s ability to carry out
strand displacement synthesis using 13 (Scheme 7) and dTTP,
it had no effect on the Klenow fragment’s activity under the
same conditions.38 However, the efficacy of the 1,4-dicarbonyl
containing inhibitor (1a) was compromised by thiols,
presumably due to nucleophilic addition to the ring-opened
1,4-dicarbonyl form (Scheme 3, Figure 4). For instance,
preincubation (20 min) of 1a (50 μM) with glutathione (5
mM) resulted in Pol β activity similar to that of 25 μM
inhibitor in the absence of thiol.38
a
Key: a) Pb(OAc)4. b) H2/Rh. c) Phosphitylation. d) i. S-Ethyl-1H-
tetrazole, 20; ii. t-BuOOH. e) N2H4. f) 2,6-Dichloro-3-hydroxy-4-
methoxybenzaldehyde, AcOH. g) Demethylation.
genation, 18 was coupled with the methyl phosphoramidite
obtained from 9. Selective cleavage of the phthalimide group
yielded alkoxyamine 21, which was conjugated to 2,6-dichloro-
3-hydroxy-4-methoxybenzaldehyde prior to revealing the
phosphate diester (18). As expected, 18 had no effect on Pol
β lyase activity because the electrophilic carbonyl groups are
masked (data not shown).
The ability of 18 to provide more effective inhibition than 3a
directly was examined in prostate cancer cell (DU145) lysates.
TLC analysis revealed that the bisacetate was converted to 3a
within 5 min in DU145 lysate. The ability of 3a and 18 to
inhibit DU145 lysate lyase activity was then examined using
3′-32P-15 as substrate (Figure 5). Lyase activity inhibition was
significantly greater by bisacetate 18 than 3a. Almost complete
inhibition was achieved using 150 μM of 18, while more than
30% lyase activity remained following incubation with the same
concentration of 3a. Varying the preincubation time of 18 with
the cell lysate between 5 and 60 min showed that inactivation
was complete by 15 min.
Design and Synthesis of a Proinhibitor. The adverse
effect of glutathione on 1a led us to design bisacetate 18 as a
potential proinhibitor. We postulated that 18 would be
converted into 3a by cellular esterases.42,43 Proinhibitor 18
Selective Targeting of Pol β in Cell Lysates. The
selectivity of 3a for Pol β was examined further by incubating
various cell lysates of mouse embryonic fibroblasts (MEFs)
−/−
−/−
with proinhibitor 18. Pol λ
and Pol β
lysates were
obtained from the MEFs of mice (in a C57BL/6 background)
that were established in two different laboratories.44,45 Pol β
−/−/Pol λ−/− lysates were obtained from the MEFs of double
+/−
knockout mice that were generated by breeding Pol β
pol λ
and
heterozygote mice together.21 Pol λ WT and pol β
+/−
WT cells were matched to Pol λ null and Pol β null,
respectively. Cell lines that differ from wild-type by the
presence or absence of these polymerases are of interest
because there is evidence that Pol λ acts as a back-up for Pol β
during BER, although its lyase activity in vitro is considerably
weaker than that of Pol β.6,21,22 Consequently, it would be
useful to know if 3a distinguished between the two enzymes.
The lyase activity on 3′-32P-15 of various MEF cell lysates was
examined in the absence and presence of 18 (50 μM). Lyase
reaction rates on 3′-32P-15 were measured following
preincubation of the lysate with 18 (or buffer). Preincubation
with proinhibitor 18 reduced the rate of the lyase reaction
almost 2-fold in lysates obtained from wild-type cells. As shown
Figure 4. Effect of glutathione (GSH) on Pol β inhibition by 1a. (A)
No GSH. (B) [GSH] = 5 mM. [1a] (μM): 0, filled circle; 5, open
square; 10, gray circle; 25, gray square; 50, filled square.
E
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importantly, 18 had a much smaller effect in Pol β-deficient
cells. The lyase activity was reduced less than 30% in the
presence of 18. The effect of 18 on lyase activity in the double
knockout (Pol β/λ -; Pol β −/−/Pol λ−/−) cells was within
experimental error of that in the Pol β-deficient (Pol β -; Pol β
−/−/Pol λ+/+) cells, providing additional evidence that the
inhibitor is selective for Pol β over Pol λ. However, the
observation that 18 has even a small effect on the lyase reaction
in the double knockout or Pol β-deficient cell lysates indicates
that one or more other enzymes are affected by the proinhibitor
(Figure 6).
The Effects of 3a and 18 in Prostate Cancer Cells
(DU145). The superior performance of 18 compared to 3a in
cell lysates was also evident in studies using DU145 cells. For
instance, ∼0.01% of the DU145 cells survived treatment with
40 μM 18, whereas 12% of the cells survived treatment with the
same concentration of 3a (Figure 7A). We postulate that
greater stability of 18 than 3a to nucleophiles in the
extracellular matrix, resulting in a higher concentration of
molecule delivered to the cell, is one source of its greater
Figure 5. Effect of 3a and proinhibitor 18 on DU145 lysate lyase
activity. (A) Comparison of 3a and 18 as a function of concentration.
Preincubation time: 1 h, [32P-15] = 200 nM. (B) Effect of
preincubation time on the ability of 18 (50 μM) to inhibit lyase
activity.
in Figure 6 this effect was similar in both WT cells as expected
(even though their individual rates were different).38 Also the
Figure 6. Effect of proinhibitor 18 (50 μM) on lyase activity (3′-32P-
15) of various cell lysates from mouse embryonic fibroblasts.
effect of 18 on lyase activity in cells lacking Pol λ (Pol λ -; Pol λ
−/−/Pol β +/+) was statistically indistinguishable (p = 0.54) from
reactivity in the wild-type cells, suggesting that the inhibitor
does not signficantly inhibit this enzyme.
The effect of 18 in lysates lacking Pol β (Pol β -; Pol β
−/−/Pol λ +/+) was clearly different (p < 0.05). The overall lyase
activity in lysates lacking Pol β (in the absence of 18) was
signficantly lower than in those obtained from the wild-type or
Pol λ-deficient cells, indicating that Pol β enzyme was the major
contributor to the lyase reaction with 3′-32P-15. More
Figure 7. Effect of 3a and proinhibitor 18 on DU145 prostate cancer
cells. (A) Survival fraction as a function of inhibitor concentration. (B)
Effect of 18 (20 μM) on methyl methanesulfonate (MMS)
cytotoxicity. (C) Potentiation of MMS cytotoxicity by 18 (20 μM).
F
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efficacy. The above cell lysate experiments suggesting that the
small-molecule DOB mimics inhibit Pol β lyase activity,
combined with the encouraging intracellular activity of 18 led
us to examine its ability to potentiate the cytotoxicity of a DNA
damaging agent whose effects would require repair by Pol β.
BER of DNA alkylated by methyl methanesulfonate (MMS)
proceeds through an abasic site (AP, Scheme 1). Consequently,
DU145 cell survival was measured as a function of MMS
concentration, without and with 18 at a concentration (20 μM)
where the proinhibitor itself results in ∼45% cell death. After
normalizing the fraction of surviving DU145 cells by taking into
account the cytotoxicity of 18, plotting cell survival as a
function of MMS concentration (Figure 7B) reveals a clear
potentiation (Figure 7C) of the alkylating agent’s cytotoxicity at
0.2 mM and above. The cytotoxicity of 18 (20 μM) and MMS
(200 μM) is more than 2-fold greater than one would expect if
the two agents were not acting synergistically. The synergistic
effect of 18 and MMS is even greater at higher MMS
concentrations but is difficult to quantify above 0.3 mM MMS
where one observes a 5-fold potentiation, due to the small
numbers of surviving cells. Interestingly, the level of
potentiation observed by 18 is comparable and even slightly
greater than that seen in cells in which either the Pol β gene is
removed or its expression is knocked down using siRNA.46,47
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for all experiments, cell lysate kinetics,
fluorescence assays of inhibition, chart of aldehydes used to
generate library, NMR spectra of new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
mgreenberg@jhu.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for generous financial support from the
National Institute of General Medical Sciences (GM-063028)
to MMG and the National Cancer Institute (CA-058236) to
TLD. We thank Professor James Stivers (JHU) for providing
access to the aldehyde library and for helpful discussions, and
Dr. Sam Wilson (NIEHS) and Dr. Julie Horton (NIEHS) for
providing the mouse embryonic fibroblasts.
REFERENCES
■
CONCLUSIONS
(1) Friedberg, E. C.; Walker, G. C.; Siede, W.; Wood, R. D.; Schultz,
R. A.; Ellenberger, T. DNA Repair and Mutagenesis, 2nd ed.; ASM
Press: Washington, DC, 2006.
■
The kinetic experiments described above demonstate that small
molecules containing a 1,4-dicarbonyl, the same functional
group present in DNA lesions that is responsible for
inactivating Pol β (and Pol λ), irreversibly inhibit the lyase
activity of this enzyme.17 To our knowledge, these are the first
suicide inhibitors that target the lyase activity of Pol β. Due to
the lack of MS evidence, we cannot unequivocally state that the
inhibitor modifies the lysine(s) involved in Schiff-base
formation. Furthermore, the IC50 of 3a is comparable to the
best of previously reported Pol β inhibitors.17 Importantly,
experiments in cell lysates derived from a variety of mouse
embryonic fibroblasts lacking neither, one, or both Pol β or Pol
λ demonstrate that 18, which is enzymatically converted to 3a
in lysates, selectively inhibits the lyase activity of the former
over the latter. Although other enzymes that contribute to the
lyase reaction of 32P-15 are affected more weakly by 18, overall
the experiments involving mouse embryonic fibroblast lysates
support inhibition of the targeted Pol β in cells. Pol λ is
believed to back up BER by Pol β. Hence, developing
molecules that selectively inhibit one of these enzymes over
the other is useful for probing the enzymes’ roles in DNA repair
in cells.48,49 This suggests that 18 could be a useful tool for
examining the effects of Pol β in cells. Furthermore, 18
functions as well or better than other molecules at potentiating
the effects of a DNA damaging agent (MMS) in cells.3 To our
knowledge, this is the first example of an irreversible inhibitor
of Pol β that potentiates the cytotoxicity of a DNA damaging
agent.3,17 Future generations of DNA repair inhibitors that
potentiate MMS and/or other DNA damaging agents at even
lower concentrations are desirable. Finally, there is a resurgence
in interest in molecules that covalently modify their biological
targets.50−52 The approach described here may be useful for
inhibiting other DNA repair processes.53
(2) Stivers, J. T.; Jiang, Y. L. Chem. Rev. 2003, 103, 2729−2759.
(3) Horton, J. K.; Watson, M.; Stefanick, D. F.; Shaughnessy, D. T.;
Taylor, J. A.; Wilson, S. H. Cell Res. 2008, 18, 48−63.
(4) Guan, L.; Greenberg, M. M. J. Am. Chem. Soc. 2010, 132, 5004−
5005.
(5) Guan, L.; Bebenek, K.; Kunkel, T. A.; Greenberg, M. M.
Biochemistry 2010, 49, 9904−9910.
(6) Stevens, A. J.; Guan, L.; Bebenek, K.; Kunkel, T. A.; Greenberg,
M. M. Biochemistry 2013, 52, 975−983.
(7) Jacobs, A. C.; Kreller, C. R.; Greenberg, M. M. Biochemistry 2011,
50, 136−143.
(8) Matsumoto, Y.; Kim, K. Science 1995, 269, 699−702.
(9) Matsumoto, Y.; Kim, K.; Katz, D. S.; Feng, J.-A. Biochemistry
1998, 37, 6456−6464.
(10) Prasad, R.; Beard, W. A.; Strauss, P. S.; Wilson, S. H. J. Biol.
Chem. 1998, 273, 15263−15270.
(11) Deterding, L. J.; Prasad, R.; Mullen, G. P.; Wilson, S. H.; Tomer,
K. B. J. Biol. Chem. 2000, 275, 10463−10471.
(12) Prasad, R.; Batra, V. K.; Yang, X. P.; Krahn, J. M.; Pedersen, L.
C.; Beard, W. A.; Wilson, S. H. DNA Repair 2005, 4, 1347−1357.
(13) Prasad, R.; Beard, W. A.; Chyan, J. Y.; Maciejewski, M. W.;
Mullen, G. P.; Wilson, S. H. J. Biol. Chem. 1998, 273, 11121−11126.
(14) Feng, J.-A.; Crasto, C. J.; Matsumoto, Y. Biochemistry 1998, 37,
9605−9611.
(15) Husain, I.; Arteaga, C. L.; Srivastava, D. K.; Wilson, S. H.
Carcinogenesis 1999, 20, 1049−1054.
(16) Starcevic, D.; Dalal, S.; Sweasy, J. B. Cell Cycle 2004, 3, 998−
1001.
(17) Barakat, K.; Gajewski, M.; Tuszynski, J. A. Curr. Top. Med.
Chem. 2012, 12, 1376−1390.
(18) Donigan, K. A.; Sun, K.-w.; Nemec, A. A.; Murphy, D. L.; Cong,
X.; Northrup, V.; Zelterman, D.; Sweasy, J. B. J. Biol. Chem. 2012, 287,
23830−23839.
(19) Nemec, A. A.; Donigan, K. A.; Murphy, D. L.; Jaeger, J.; Sweasy,
J. B. J. Biol. Chem. 2012, 287, 23840−23849.
(20) Donigan, K. A.; Hile, S. E.; Eckert, K. A.; Sweasy, J. B. DNA
Repair 2012, 11, 381−390.
G
dx.doi.org/10.1021/ja411733s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(21) Braithwaite, E. K.; Kedar, P. S.; Stumpo, D. J.; Bertocci, B.;
Freedman, J. H.; Samson, L. D.; Wilson, S. H. PLoS One 2010, 5,
e12229.
(22) Braithwaite, E. K.; Prasad, R.; Shock, D. D.; Hou, E. W.; Beard,
W. A.; Wilson, S. H. J. Biol. Chem. 2005, 280, 18469−18475.
(23) Gao, Z.; Maloney, D. J.; Dedkova, L. M.; Hecht, S. M. Bioorg.
Med. Chem. 2008, 16, 4331−4340.
(52) Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L.
H.; Gray, N. S. Chem. Biol. 2013, 20, 146−159.
(53) Helleday, T.; Petermann, E.; Lundin, C.; Hodgson, B.; Sharma,
R. A. Nat. Rev. Cancer 2008, 8, 193−204.
(24) Nakamura, R.; Takeuchi, R.; Kuramochi, K.; Mizushina, Y.;
Ishimaru, C.; Takakusagi, Y.; Takemura, M.; Kobayashi, S.; Yoshida,
H.; Sugawara, F.; Sakaguchi, K. Org. Biomol. Chem. 2007, 5, 3912−
3921.
(25) Strittmatter, T.; Bareth, B.; Immel, T. A.; Huhn, T.; Mayer, T.
U.; Marx, A. ACS Chem. Biol. 2011, 6, 314−319.
(26) Wilson, S. H.; Beard, W. A.; Shock, D. D.; Batra, V. K.;
Cavanaugh, N. A.; Prasad, R.; Hou, E. W.; Liu, Y. A.; Asagoshi, K.;
Horton, J. K.; Stefanick, D. F.; Kedar, P. S.; Carrozza, M. J.; Masaoka,
A.; Heacock, M. L. Cell. Mol. Life Sci. 2010, 67, 3633−3647.
́
(27) Pitie, M.; Pratviel, G. Chem. Rev. 2010, 110, 1018−1059.
(28) Goldberg, I. H. Acc. Chem. Res. 1991, 24, 191−198.
(29) Jiang, Y. L.; Krosky, D. J.; Seiple, L.; Stivers, J. T. J. Am. Chem.
Soc. 2005, 127, 17412−17420.
(30) Chung, S.; Parker, J. B.; Bianchet, M.; Amzel, L. M.; Stivers, J. T.
Nat. Chem. Biol. 2009, 5, 407−413.
(31) Mootoo, D. R.; Date, V.; Fraser-Reid, B. J. Am. Chem. Soc. 1988,
110, 2662−2663.
(32) Chen, F.; Gaucher, E. A.; Leal, N. A.; Hutter, D.; Havemann, S.
A.; Govindarajan, S.; Ortlund, E. A.; Benner, S. A. Proc. Natl. Acad. Sci.
U.S.A. 2010, 107, 1948−1953.
(33) Kodama, T.; Greenberg, M. M. J. Org. Chem. 2005, 70, 9916−
9924.
(34) Bournaud, C.; Marchal, E.; Quintard, A.; Sulzer-Mosse,
́
S.;
Alexakis, A. Tetrahedron: Asymmetry 2010, 21, 1666−1673.
(35) Boeckman, R. K.; Miller, J. R. Org. Lett. 2009, 11, 4544−4547.
(36) Liu, J.; Wong, C.-H. Tetrahedron Lett. 2002, 43, 4037−4039.
(37) Dorjsuren, D.; Wilson, D. M.; Beard, W. A.; McDonald, J. P.;
Austin, C. P.; Woodgate, R.; Wilson, S. H.; Simeonov, A. Nucleic Acids
Res. 2009, 37, e128−e128.
(38) See Supporting Information.
(39) Silverman, R. B. The Organic Chemistry of Enzyme-Catalyzed
Reactions; Academic Press: San Diego, 2000.
(40) Brandt, G. S.; Nemeria, N.; Chakraborty, S.; McLeish, M. J.;
Yep, A.; Kenyon, G. L.; Petsko, G. A.; Jordan, F.; Ringe, D.
Biochemistry 2008, 47, 7734−7743.
(41) Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen,
M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frodin, M.;
̈
Taunton, J. Nat. Chem. Biol. 2012, 8, 471−476.
(42) Ora, M.; Taherpour, S.; Linna, R.; Leisvuori, A.; Hietamaki, E.;
Poijarvi-Virta, P.; Beigelman, L.; Lonnberg, H. J. Org. Chem. 2009, 74,
̈
̈
̈
4992−5001.
(43) Jessen, H. J.; Schulz, T.; Balzarini, J.; Meier, C. Angew. Chem.,
Int. Ed. 2008, 47, 8719−8722.
(44) Sobol, R. W.; Horton, J. K.; Kuhn, R.; Gu, H.; Singhal, R. K.;
Prasad, R.; Rajewsky, K.; Wilson, S. H. Nature 1996, 379, 183−186.
(45) Bertocci, B.; De Smet, A.; Flatter, E.; Dahan, A.; Bories, J.-C.;
Landreau, C.; Weill, J.-C.; Reynaud, C.-A. S. J. Immunol. 2002, 168,
3702−3706.
(46) Horton, J. K.; Joyce-Gray, D. F.; Pachkowski, B. F.; Swenberg, J.
A.; Wilson, S. H. DNA Repair 2003, 2, 27−48.
(47) Polosina, Y. Y.; Rosenquist, T. A.; Grollman, A. P.; Miller, H.
DNA Repair 2004, 3, 1469−1474.
(48) Strittmatter, T.; Brockmann, A.; Pott, M.; Hantusch, A.;
Brunner, T.; Marx, A. ACS Chem. Biol. 2014, 9, 282−290.
(49) Kuriyama, I.; Miyazaki, A.; Tsuda, Y.; Yoshida, H.; Mizushina, Y.
Bioorg. Med. Chem. 2013, 21, 403−411.
(50) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. Nat. Rev. Drug
Discovery 2011, 10, 307−317.
(51) Kwarcinski, F. E.; Fox, C. C.; Steffey, M. E.; Soellner, M. B. ACS
Chem. Biol. 2012, 7, 1910−1917.
H
dx.doi.org/10.1021/ja411733s | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX