Expanding the scope of human DNA polymerase and inhibitors

Article abstract of DOI:10.1021/cb4007562

The exact biological functions of individual DNA polymerases still await clarification, and therefore appropriate reagents to probe their respective functions are required. In the present study, we report the development of a highly potent series of human DNA polymerase γand β(pol γandβ ) inhibitors based on the rhodanine scaffold. Both enzymes are involved in DNA repair and are thus considered as future drug targets. We expanded the chemical diversity of the small-molecule inhibitors arising from a high content screening and designed and synthesized 30 novel analogues. By biochemical evaluation, we discovered 23 highly active compounds against polβ . Importantly, 10 of these small-molecules selectively inhibited polβ and not the homologous polβ . We discovered 14 small-molecules that target polγ and found out that they are more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells toward DNAdamaging reagents. Thus, we cotreated human colorectal cancer cells (Caco-2) with the small-molecule inhibitors and hydrogen peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic agents in a DNA repair pathway-dependent manner.2013 American Chemical Society.

Full text of DOI:10.1021/cb4007562

Articles
pubs.acs.org/acschemicalbiology
Expanding the Scope of Human DNA Polymerase λ and β Inhibitors
Tobias Strittmatter, Anette Brockmann, Moritz Pott, Annika Hantusch, Thomas Brunner,
and Andreas Marx*
Departments of Chemistry and Biology, Konstanz Research School Chemical Biology, University of Konstanz, Universitatsstrasse 10,
̈
78457 Konstanz, Germany
S
* Supporting Information
ABSTRACT: The exact biological functions of individual DNA
polymerases still await clarification, and therefore appropriate
reagents to probe their respective functions are required. In the
present study, we report the development of a highly potent series of
human DNA polymerase λ and β (pol λ and β) inhibitors based on
the rhodanine scaffold. Both enzymes are involved in DNA repair and
are thus considered as future drug targets. We expanded the chemical
diversity of the small-molecule inhibitors arising from a high content
screening and designed and synthesized 30 novel analogues. By
biochemical evaluation, we discovered 23 highly active compounds
against pol λ. Importantly, 10 of these small-molecules selectively
inhibited pol λ and not the homologous pol β. We discovered 14 small-molecules that target pol β and found out that they are
more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells toward DNA-
damaging reagents. Thus, we cotreated human colorectal cancer cells (Caco-2) with the small-molecule inhibitors and hydrogen
peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic
agents in a DNA repair pathway-dependent manner.
ach living cell is exposed to DNA-damaging agents 24
primary sequence of the catalytic core in the C-terminal part of
Ehours a day. To maintain the genetic integrity of its pol λ (residues 244−575) are highly homologous to pol β.9,10
Because of its ability to remove the 5′-deoxyribose phosphate
(dRP) generated after incision by an abasic (AP) endonuclease
(dRP-lyase activity) and its DNA synthesis specificity for short
gaps, pol β is the prime DNA polymerase involved in
BER.2−5,11,12 Additionally, pol β is able to associate with
other downstream enzymes of the BER pathway like DNA
ligase I, AP endonuclease, and XRCC1-DNA ligase III.2,11
Extensive studies show that pol β is able to bypass several DNA
lesions via translesion synthesis (TLS), for example, AP sites13
and cisplatin adducts.14 Pol λ, the other enzyme of interest
here, is capable of synthesizing DNA de novo and template-
dependent. Furthermore, it displays terminal deoxynucleotidyl
transferase (TdT) as well as dRP-lyase activity.15−19 Pol λ is
implicated in V(D)J recombination,20,21 TLS,13,22,23 and
BER.15,24,25 Moreover, studies with eukaryotic cells and reactive
oxygen species (ROS) indicate that pol λ functions as a backup
for pol β in BER26 and protects cells from oxidative
damage.24,27 There is also evidence that pol λ is required for
cell cycle progression and is functionally connected to the S
phase DNA damage response machinery in cancer cells.27
A recent investigation of pol β and λ expression patterns in
68 tumor samples indicated overexpression of either one in
30% of the assayed cases.28 The regulation of these two DNA
genome, an elaborate set of sophisticated repair mechanisms
have evolved. Base excision repair (BER), for example, is
responsible for the accurate removal of DNA nucleobase
damage generated by oxidation, hydrolytic reactions, or
alkylation.1−3 Errors in DNA damage repair pathways can
lead to severe developmental defects, cancer, or even death.1−5
Key enzymes in DNA metabolism are DNA polymerases. They
are involved in the duplication of the genome, repair of DNA
lesions, and DNA recombination.3−5 To date 15 different DNA
polymerases have been discovered in human cells.3,5 The
functions of some of these 15 enzymes are known, but for the
majority, for example, repair enzymes, the exact roles still await
clarification. Therefore, appropriate reagents to probe their
respective functions are required. Given their fast mode of
action, cell-permeable small-molecule inhibitors are ideally
suited to interfere in this highly dynamic process. These
molecular tools not only might be of great value for basic
science but also may open up novel avenues for the treatment
of diseases related to genome integrity.2−4,6,7
In this study, we focused on the inhibition of the
nonreplicative human DNA polymerases β (pol β)8 and λ
(pol λ),9 both members of the DNA polymerase X family.2−5
The exonuclease-deficient pol λ (64 kDa) contains all the
important residues required for DNA binding, nucleotide
binding and selection, and catalysis of DNA polymerization,
which are conserved in pol β (39 kDa), the smallest known
human DNA polymerase. Hence, the 3D-structure and the
Received: October 1, 2013
Accepted: October 30, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
polymerases could be fundamental in cancer treatment, since
many chemotherapeutic regimes in use depend at least in part
on the artificial induction of DNA damage. The clinical efficacy
of anticancer drugs like cisplatin and monofunctional alkylating
agents is often reduced by cellular DNA repair mecha-
nisms.1−5,12,29 Consequently, both DNA polymerases involved
in DNA repair are discussed as promising future drug targets, to
reduce the dosage of DNA-damaging agents while improving
their activity via inhibition of their respective DNA repair
pathways.2−4,12,29
In order to discover and evaluate inhibitors of DNA
polymerases, high content screening (HCS) strategies and in
vitro enzyme assays were developed.14,30−34 By application of a
recently reported fluorescence based screening technique and
radioactive primer extension (PEX) assays, several small-
molecule inhibitors of human pol λ were discovered.32 The
rhodanine-based compounds, classified as a privileged drug
scaffold,35−38 were found to be the most active inhibitor class
(Figure 1).
tumor cell cytotoxicity of chemotherapeutics for the treatment
of colorectal cancers.33,40−42
RESULTS
■
Compound Design and Chemical Synthesis. To further
expand the chemical diversity of pol λ and pol β small-molecule
inhibitors, we designed and synthesized 30 (3−32) novel
analogues of 1 and 2 (Table 1). Molecule 2 was resynthesized
because of its highest activity against pol β in the initial
screen.32 For this compound series, 1 and 2 were subdivided
into the previously described molecular scaffold.32 The scaffold
consists of three variable parts, R1, R2, and R3, which are
connected via an aromatic core and a variable Z-linkage (Table
1). To generate the next generation of small-molecule
inhibitors, we further derivatized 1 and 2 in all these parts.
Reportedly, the heterocyclic rhodanine, moiety A in R1, proved
to be highly important for the inhibitory activity against pol λ.32
However, it could be shown that moiety A can be replaced in
some cases by the 2,4-thiazolidinone, moiety B. Motivated by
this fact, we investigated the synthesis of heterocyclic molecules
3−9 bearing moiety B and the further well-known pharmaco-
phoric moieties C, D, E, F, G and H. The thioether-Z-linkage
proved not to be essential for high activity toward pol λ and
could be replaced by ester-, benzyl phenyl ether-, or diphenyl
ether-Z-linkages.32 Thus, this modification site was not in our
main focus herein, and we only tested a sulfone as the Z-linkage
(10). Importantly, we could show that the variation of the
substituents in R2 and R3 can influence the inhibitory activity
against pol λ. On the other hand there were indications that
some of these changes in R2 and R3 boosted the inhibitory
activity against pol β.32 To further expand the compound series
with modifications in part R2 and R3, we designed and
synthesized analogues 11−30. In the initial screen (Z)-5-(5-
nitro-2-(p-tolylthio)benzylidene)-2-thioxothiazolidin-4-one also
proved to be a pol λ inhibitor in the low micromolar range
(IC50 = 10.0 μM).32 For that reason, we “combined” this
compound (apart from the nitro groups) with 1 to generate
molecule 31. Lastly, we investigated the inhibitory potential of
the first compound with a heterocyclic aromatic core structure
and created 32.
Figure 1. Chemical structures of the initial screening hits 1 and 2.
The most potent pol λ inhibitor obtained was compound 1,
with a half-maximal inhibitory concentration (IC50) of 5.9 μM
for its DNA polymerization function and 4.5 μM for its TdT
function. Interestingly, 1 is up to 10 times less active against the
highly homologous pol β (IC50 = 64.4 μM)32 and thus could be
a useful probe for specifically investigating the biological
functions of pol λ. Additionally, 1 did not inhibit (IC50 > 100
μM) the DNA polymerases 9°N, Therminator, Pfu, and Dpo4
(unpublished data). During the course of evaluating the
inhibitory potential of the initial compound library, further
rhodanine-based inhibitors were found whose properties are
comparable with 1.32 So far a side-by-side comparison of 1 with
the most active known pol λ inhibitor (epigallocatechin
gallate39) has been investigated, and it was found that the
rhodanines are currently the strongest known pol λ inhibitors.
Furthermore, there were early indications that some com-
pounds (e.g., 2) also showed activity against pol β.32 Based on
these findings, systematic synthetic optimization has been
undertaken by us in order to further expand the chemical
diversity and to find novel and more potent small-molecule
inhibitors of pol β and λ.
Because pol β and λ are implicated as prime targets for
improving the response to genotoxic agents and the rhodanines
showed moderate toxicity in human cervix carcinoma (HeLa
S3) and hepatocellular carcinoma (Hep G2) cell lines,32 we
explored the cotreatment of these kinds of DNA-damaging
agents with rhodanine probes. The anticancer regime was
studied with the approved monofunctional alkylating drug
temozolomide (TMZ) and the model ROS inducer hydrogen
peroxide (H2O2) on the human colon carcinoma cell line Caco-
2. This cell line is especially interesting since there is an urgent
need for the development of novel approaches to enhance
For the chemical synthesis of the small-molecule entities 2−
32, we followed the reported synthesis strategy, which can be
divided into two parts (Scheme 1).32
In the first part, we built up the precursor aldehydes 33−35
and 37−58 and for compound 8 a precursor acetophenone 36.
To synthesize the new precursors 34−57 in good to excellent
yields, we applied the nucleophilic aromatic substitution (SNAr)
under basic conditions in dimethylformamide (DMF) to
displace activated halides (F, Cl, or Br) at the aromatic core
by aromatic or aliphatic thiols (Scheme 1A). To synthesize the
3-nitro-4-tosylbenzaldehyde precursor 58 in a three step
sequence, we assigned a literature-known synthesis strategy43
starting from 3-nitro-4-(p-tolylthio)benzaldehyde 33.32 There-
fore, 33 was reduced with sodium borohydride (NaBH4) in
methanol to yield the (3-nitro-4-(p-tolylthio)phenyl)methanol
intermediate in 98%. Next, the intermediate was refluxed
together with H2O2 in acetic acid to give (3-nitro-4-
tosylphenyl)methanol 59 in 61% yield. In the last step, 59
was oxidized with Dess−Martin periodinane (DMP) to furnish
precursor 58 in 57% yield (Scheme 1A).
In the second part, we performed Knoevenagel condensa-
tions by fusing the precursor molecules together with varying
heterocycles (Scheme 1B). In doing so, an exocyclic double
B
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
a
Table 1. Small-Molecules 1−32 and Their Biochemical Evaluation
Pol λ conversion [%],
Pol λ conversion [%],
Pol λ IC
Pol β conversion [%],
Pol β IC
a50
a
a
a50
a
no.
R1
R2
R3
Z
20 μM compound
10 μM compound
[μM]
50 μM compound
[μM]
b
b
b
b
b
1
2
A
A
B
C
D
E
-NO2 4-Me-Ph-
-NO2 cyclohexane-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-NO2 4-Me-Ph-
-S-
3
8
5.9
<10
92
64.4
<50
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-SO2-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
4
4
19
26
32
90
88
99
93
98
38
100
96
37
3
3
<10
>50
4
80
100
91
97
5
5
6
7
F
8
G
H
A
A
A
A
A
A
A
A
A
A
A
A
19
<10
<50
9
46
23
3
10
11
12
13
14
15
16
17
18
19
20
21
<20
<10
<10
5.7
-H
4-Me-Ph-
4-Me-Ph-
4-Me-Ph-
4-Me-Ph-
4-Me-Ph-
4-Me-Ph-
31
14
9
<50
38.7
>50
>50
28.1
-F
2
-Cl
-Br
-CF3
-CN
2
82
87
7
2
19
14
11
14
18
<10
<10
<10
<10
<10
<20
<10
<10
2
2
93
20
90
83
90
76
-NO2 4-Et-Ph-
3
<50
>50
>50
>50
>50
-NO2 4-F3C-Ph-
-NO2 4-F3CO-Ph-
-NO2 3-F,4-Me-Ph-
2
18
3
14
18
-NO2 2,3,5,6,-F; 4-
F3C-Ph-
3
22
23
24
25
26
27
28
29
30
31
A
A
A
A
A
A
A
A
A
-NO2 3-Me-Ph-
-NO2 2-Me-Ph-
-NO2 2,5-Me-Ph-
-NO2 2-naphtyl-
-NO2 cyclopentane-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
-S-
2
3
10
6
6.0
3.9
8
5
29.8
18.2
<50
2
15
19
<10
<10
<20
4.0
31
67
90
84
47
50
37
27
3
>50
17
2
>50
-Br
-H
3-F,4-Me-Ph-
2-Me-Ph-
5
21
14
15
10
>50
4
<10
<10
<10
4.0
∼ 50
∼ 50
<50
-F
2-Me-Ph-
3
-CF3
2-Me-Ph-
3
(Z)-5-(2,4-bis(p-tolylthio)benzylidene)-
2-thioxothiazolidin-4-one
2
<50
32
(Z)-5-((4-bromo-5-(p-tolylthio)
thiophen-2-yl)methylene)-2-
thioxothiazolidin-4-one
4
17
<10
22
<50
a
b
Averages of three independent experiments are shown (for details, see Supporting Information). See ref 32.
bond is generated, whereas in theory two diastereomeres E and
Z can be formed. Reportedly, the Z-configuration is
thermodynamically more stable than the E-configuration.44−46
For that reason, compounds 2−8 and 10−32 were synthesized
under thermodynamic reaction control. We refluxed the
precursor aldehydes 33−35 and 37−58 with rhodanine,
thioazolidine-2,4-dione, thiohydantoine, rhodanine-3-acetic
acid, 3-phenyl-2-thioxothiazolidin-4-one, or pseudothiohydan-
toin in a solution of anhydrous NaOAc in glacial acetic acid to
obtain the isomeric pure compounds 2−7 and 10−32 in good
according to Unangst et al.50 in a mixture of NH4OAc and
toluene heated under reflux to yield 8 in 78% (Scheme 1B).
Compound 8 shows again only one sharp signal for the 5-
ethylidene methyl group in 1H NMR implying Z-configuration.
The exclusive formation of the thermodynamically stable Z-
isomers of 2−8 and 10−32 is in agreement with various
literature reports for similar compounds.37,45,47−51 For the
synthesis of small-molecule 9, with the free rotatable
pharmacophoric moiety H, we planned to hydrogenate the
exocyclic double bond of 1. Only a few methods have been
reported to reduce the exocyclic double bond in 5-benzylidene-
2-thioxothiazolidin-4-ones. Kikelj and Peterlin Masic et al.48,49
to excellent yields (Scheme 1B).32,47 The H NMR spectra of
1
compounds 2−5, 7, and 10−32 exhibited only one signal for
the 5-methylidene proton in the range 7.50 to 7.80 ppm and for
6 at 6.50 ppm at lower field values than those expected for the
E isomers,48,49 which strongly indicates the formation of the
more stable Z isomers. To attach rhodanine to the
acetophenone precursor 36, the reaction was performed
̌
̌
successfully adopted the Hantzsch ester on silica gel method.52
We also used this chemoselective method and reduced 1 to the
racemic compound 9 in a good yield (Scheme 1B).
Biochemical Evaluation and IC50 Determination.
Holding novel small-molecule entities in hand, we tested
C
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
a
Scheme 1. Chemical Synthesis of Small-Molecules 2−32
a
Reagents and conditions: (A) Part one, (a) corresponding thiol, corresponding aldehyde (or acetophenone), K2CO3, DMF, 80 °C, up to 89%; (b)
corresponding thiol, corresponding benzaldehyde, KOH, DMF, 0 °C to rt, up to 97%; (c) NaBH4, methanol, rt, 1 h, 98%; (d) 30% H2O2, AcOH,
reflux 61%; (e) DMP, CH2Cl2, rt, overnight, 53%. (B) Part two, (f) corresponding aldehyde, rhodanine (or rather thioazolidine-2,4-dione,
thiohydantoine, rhodanine-3-acetic acid, 3-phenyl-2-thioxothiazolidin-4-one, pseudothiohydantoin), NaOAc (2.5 M in AcOH), reflux, overnight, up
to 98%;32,47 (g) NH4OAc, toluene, reflux, overnight, 78%; (i) diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate (Hantzsch ester), activated
silica gel 60, toluene, reflux for 24 h in the dark, 82%. For details, see Supporting Information.
them at 20 μM concentration in the radioactive pol λ PEX assay
in comparison to compound 1 (Figure 2, Table 1). Therefore, a
radioactively labeled 20-nucleotide primer strand was annealed
to a 33-nucleotide template strand, and all four native dNTPs
were added to start the reaction. After 30 min at 37 °C, the
reaction was quenched and analyzed via polyacrylamide gel
electrophoresis (PAGE) analysis (Supplementary Scheme S1A,
Supporting Information). In doing so, we found four inactive
compounds (4−7), four compounds (9, 10, 19, and 26) with a
moderate activity (15−50% conversion), and 23 highly active
compounds (2, 3, 8, 11−18, 20−25, and 27−32) with
conversion below 10% (Figure 2A; Table 1). Next, we studied
the 20 highly active small-molecules for their effect in pol λ
PEX assay at 10 μM to get a further selection criterion.
Interestingly, we found out that all these compounds had an
IC50 value of less than 10 μM against pol λ (Figure 2B; Table
1). The exact IC50 value was determined for the five most active
compounds (13, 22, 23, 27, and 31) with a turnover of ≤10%
in the PEX assay (Figure 2C; Table 1). Compounds 13, 22, 23,
27, and 31 inhibited dose-dependently the polymerization
function of pol λ with IC50 values of 5.7, 6.0, 3.9, 4.0, and 4.0
μM, respectively, and are thus equally or even more active than
the lead compound 1 (IC50 = 5.9 μM).32 Reportedly, human
pol λ has a TdT activity, and its involvement in recombination
events has been suggested.53,54 Thus, to test the inhibitory
potential of 13, 22, 23, 27, and 31, we investigated TdT activity
using the radioactive assay of single-stranded primer extension.
All tested compounds inhibit this function in a low micromolar
range comparable to 1 (IC50 = 4.5 μM)32 (Figure 2D, see also
Supplementary Scheme S1B, Supporting Information).
To test for selectivity and to identify inhibitors of pol β, we
screened the small-molecule series at 50 μM concentration in
the radioactive pol β PEX assay (Figure 3, Table 1). Therefore,
PEX with the same sequence context was used as described
before (Supplementary Scheme S1A, Supporting Information).
In this manner, we identified ten compounds (3, 13, 14, 16, 18,
20, 21, and 25−27) that selectively inhibited in a low
micromolar range pol λ but not pol β. Compounds that were
not (4−7) or were moderately (9, 10, 19, and 26) active
against pol λ showed no activity against pol β. The
resynthesized compound 2 belongs to the ten compounds (2,
8, 11, 17, 24, and 28−32) showing a moderate activity (15−
50% conversion) against pol β. Interestingly, we discovered
four highly active compounds (12, 15, 22, 23) with a
conversion below 10% (Figure 3A; Table 1). For the four
small-molecule, the exact IC50 values were determined. We
found out that 12, 15, 22, and 23 dose-dependently inhibited
the polymerization function of pol β with IC50 values of 38.7,
28.1, 29.8, and 18.2 μM (Figure 3B, Table 1). To further
evaluate compound 23, we used the reported pol β inhibitors
betulinic acid,55 oleanolic acid,56 and lithocholic acid57 in a side-
by-side comparison using the radioactive PEX assay. We found
that the reported pol β inhibitors are less active than 23 in this
assay (Figure 3C).
Cellular Studies. Motivated by the reports that the two
BER repair proteins pol β and λ were found to be
overexpressed in colorectal cancer tissue28 and are discussed
D
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 2. Evaluation of compounds listed in Table 1 toward pol λ. (A) Evaluation of the compounds at 20 μM in the pol λ PEX assay. Inactive
compounds are shown in red. Compounds with moderate activity (15−50% conversion) are shown in yellow. Compounds with less than 10%
conversion (green) were chosen to be analyzed at 10 μM in the pol λ PEX assay. (B) Evaluation of the compounds at 10 μM in the pol λ PEX assay.
Compounds with a conversion below 50% are shown in green. Compounds with less than 10% conversion (blue) were chosen to determine their
exact IC50 value. (C) Dose−response curves of synthesized compounds 13, 22, 23, 27, and 31, which dose-dependently inhibited the polymerization
function of pol λ with IC50 values of 5.7 1.0, 6.0 1.0, 3.9 1.1, 4.0 1.1, and 4.0 1.0 μM. In general averages of at least three independent
experiments and standard deviations are shown. (D) PAGE analysis showing the influence of 13, 22, 23, 27, and 31 on TdT function of pol λ. Lane
1, primer only; lane 2, DMSO control; lanes 3−7, compounds 13, 22, 23, 27, and 31 (each 10 μM). For details, see Supporting Information.
as appropriate cellular targets to overcome resistance of DNA-
damaging agents,2−4,12,29 we explored cotreatment of the
human colorectal tumor cell line Caco-2 with genotoxic
reagents TMZ or H2O2 in combination with small-molecule
inhibitors 1 or 23. To find suitable conditions for this proof-of-
concept study, we first adopted the previously described 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay40,58 and determined the half-maximal inhibitory concen-
tration of the cell viability (EC50) of 1, 23, TMZ, and H2O2. As
shown in Supplementary Figure S1, Supporting Information,
The data shows (Figure 4) that 1 and 23 enhanced the
sensitivity of human Caco-2 cells toward H2O2 and TMZ.
Indeed, the major pathway for oxidative and monoalkylated
DNA damage recognition and repair is the BER.1−3,12,29 The
additive or even synergistic effect of cell death induction in
Caco-2, caused by inhibitor 1 and 23 together with the ROS
inducer H2O2 or monoalkylating drug TMZ, suggests that both
small-molecules act in a way that affects the BER pathway. To
prove these facts in detail is particularly challenging, but these
findings support our efforts to further develop the reported pol
λ and β inhibitors in a cellular context.
cell viability was suppressed dose-dependently by 1 (EC50
=
58.1 μM) and 23 (EC50 = 63.8 μM) after 96 h incubation and
by H2O2 (EC50 = 540 μM) after 72 h incubation. Because of
the low cellular response, the exact EC50 value of TMZ could
not be determined (EC50 > 1000 μM). Afterward, cotreatment
experiments were performed at concentrations that yielded
responses below 30% cell death (Figure S1, Supporting
Information), to study whether 1 or 23 can sensitize Caco-2
cells to H2O2 or TMZ.
DISCUSSION AND CONCLUSION
■
By systematic synthetic optimization, we could further expand
the chemical diversity of rhodanine-based small-molecules. The
scaffold oriented synthesis of drug-like compounds 2−32 was
easily performed in two to four high-yielding steps starting from
cheap and commercially available building blocks.
Because of the evaluation of 2−32 in radioactive PEX assays,
we could not only discover several highly potent inhibitors of
E
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 3. Evaluation of compounds listed in Table 1 toward pol β. (A) Evaluation of the compounds at 50 μM in the pol β PEX assay. Compounds
inactive against pol β and pol λ are shown in gray. Inactive compounds, with a high conversion, are shown in red. Compounds with moderate activity
(15−50% conversion) are shown in yellow. Interesting compounds with less than 10% conversion (green) were chosen to determine their exact IC50
values. (B) Dose−response curves of synthesized compounds 12, 15, 22, and 23, which inhibited dose-dependently the polymerization function of
pol β with IC50 values of 38.7 1.0, 28.1 1.0, 29.8 1.0, and 18.2 1.0 μM. (C) Potency of compound 23 against pol β compared with betulinic
acid,55 oleanolic acid,56 and lithocholic acid57 using the same reaction conditions (500, 250, and 125 μM compound). In general, averages of at least
three independent experiments and standard deviations are shown. For details, see Supporting Information.
The molecular scaffold (Table 1), also ideally suited to
establish SAR, consists of three variable parts R1, R2, and R3,
which are connected via an aromatic core and a variable linkage
Z.32 The study herein clearly demonstrates the extraordinary
importance of the heterocyclic warhead for the effectiveness of
the inhibitors against pol λ and pol β. We could show that the
hydrogen atom in moiety A cannot be substituted. In line with
the recently reported substitution of the hydrogen atom to an
allyl group,32 also the replacement by an acetic acid group
(moiety C) or a phenyl group (moiety D) resulted in
completely inactive compounds (4, 5) against pol λ and β.
Moreover, the endocyclic sulfur atom in moiety A proved to be
important since the substitution with an NH group (moiety E)
in compound 6 resulted in a loss of activity. In contrast,
modifications at the exocyclic double bond (moieties G and H)
were tolerated. In consequence of methylation of the double
bond in 8 (moiety G), the high potency against pol λ was
conserved, but resulted in a compound with moderate activity
against pol β. The hydrogenation of 1 to 9 was attended by the
Figure 4. Compounds 1 and 23 sensitize colorectal cancer cells to the
change of the hybridization state of the exo- and endocyclic
ROS inducer H2O2 and monoalkylating drug TMZ. Caco-2 cells were
carbon atoms (sp3 to sp2). Pol λ could be inhibited by 9, but
preincubated with 25 μM 1 or 23. After 24 h, cells were treated with
with a lower effectiveness than 1. The replacement of the
125 μM H2O2 (A) or 500 μM TMZ (B) for 72 h. Then, cell death
exocyclic sulfur of moiety A via an oxygen atom (moiety B) or
an NH group (moiety F) resulted in indifferent findings.
Moiety F containing 7 was completely inactive against both
family X DNA polymerases. Interestingly, by substitution of
moiety A with moiety B, we generated 3 showing the same
inhibitory properties as lead compound 1, but with a 2,4-
thiazolidinone warhead.
induction was measured by the MTT assay. Averages of at least seven
independent experiments and standard deviations are shown. For
details, see Supporting Information.
pol λ and β but also confirm and significantly extend the
recently reported basic structure−activity relationships (SAR).
F
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
In the initial screening, (Z)-5-(5-nitro-2-(p-tolylthio)-
benzylidene)-2-thioxothiazolidin-4-one proved to be a pol λ
inhibitor in the low micromolar range (IC50 = 10.0 μM).32 For
that reason, we “combined” this compound (apart from the
nitro groups) with 1 and generated the very potent pol λ
inhibitor 31 with an IC50 value of 4.0 μM. Inhibitor 31 was also
moderately active against pol β.
As mentioned above, the thioether used as Z linkage proved
not to be essential for high activities or selectivity and could be
replaced by ester, benzyl phenyl ether, or diphenyl ether
linkages,32 and so herein we installed only the sulfone linkage in
compound 10. By oxidation of the thioether to the sulfone Z
linkage, the pol λ selective inhibitor 10 with a lower activity
than 1 was generated.
In conclusion, we report the discovery and development of a
highly potent inhibitor series based on a rhodanine scaffold
arising from a fluorescence based HCS.32 We expanded the
chemical diversity of pol λ and pol β small-molecule inhibitors
and designed and synthesized 30 (3−32) novel analogues.
By biochemical evaluation of the small-molecule entities, we
discovered 23 highly active compounds against pol λ (2, 3, 8,
11−18, 20−25, 27−32) with IC50 values less than 10 μM.
Interestingly, 10 of these small-molecules (3, 13, 14, 16, 18, 20,
21, 25−27) selectively inhibited pol λ in a low micromolar
range but not pol β. The exact IC50 values were determined for
the five most active compounds. Compounds 13, 22, 23, 27,
and 31 dose-dependently inhibited the polymerization function
of pol λ with IC50 values of 5.7, 6.0, 3.9, 4.0, and 4.0 μM and are
thus equally or even more active than lead compound 1 (IC50
=
Comparing compounds 11−30, it is evident that the
variation of the substituents in position R2 and R3 of the
scaffold is also able to influence the activity against pol λ and
pol β. With the introduction of several substituents in position
R2 (like H, F, Cl, Br, CF3, CN, NO2, or MeO32), high
inhibitory activity was maintained against pol λ without
exception. Interestingly variation of the R2 substituent to Cl
(13), Br (14), and CN (16), beside the known NO2 group
(1),32 led to compounds that are able to discriminate between
pol λ and pol β. By analyzing 11, 12, 15, 28−30, we found that
the activity rose considerably against pol β in the specified
order H ≤ F < CF3. As we reported previously, R3 belongs to
the scaffold and therefore it cannot be waived. Looking at the
compound series with variations in R3, we found that all tested
compounds (except R3 = 2-pyridine32) were able to inhibit pol
λ. It is noteworthy that the R3 modifications 4-F3C-Py-32 or 4-
F3C-Ph- to 4-F3CO-Ph- and cyclohexane to cyclopentane led to
moderately active pol λ inhibitors. A particularly interesting
discovery is that the alkylation pattern of the aromatic ring in
R3 not only affects the activity against pol λ but also against pol
β. A close look at 1, 17, 22−24, and 28−30 reveals that the
IC50 value against pol λ decreases in the same order (4-Me-Ph
> 3-Me-Ph > 4-Me-Ph), as the activity against pol β is
modulated (4-Me-Ph ≪ 4-Et-Ph = 3-Me-Ph < 4-Me-Ph).
Lastly, we analyzed 32, the first compound with a heterocyclic
aromatic core structure. As a result of the bioisosteric thiophene
ring, the activity against pol λ was preserved, but the selectivity
dropped.
5.9 μM).32 To our knowledge, the herein reported small-
molecules are currently the strongest inhibitors for pol λ.
Furthermore, we discovered 14 small-molecules (2, 8, 11, 12,
15, 17, 22−24, 28−32) that target pol β. The exact IC50 value
was determined for the four most active compounds.
Compounds 12, 15, 22, and 23 inhibited dose-dependently
the polymerization function of pol β with IC50 values of 38.7,
28.1, 29.8, and 18.2 μM. To further evaluate pol β inhibitors,
we compared 23 with betulinic acid,55 oleanolic acid,56 and
lithocholic acid57 in a side-by-side comparison using the
radioactive pol β PEX assay and found that the reported pol
β inhibitors are less active. Out of the in vitro data, we could
additionally extend and confirm the recently reported SAR.32
In addition, we explored whether the discovered compounds
sensitize cancer cells toward DNA-damaging reagents. Thus, we
cotreated human colorectal cancer cells (Caco-2) with the
discovered inhibitors and ROS inducer H2O2 or the approved
drug TMZ. Importantly, the tested small-molecules 1 and 23
were pharmacologically active and sensitized Caco-2 cells
toward both genotoxic agents. The resulting data indicates that
the compounds act in a way that influences the BER pathway
and supports our efforts to further develop the reported pol λ
and β inhibitors in vitro and in a cellular context.
METHODS
■
Full experimental details are given in the Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
In general, rhodanines are classified as nonmutagenic,59 and a
long-term study on the clinical effects of the rhodanine-based
epalrestat demonstrated that it is well tolerated by patients.60
Because chemotherapies depend frequently in part on the
artificial induction of DNA damage, consideration of targeting
DNA repair capacity is an ongoing concern in improving
responses to treatments. Like other gene products involved in
DNA damage repair, the regulation of pol β and λ, which are
overexpressed in cancer tissue,28 could be fundamental in
cancer treatment.2−4,12,29 The herein reported cellular studies
support this notion, as 1 and 23 enhanced the sensitivity of
human colorectal cancer cells toward the genotoxic H2O2 and
TMZ considerably. The additive or even synergistic effect
suggest that 1 and 23 have the potential to reduce the dosage of
DNA damaging reagents, while improving their activity via
inhibition of their respective DNA repair pathways. For this
reason, the discovered small-molecules not only might be of
great value for basic science but may also serve as lead
structures for the development of novel avenues for the
treatment of diseases related to genome integrity.
■
S
Schematic of PEX assays for polymerase and TdT inhibitor
activity of pol β and λ, full experimental details, measured cell
death curves of compounds 1 and 23, H2O2, and TMZ, and 1H
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Andreas.Marx@uni-konstanz.de. Fax: +49 7531 88
5140. Tel: +49 7531 88 5139.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge funding and scientific support by
the Konstanz Research School Chemical Biology. We thank N.
Hardt and M. Drum for carefully reading the manuscript and
helpful discussions.
G
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
DNA end joining in human nuclear extracts. J. Biol. Chem. 279, 805−
811.
REFERENCES
■
(1) Scharer, O. D. (2003) Chemistry and biology of DNA repair.
Angew. Chem., Int. Ed. 42, 2946−2974.
(2) Maga, G., and Hubscher, U. (2008) Repair and translesion DNA
polymerases as anticancer drug targets. Anti-Cancer Agents Med. Chem.
8, 431−447.
(21) Nick McElhinny, S. A., Havener, J. M., Garcia-Diaz, M., Juarez,
R., Bebenek, K., Kee, B. L., Blanco, L., Kunkel, T. A., and Ramsden, D.
A. (2005) A gradient of template dependence defines distinct
biological roles for family X polymerases in nonhomologous end
joining. Mol. Cell 19, 357−366.
̈
(3) Lange, S. S., Takata, K.-i., and Wood, R. D. (2011) DNA
polymerases and cancer. Nat. Rev. Cancer 11, 96−110.
(4) Loeb, L. A., and Monnat, R. J., Jr. (2008) DNA polymerases and
human disease. Nat. Rev. Genet. 9, 594−604.
(22) Picher, A. J., and Blanco, L. (2007) Human DNA polymerase
lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-
oxoguanine. DNA Repair 6, 1749−1756.
(23) Maga, G., Villani, G., Crespan, E., Wimmer, U., Ferrari, E.,
(5) Hubscher, U., Spadari, S., Villani, G., and Maga, G. (2010) DNA
̈
Bertocci, B., and Hubscher, U. (2007) 8-Oxo-guanine bypass by
̈
Polymerases: Discovery, Characterization and Functions in Cellular DNA
human DNA polymerases in the presence of auxiliary proteins. Nature
447, 606−608.
Transactions; World Scientific: Singapore.
(6) Mayer, T. U. (2003) Chemical genetics: Tailoring tools for cell
biology. Trends Cell Biol. 13, 270−277.
(7) Florian, S., Hummer, S., Catarinella, M., and Mayer, T. U. (2007)
Chemical genetics: Reshaping biology through chemistry. HFSP J. 1,
104−114.
(8) Chang, L. M. S., and Bollum, F. J. (1971) Low molecular weight
deoxyribonucleic acid polymerase in mammalian cells. J. Biol. Chem.
246, 5835−5837.
(9) Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, L. A., de
Lera, L. T., Saniger, M. L., Ruiz, J. F., Parraga, M., Garcia-Ortiz, M. J.,
Kirchhoff, T., del Mazo, J., Bernad, A., and Blanco, L. (2000) DNA
polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase
with a potential role in meiosis. J. Mol. Biol. 301, 851−867.
(10) Garcia-Diaz, M., Bebenek, K., Krahn, J. M., Kunkel, T. A., and
Pedersen, L. C. (2005) A closed conformation for the Pol lambda
catalytic cycle. Nat. Struct. Mol. Biol. 12, 97−98.
(24) van Loon, B., and Hubscher, U. (2009) An 8-oxo-guanine repair
̈
pathway coordinated by MUTYH glycosylase and DNA polymerase
lambda. Proc. Natl. Acad. Sci. U. S. A. 106, 18201−18206.
(25) Markkanen, E., van Loon, B., Ferrari, E., Parsons, J. L., Dianov,
G. L., and Hubscher, U. (2012) Regulation of oxidative DNA damage
̈
repair by DNA polymerase lambda and MutYH by cross-talk of
phosphorylation and ubiquitination. Proc. Natl. Acad. Sci. U. S. A. 109,
437−442.
(26) Braithwaite, E. K., Kedar, P. S., Stumpo, D. J., Bertocci, B.,
Freedman, J. H., Samson, L. D., and Wilson, S. H. (2010) DNA
polymerases β and λ mediate overlapping and independent roles in
base excision repair in mouse embryonic fibroblasts. PLoS One 5,
No. e12229.
(27) Zucca, E., Bertoletti, F., Wimmer, U., Ferrari, E., Mazzini, G.,
Khoronenkova, S., Grosse, N., van Loon, B., Dianov, G., Hubscher, U.,
̈
and Maga, G. (2013) Silencing of human DNA polymerase lambda
causes replication stress and is synthetically lethal with an impaired S
phase checkpoint. Nucleic Acids Res. 41, 229−241.
(11) Almeida, K. H., and Sobol, R. W. (2007) A unified view of base
excision repair: Lesion-dependent protein complexes regulated by
post-translational modification. DNA Repair (Amst) 6, 695−711.
(12) Barakat, K., Gajewski, M., and A. Tuszynski, J. (2012) DNA
repair inhibitors: The next major step to improve cancer therapy. Curr.
Top. Med. Chem. 12, 1376−1390.
(28) Albertella, M. R., Lau, A., and O’Connor, M. J. (2005) The
overexpression of specialized DNA polymerases in cancer. DNA Repair
4, 583−593.
(29) Fu, D., Calvo, J. A., and Samson, L. D. (2012) Balancing repair
and tolerance of DNA damage caused by alkylating agents. Nat. Rev.
Cancer 12, 104−120.
(13) Blanca, G., Villani, G., Shevelev, I., Ramadan, K., Spadari, S.,
Hubscher, U., and Maga, G. (2004) Human DNA polymerases λ and β
̈
show different efficiencies of translesion DNA synthesis past abasic
sites and alternative mechanisms for frameshift generation. Bio-
chemistry 43, 11605−11615.
(14) Boudsocq, F., Benaim, P., Canitrot, Y., Knibiehler, M., Ausseil,
F., Capp, J. P., Bieth, A., Long, C., David, B., Shevelev, I., Frierich-
(30) Summerer, D., and Marx, A. (2002) A molecular beacon for
quantitative monitoring of the DNA polymerase reaction in real-time.
Angew. Chem., Int. Ed. 41, 3620−3622.
(31) Locatelli, G. A., Di Santo, R., Crespan, E., Costi, R., Roux, A.,
Hubscher, U., Shevelev, I., Blanca, G., Villani, G., Spadari, S., and
̈
Heinecken, E., Hubscher, U., Amalric, F., Massiot, G., Hoffmann, J. S.,
̈
Maga, G. (2005) Diketo hexenoic acid derivatives are novel selective
non-nucleoside inhibitors of mammalian terminal deoxynucleotidyl
transferases, with potent cytotoxic effect against leukemic cells. Mol.
Pharmacol. 68, 538−550.
and Cazaux, C. (2005) Modulation of cellular response to cisplatin by
a novel inhibitor of DNA polymerase β. Mol. Pharmacol. 67, 1485−
1492.
(15) Garcia-Diaz, M., Bebenek, K., Kunkel, T. A., and Blanco, L.
(2001) Identification of an intrinsic 5′-deoxyribose-5-phosphate lyase
activity in human DNA polymerase λ. J. Biol. Chem. 276, 34659−
34663.
(32) Strittmatter, T., Bareth, B., Immel, T. A., Huhn, T., Mayer, T.
U., and Marx, A. (2011) Small molecule inhibitors of human DNA
polymerase λ. ACS Chem. Biol. 6, 314−319.
(33) Jaiswal, A. S., Banerjee, S., Aneja, R., Sarkar, F. H., Ostrov, D. A.,
and Narayan, S. (2011) DNA polymerase β as a novel target for
chemotherapeutic intervention of colorectal cancer. PLoS One 6,
No. e16691.
(34) Coggins, G. E., Maddukuri, L., Penthala, N. R., Hartman, J. H.,
Eddy, S., Ketkar, A., Crooks, P. A., and Eoff, R. L. (2013) N-Aroyl
indole thiobarbituric acids as inhibitors of DNA repair and replication
stress response polymerases. ACS Chem. Biol. 8, 1722−1729.
(35) Tomasic, T., and Masic, L. P. (2009) Rhodanine as a privileged
scaffold in drug discovery. Curr. Med. Chem. 16, 1596−1629.
(36) Welsch, M. E., Snyder, S. A., and Stockwell, B. R. (2010)
Privileged scaffolds for library design and drug discovery. Curr. Opin.
Chem. Biol. 14, 347−361.
(16) Shimazaki, N., Yoshida, K., Kobayashi, T., Toji, S., Tamai, K.,
and Koiwai, O. (2002) Over-expression of human DNA polymerase
lambda in E. coli and characterization of the recombinant enzyme.
Genes Cells 7, 639−651.
(17) Ramadan, K., Shevelev, I. V., Maga, G., and Hubscher, U.
̈
(2002) DNA polymerase lambda from calf thymus preferentially
replicates damaged DNA. J. Biol. Chem. 277, 18454−18458.
(18) Ramadan, K., Maga, G., Shevelev, I. V., Villani, G., Blanco, L.,
and Hubscher, U. (2003) Human DNA polymerase lambda possesses
̈
terminal deoxyribonucleotidyl transferase activity and can elongate
RNA primers: Implications for novel functions. J. Mol. Biol. 328, 63−
72.
(19) Ramadan, K., Shevelev, I., and Hubscher, U. (2004) The DNA-
̈
(37) Mendgen, T., Steuer, C., and Klein, C. D. (2012) Privileged
scaffolds or promiscuous binders: A comparative study on rhodanines
and related heterocycles in medicinal chemistry. J. Med. Chem. 55,
743−753.
(38) Toma, T., Peterlin, M., and Lucija (2012) Rhodanine as a
scaffold in drug discovery: A critical review of its biological activities
polymerase-X family: Controllers of DNA quality? Nat. Rev. Mol. Cell.
Biol. 5, 1038−1043.
(20) Lee, J. W., Blanco, L., Zhou, T., Garcia-Diaz, M., Bebenek, K.,
Kunkel, T. A., Wang, Z., and Povirk, L. F. (2004) Implication of DNA
polymerase lambda in alignment-based gap filling for nonhomologous
H
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
and mechanisms of target modulation. Expert Opin. Drug Discovery 7,
549−560.
differentially coordinated by proliferating cell nuclear antigen and
replication protein A. J. Biol. Chem. 280, 1971−1981.
(39) Mizushina, Y., Saito, A., Tanaka, A., Nakajima, N., Kuriyama, I.,
Takemura, M., Takeuchi, T., Sugawara, F., and Yoshida, H. (2005)
Structural analysis of catechin derivatives as mammalian DNA
polymerase inhibitors. Biochem. Biophys. Res. Commun. 333, 101−109.
(54) Ramadan, K., Shevelev, I. V., Maga, G., and Hubscher, U.
̈
(2004) De novo DNA synthesis by human DNA polymerase lambda,
DNA polymerase mu and terminal deoxyribonucleotidyl transferase. J.
Mol. Biol. 339, 395−404.
(55) Ma, J., Starck, S. R., and Hecht, S. M. (1999) DNA polymerase
β inhibitors from Tetracera boiviniana. J. Nat. Prod. 62, 1660−1663.
(56) Gao, Z., Maloney, D. J., Dedkova, L. M., and Hecht, S. M.
(2008) Inhibitors of DNA polymerase β: Activity and mechanism.
Bioorg. Med. Chem. 16, 4331−4340.
(57) Ogawa, A., Murate, T., Suzuki, M., Nimura, Y., and Yoshida, S.
(1998) Lithocholic acid, a putative tumor promoter, inhibits
mammalian DNA polymerase β. Cancer Sci. 89, 1154−1159.
(58) Corazza, N., Jakob, S., Schaer, C., Frese, S., Keogh, A., Stroka,
D., Kassahn, D., Torgler, R., Mueller, C., Schneider, P., and Brunner,
T. (2006) TRAIL receptor-mediated JNK activation and Bim
phosphorylation critically regulate Fas-mediated liver damage and
lethality. J. Clin. Invest. 116, 2493−2499.
(59) Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., Mortelmans,
K., and Speck, W. (1987) Salmonella mutagenicity tests: III. Results
from the testing of 255 chemicals. Environ. Mutagen. 9 (Suppl. 9), 1−
109.
(60) Hotta, N., Akanuma, Y., Kawamori, R., Matsuoka, K., Oka, Y.,
Shichiri, M., Toyota, T., Nakashima, M., Yoshimura, I., Sakamoto, N.,
and Shigeta, Y. (2006) Long-term clinical effects of epalrestat, an
aldose reductase inhibitor, on diabetic peripheral neuropathy: the 3-
year, multicenter, comparative aldose reductase inhibitor-diabetes
complications trial. Diabetes Care 29, 1538−1544.
(40) Muller, J., Sidler, D., Nachbur, U., Wastling, J., Brunner, T., and
̈
Hemphill, A. (2008) Thiazolides inhibit growth and induce
glutathione-S-transferase Pi (GSTP1)-dependent cell death in
human colon cancer cells. Int. J. Cancer 123, 1797−1806.
(41) Martin, S. A., McCabe, N., Mullarkey, M., Cummins, R.,
Burgess, D. J., Nakabeppu, Y., Oka, S., Kay, E., Lord, C. J., and
Ashworth, A. (2010) DNA polymerases as potential therapeutic targets
for cancers deficient in the DNA mismatch repair proteins MSH2 or
MLH1. Cancer Cell 17, 235−248.
(42) Sidler, D., Brockmann, A., Mueller, J., Nachbur, U., Corazza, N.,
Renzulli, P., Hemphill, A., and Brunner, T. (2012) Thiazolide-induced
apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim
axis and reveals glutathione-S-transferase P1 as Achilles’ heel. Oncogene
31, 4095−4106.
(43) Ladduwahetty, T., Gilligan, M., Humphries, A., Merchant, K. J.,
Fish, R., McAlister, G., Ivarsson, M., Dominguez, M., O’Connor, D.,
and MacLeod, A. M. (2010) Non-basic ligands for aminergic GPCRs:
The discovery and development diaryl sulfones as selective, orally
bioavailable 5-HT2A receptor antagonists for the treatment of sleep
disorders. Bioorg. Med. Chem. Lett. 20, 3708−3712.
(44) Ishida, T., In, Y., Inoue, M., Ueno, Y., Tanaka, C., and
Hamanaka, N. (1989) Structural elucidation of epalrestat(ONO-
2235), a potent aldose reductase inhibitor, and isomerization of its
double bonds. Tetrahedron Lett. 30, 959−962.
(45) Xia, Z., Knaak, C., Ma, J., Beharry, Z. M., McInnes, C., Wang,
W., Kraft, A. S., and Smith, C. D. (2009) Synthesis and evaluation of
novel inhibitors of Pim-1 and Pim-2 protein kinases. J. Med. Chem. 52,
74−86.
(46) Anderluh, M., Jukic, M., and Petric, R. (2009) Three-
̌ ̌
component one-pot synthetic route to 2-amino-5-alkylidene-thiazol-
4-ones. Tetrahedron 65, 344−350.
́ ́
(47) Vickers, C. J., Gonzalez-Paez, G. E., Umotoy, J. C., Cayanan-
Garrett, C., Brown, S. J., and Wolan, D. W. (2013) Small-molecule
procaspase activators identified using fluorescence polarization.
ChemBioChem 14, 1419−1422.
(48) Tomasi
̌
c,
́
T., Zidar, N., Kovac,
M., Grdadolnik, S. G., Zega, A.,
Anderluh, M., Gobec, S., Kikelj, D., and Peterlin Masic, L. (2010) 5-
̌ ̌ ̌
A., Turk, S., Simcic, M., Blanot,
D., Muller-Premru, M., Filipic,
̌
̈
̌
̌
Benzylidenethiazolidin-4-ones as multitarget inhibitors of bacterial
Mur ligases. ChemMedChem 5, 286−295.
̌ ́ ̌ ̌
(49) Zidar, N., Tomasic, T., sink, R., Rupnik, V., Kovac, A., Turk, S.,
Patin, D., Blanot, D., Contreras Martel, C., Dessen, A. a., Muller
̈
̌ ̌
Premru, M., Zega, A., Gobec, S., Peterlin Masic, L., and Kikelj, D.
(2010) Discovery of novel 5-benzylidenerhodanine and 5-benzylide-
nethiazolidine-2,4-dione inhibitors of MurD ligase. J. Med. Chem. 53,
6584−6594.
(50) Unangst, P. C., Connor, D. T., Cetenko, W. A., Sorenson, R. J.,
Kostlan, C. R., Sircar, J. C., Wright, C. D., Schrier, D. J., and Dyer, R.
D. (1994) Synthesis and biological evaluation of 5-[[3,5-bis(1,1-
dimethylethyl)-4-hydroxyphenyl]methylene]oxazoles, -thiazoles, and
-imidazoles: novel dual 5-lipoxygenase and cyclooxygenase inhibitors
with antiinflammatory activity. J. Med. Chem. 37, 322−328.
(51) Kottakota, S. K., Benton, M., Evangelopoulos, D., Guzman, J. D.,
Bhakta, S., McHugh, T. D., Gray, M., Groundwater, P. W., Marrs, E. C.
L., Perry, J. D., and Harburn, J. J. (2012) Versatile routes to marine
sponge metabolites through benzylidene rhodanines. Org. Lett. 14,
6310−6313.
(52) Nakamura, K., Fujii, M., Ohno, A., and Oka, S. (1984)
NAD(P)+-NAD(P)H model. 52. Reduction of olefins by hantzsch
ester on silica gel. Tetrahedron Lett. 25, 3983−3986.
(53) Maga, G., Ramadan, K., Locatelli, G. A., Shevelev, I., Spadari, S.,
and Hubscher, U. (2005) DNA elongation by the human DNA
̈
polymerase lambda polymerase and terminal transferase activities are
I
dx.doi.org/10.1021/cb4007562 | ACS Chem. Biol. XXXX, XXX, XXX−XXX