4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors

Article abstract of DOI:10.1021/jm901061s

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes the hydrolysis of 3′-phosphotyrosyl bonds. Such linkages form in vivo when topoisomerase I (Top1) processes DNA. For this reason, Tdp1 has been implicated in the repair of irreversible Top

Full text of DOI:10.1021/jm901061s

7122 J. Med. Chem. 2009, 52, 7122–7131
DOI: 10.1021/jm901061s
4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and Related Novel Steroid
Derivatives as Tyrosyl-DNA Phosphodiesterase (Tdp1) Inhibitors
Thomas S. Dexheimer,† Lalji K. Gediya,‡,§ Andrew G. Stephen, Iwona Weidlich,^,¥ Smitha Antony,† Christophe Marchand,†
Heidrun Interthal,# Marc Nicklaus,^ Robert J. Fisher, Vincent C. Njar,‡,§ and Yves Pommier*,†
†Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20892, ‡Department of Pharmacology and Experimental Therapeutics, School of Medicine and the Greenebaum Cancer Center,
University of Maryland, Baltimore, Maryland 21201, §Department of Pharmaceutical Sciences, Jefferson School of Pharmacy, Thomas Jefferson
University, 130 South Ninth Street, Philadelphia, Pennsylvania 19107, Protein Chemistry Laboratory, Advanced Technology Program, SAIC;
Frederick, Inc., NCI Frederick, Frederick, Maryland 21702, ^Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Frederick, Maryland 21702, and #Institute of Cell Biology, University of Edinburgh, Edinburgh, U.K.
¥ On leave from the Poznanꢀ University of Medical Sciences, Faculty of Pharmacy, Poland.
Received July 17, 2009
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes the hydrolysis of 30-phosphotyro-
syl bonds. Such linkages form in vivo when topoisomerase I (Top1) processes DNA. For this reason,
Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes. Tdp1 inhibitors
have been regarded as potential therapeutics in combination with Top1 inhibitors, such as the
camptothecin derivatives, topotecan, and irinotecan, which are used to treat human cancers. Using a
novel high-throughput screening assay, we have identified the C21-substituted progesterone derivative,
NSC 88915 (1), as a potential Tdp1 inhibitor. Secondary screening and cross-reactivity studies with
related DNA processing enzymes confirmed that compound 1 possesses specific Tdp1 inhibitory
activity. Deconstruction of compound 1 into discrete functional groups reveals that both components
are required for inhibition of Tdp1 activity. Moreover, the synthesis of analogues of compound 1 has
provided insight into the structural requirements for the inhibition of Tdp1. Surface plasmon resonance
shows that compound 1 binds to Tdp1, whereas an inactive analogue fails to interact with the enzyme.
On the basis of molecular docking and mechanistic studies, we propose that these compounds are
competitive inhibitors, which mimics the oligonucleotide-peptide Tdp1 substrate. These steroid
derivatives represent a novel chemotype and provide a new scaffold for developing small molecule
inhibitors of Tdp1.
Introduction
DNA lesions (for reviews, see refs 10-12), such as strand
breaks, abasic sites, base mismatches, and specific oxidized or
modified bases. Top1-DNA cleavage complexes caused by
DNA lesions have the propensity to self-sufficiently yield
abortive Top1 cleavage complexes, whereas the reversible
drug-stabilized Top1 cleavage complexes require conversion
to Top1-linked DNA strand breaks by collision of DNA and
RNA polymerases during replication and transcription, re-
spectively (for reviews, see refs 1 and 10). Consequently, these
irreversible Top1-DNA lesions confer a unique barrier for the
DNA repairmachinery, since the DNAstrandbreak isencumbered
with a 30-protein adduct.
Topoisomerase I (Top1a) inhibitorsrepresent animportant
class of chemotherapeutic drugs with distinct mechanisms of
damaging DNA (for review, see ref 1). Among these are the
camptothecin (CPT) derivatives topotecan and irinotecan,
which are used clinically in cancer chemotherapy. Currently,
newerCPTderivatives, suchasgimatecan (Novartis),2 S39625
(Servier),3 and diflomotecan,4 are in clinical development. In
addition, several non-CPT Top1 inhibitors including the
indenoisoquinolines, the phenanthrolines, and the indolocar-
bazoles, have been identified and are currently undergoing
clinical development (for reviews, see refs 5-7).
Tyrosyl-DNA phosphodiesterase (Tdp1) has been asso-
ciated with the repair of Top1 cleavage complexes by virtue
of its ability to hydrolyze the phosphodiester linkage between
a tyrosine residue and a DNA 30-phosphate.13,14 Besides the
Top1-derived phosphotyrosyl bond, Tdp1 has been shown to
hydrolyze other covalently linked 30-blocking lesions,
although less efficiently than 30-phosphotyrosyl ends.15 For
example, Tdp1 has been shown to cleave 30-terminal phos-
phoglycolate diester linkages, which are commonly generated
by oxidative DNA damage.16 Interestingly, cells harboring
the disease-associated Tdp1 SCAN1 (spinocerebellar ataxia
with axonal neuropathy-1) mutation are hypersensitive to
Mechanistically, Top1 inhibitors selectively bind to the
Top1-DNA interface and damage DNA by trapping the
cleavage complex between the Top1 catalytic tyrosine and the
30-end of the broken DNA.8,9 Likewise, Top1 cleavage com-
plexes have also been shown to accumulate at preexisting
*To whom correspondence should be addressed. Phone: 301-496-
5944. Fax: 301-402-0752. E-mail: pommier@nih.gov.
a Abbreviations: Tdp1, tyrosyl-DNA phosphodiesterase; Top1, to-
poisomerase I; CPT; camptothecin; SCAN1, spinocerebellar ataxia with
axonal neuropathy-1; IC50, half maximal (50%) inhibitory concentra-
tion.
r
pubs.acs.org/jmc
Published on Web 11/02/2009
2009 American Chemical Society

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7123
Figure 1. Tdp1 inhibition by 1. (A) Chemical structure of 1. (B) Schematic representation of the Tdp1 gel-based biochemical assay. Tdp1
hydrolyzes the 30-phosphotyrosine bond and converts N14Y to an oligonucleotide containing a 30-phosphate (N14P). (C) Representative gel
demonstrating dose-dependent inhibition of Tdp1 by 1. (D) Graphical representation of the percent inhibition of Tdp1 by 1. Each point
represents the mean ( SEM of three independent experiments.
both CPT and oxidative stress (i.e., H2O2 and ionizing
radiation).17-20 Cell extracts from SCAN1 cells have also
been shown to be deficient in processing 30-phosphoglyco-
lates.21,22 Moreover, CPT-treated skin fibroblasts from
SCAN1 patients have been shown to accumulate Tdp1-
DNA intermediates wherein the mutant form of Tdp1
(H493R) becomes covalently linked to DNA, which provides
in vivo evidence for the involvement of Tdp1 in the removal of
drug-induced Top1-DNA cleavage complexes.23
In addition to studies performed with the physiologically
relevant SCAN1 Tdp1 mutant, the recent generation of Tdp1
knockout mice further establishes the function of Tdp1 in the
repair of Top1-DNA cleavage complexes and oxidative
DNA damage. Specifically, primary neural cells from
Tdp1-/- mice have been shown to accrue more total DNA
strand breaks than wild-type cells after treatment with CPT,
H2O2 or ionizing radiation.24 Both Tdp1-/- mice and cells
derived from Tdp1-/- mice are hypersensitive to the Top1
inhibitors.23,24 Taken together, these studies demonstrate that
a single defect in Tdp1 activity is sufficient for Top1 inhibitor
hypersensitivity.
In corroboration, two independent studies have shown that
overexpression of wild-type Tdp1 in human cells protects
against CPT-induced cell death,25,26 whereas the catalytically
inactive Tdp1 mutant does not.25 A recent study has also
observed an increase in expression and activity of Tdp1 in
greater than 50% of the non-small cell lung cancer tissue
samples analyzed compared to non-neoplastic tissues.27 Thus,
the presence and activity of Tdp1 are consistent with a role for
the enzyme in protecting cells against the cytotoxic effects of
Top1 inhibitors. It is therefore logical to develop inhibitors of
Tdp1 to counteract the inherited resistance to Top1 inhibitors
caused by the Tdp1-mediated repair of Top1-DNA lesions.
Tdp1 inhibitors may possibly augment current radiotherapy
as well.
At present, only a small number of Tdp1 inhibitors have
been characterized. Although unattractive as pharmacologi-
cal inhibitors of Tdp1, both vanadate and tungstate, which
inhibit Tdp1 at millimolar concentrations, have been useful in
cocrystallization studies of Tdp1.28,29 The aminoglycoside
antibiotic, neomycin B, has also been examined as a potential
Tdp1 inhibitor based on its ability to target members of the
phospholipase D superfamily.30 In addition, recent high-
throughput screening efforts have identified furamidine31
and several phosphotyrosine mimetics as Tdp1 inhibitors.32
In this report, we characterize a new chemotype of fully
synthetic small molecule inhibitors of Tdp1 that were origin-
ally identified in a high-throughput screen.31,33 We demon-
strate that the lead compound (1; see Figure 1A) blocks the
formation of the initial Tdp1-DNA covalent intermediate
through the use of the SCAN1 Tdp1 mutant enzyme. In
addition, molecular docking of the inhibitor into the active
site of Tdp1 suggests that it competes for binding by mimick-
ing the Tdp1 substrate.
Experimental Section
Chemistry. General procedures and techniques were identical
with those previously reported.34 1H NMR spectra were re-
corded in CDCl3 or DMSO-d6 at 500 MHz with Me4Si as an
internal standard using a Varian Inova 500 MHz spectrometer.
High-resolution mass spectra (HRMS) were determined on a
Bruker 12 T APEX-Qe FTICR-MS by positive ion ESI mode by
Susan A. Hatcher, Facility Director, College of Sciences Major
InstrumentationCluster, OldDominionUniversity, Norfolk, VA.

7124 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Dexheimer et al.
Scheme 1. Synthesis of Deoxycorticosteronebenzene Sulfonates (1-6)a
a Reagents and conditions: (i) THF/Et3N, ArSO2Cl, 0 ꢀC, then 4-8 ꢀC, 24 h.
Deoxycorticosterone and compound 7 were obtained from
Steraloids, Inc., Newport, RI. All other reagents were purchased
from Sigma-Aldrich. Compounds were stored in an atmosphere
of argon and in the cold (-20 or -80 ꢀC).
(s, 1H, C4-H), 7.23 (d, 2H, J = 8.5 Hz, 3050Ar-Hs), 8.00 (d, 2H,
J = 7.5 Hz, 2060Ar-Hs). HRMS calcd 511.1924 (C27H33-
FO5SNaþ), found 511.1912.
4-Pregnen-21-ol-3,20-dione-21-(4-chlorobenzenesufonate) (6).
Mp: 134-135 ꢀC. IR (CHCl3): 1730, 1663, 1577, 1372, 1178, 679
cm-1. 1H NMR (CDCl3) δ 0.65 (s, 3H, 18 -CH3), 1.18 (s, 3H,
19-CH3), 2.59 (m, 2H, 2-CH2), 4.66 (m, 2H, 21-CH2), 5.73 (s,
1H, C4-H), 7.55 (d, 2H, J = 8.5 Hz, 3050Ar-Hs), 7.90 (d, 2þH,
J = 8.5 Hz, 2060Ar-Hs). HRMS calcd 499.2818 (C31H40O4Na ),
found 499.2822.
General Method for the Preparation of Deoxycorticosterone-
21-sulfonates (1-6).35,36 Compounds 1-6 were synthesized
from commercially available deoxycorticosterone using a
simple general procedure as outlined in Scheme 1. To a 0.1 M
solution of the deoxycorticosterone in anhydrous tetrahydro-
furan at 0 ꢀC was added 1.5 equiv of triethylamine, and 1.5 equiv
of 4-substituted benzenesulfonyl chloride was added over 10
min. The reaction mixture (solution) was allowed to stand at
4-8 ꢀC for 24 h. The cold solution was evaporated to dryness,
and to this was added water containing sodium bicarbonate and
extracted with ethyl acetate (50 ꢀ 2 mL). The extract was washed
with brine and dried over sodium sulfate. The solvent was
evaporated and crude product was purified using flash column
chromatograpy [FCC, petroleum ether/EtOAc (7:3, v/v)] to
obtain the various sulfonates in 60-70% yields. The purities
of all final products (compounds 1-6) were confirmed by HPLC
in two solvent systems and high resolution mass spectral data.
All compounds were found to be >95% pure (see Supporting
Information).
Oligonucleotides. The N14Y oligonucleotide (50-GATCTAA-
AAGACTTY-30), which contains a 30-phosphotyrosine (Y),
was synthesized by Midland Certified Reagents Company
(Midland, TX). All of the other DNA oligonucleotides were
synthesized by Integrated DNA Technologies (Coralville, IA).
End-Labeling and Preparation of DNA Substrates. Oligonu-
cleotides were 50-end labeled using T4 polynucleotide kinase and
[γ-32P]ATP. Unincorporated radioactive nucleotides were re-
moved using a mini Quick Spin Oligo column (Roche Diag-
nostics, Indianapolis, IN) after inactivation of the kinase by
heating for 5 min at 95 ꢀC. For construction of double-stranded
DNA substrates, labeled single-stranded oligonucleotides were
annealed with a complementary strand by heating for 5 min at
95 ꢀC and slowly cooling to 25 ꢀC.
4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (1).
Mp: 147-149 ꢀC (lit. 148-150 ꢀC). IR (CHCl3): 1731, 1661,
Expression and Purification of Tdp1. Wild-type and mutant
(H493R) human Tdp1 enzymes were expressed in E. coli BL21
(DE3) cells and purified as described previously.31
1
1576, 1376, 1188, 609 cm-1. H NMR (CDCl3) δ 0.65 (s, 3H,
19-CH3), 1.18 (s, 3H, 18-CH3), 2.59 (m, 2H, 2-CH2), 4.56 (m,
2H, 21-CH2), 5.73 (s, 1H, C4-H), 7.71 (d, 2H, J = 8.5 Hz, 3050Ar-
Hs), 7.81 (d, 2H, J = 8.6 Hz, 2060Ar-Hs). HRMS calcd 571.1124
(C27H33BrO5SNaþ), found 571.1115.
Tdp1 Gel-Based Assay. A 1 nM 50-32P-labeled DNA substrate
was incubated with 0.1 nM recombinant Tdp1 in the absence or
presence of inhibitor for 20 min at 25 ꢀC in a reaction buffer
containing 50 mM Tris-HCl (pH 7.5), 80 mM KCl, 2 mM
EDTA, and 40 μg/mL bovine serum albumin (BSA). Reactions
were terminated by the addition of two volumes of gel loading
buffer (96% (v/v) formamide, 10 mM EDTA, 1% (w/v) xylene
cyanol, and 1% (w/v) bromphenol blue). The samples were
subsequently heated to 95 ꢀC for 5 min and subjected to 18%
sequencing gel electrophoresis. A concentration of 100 nM was
used when employing the SCAN1 mutant H493R. In addition,
H493R reactions were divided in half. One-half of the reaction
was run on a sequencing gel, while the other half was analyzed by
4-20% SDS-PAGE electrophoresis. Imaging and quantifica-
tion were preformed using the Typhoon 8600 and ImageQuant
software (Molecular Dynamics), respectively.
4-Pregnen-21-ol-3,20-dione-21-(4-benzenesufonate) (2). Mp:
166-168 ꢀC. IR (CHCl3): 1730, 1660, 1579, 1380, 1177 cm-1
.
1H NMR (CDCl3) δ 0.64 (s, 3H, 18-CH3), 1.17 (s, 3H, 19-CH3),
2.63 (m, 2H, 2-CH2), 4.58 (m, 2H, 21-CH2), 5.73 (s, 1H, C4-H),
7.59 (m, 2H, 3050Ar-Hs), 7.69 (s, 1H, 40Ar-H), 7.95 (d, 2H, Jþ=
7.0 Hz, 2060Ar-Hs). HRMS calcd 493.2019 (C27H34O5SNa ),
found 493.2005.
4-Pregnen-21-ol-3,20-dione-21-(4-methylbenzenesufonate) (3).
Mp: 160-161 ꢀC, IR (CHCl3): 1731, 1660, 1570, 1384, 1191
cm-1. 1H NMR (CDCl3) δ 0.64 (s, 3H, 18-CH3), 1.17 (s, 3H, 19-
CH3), 2.45 (s, 3H, 40-CH3), 2.65 (m, 2H, 2-CH2), 4.58 (m, 2H, 21-
CH2), 5.73 (s, 1H, C4-H), 7.37 (d, 2H, J = 8.5 Hz, 3050Ar-Hs),
7.83 (d, 2H, J = 8.0 Hz, 2060Ar-Hs). HRMS calcd 507.2175
(C28H36O5SNaþ), found 507.2168.
4-Pregnen-21-ol-3,20-dione-21-(4-nitrobenzenesufonate) (4).
Mp: 137-138 ꢀC, IR (CHCl3): 1733, 1657, 1569, 1373, 1197
cm-1. 1H NMR (CDCl3) δ 0.66 (s, 3H, 18 -CH3), 1.18 (s, 3H,
19-CH3), 2.56 (m, 2H, 2-CH2), 4.78 (m, 2H, 21-CH2), 5.73 (s,
1H, C4-H), 8.17 (d, 2H, J = 7.5 Hz, 3050Ar-Hs), 8.42 (d, 2H, Jþ=
8.0 Hz, 2060Ar-Hs). HRMS calcd 538.1869 (C27H33NO7SNa ),
found 538.1857.
Counterscreen Assays. a. APE1 Assay. A sample of 20 nM
50-32P labeled 21-mer double-stranded DNA containing a single
AP site was incubated with 0.1 units of APE1 (New England
Biolabs, Ipswich, MA) in NEBuffer 4 [20 mM Tris-acetate (pH
7.9), 50 mM potassium acetate, 10 mM magnesium acetate,
1 mM DTT, pH 7.9] for 30 min at 25 ꢀC in a total volume of
10 μL. Reactions were terminated and run analogous to the
Tdp1 gel-based assay described above. Cleavage at the AP site
by APE1 was detected by the appearance of a predominant
second band of higher mobility on the gel.
4-Pregnen-21-ol-3,20-dione-21-(4-flourobenzenesufonate) (5).
Mp: 156-157 ꢀC. IR (CHCl3): 1729, 1665, 1566, 1379, 1177,
1
1011 cm-1. H NMR (CDCl3) δ 0.65 (s, 3H, 18-CH3), 1.18 (s,
3H, 19-CH3), 2.60 (m, 2H, 2-CH2), 4.66 (m, 2H, 21-CH2), 5.73
b. Top1 Relaxation Assay. Recombinant Top1 was incubated
with 0.25 μg of supercoiled ΦX174 DNA for 30 min at 37 ꢀC in a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7125
reaction buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM
KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 μg/mL BSA.
Reactions were terminated by the addition of 0.5% SDS and
0.5 mg/mL proteinase K (final concentrations), followed by
incubation for 30 min at 50 ꢀC. Next, loading dye was added and
the samples were subjected to 1% agarose gel electrophoresis.
After electrophoresis, the gels were stained with 1ꢀ buffer
solution containing 10 μg/mL of ethidium bromide and the
DNA was visualized by transillumination with UV light.
Surface Plasmon Resonance. Direct binding experiments were
performed on a Biacore 2000 instrument (GE, Piscatawy, NJ).
Recombinant Tdp1 was coupled to the carboxylmethylated
dextran surface of a CM5 chip (GE, Piscatawy, NJ) using
standard amine coupling chemistry. Briefly, the surface was
activated with 0.1 M N-hydroxysuccinimide and 0.4 M N-ethyl-
N0-(3-dimethylaminopropyl)carbodiimide at a flow rate of 20
μL/min. Tdp1 was diluted in 10 mM sodium acetate (pH 4.5)
and injected until a density of 1000 RU was attached. Activated
Figure 2. Counterscreening of 1 against apurinic/apyrimidinic endonuclease enzyme (APE1) and topoisomerase I (Top1). (A) Schematic
representation of the APE1 gel-based biochemical assay. APE1 hydrolyzes the DNA phosphodiester backbone 50 of the abasic site (/)
generating an 8-mer oligonucleotide. (B) Representative gel showing that 1 is inactive against APE1. (C) Representative gel showing that 1 is
inactive in a Top1 DNA relaxation assay.
Figure 3. Both the steroid and phenylsulfonyl ester moieties of 1 are required for Tdp1 inhibition. (A) Representative gel demonstrating
dose-dependent inhibition of Tdp1 by 1 and its two parts: progesterone and methyl-p-toluene sulfonate (chemical structures shown above gel).
(B) Graphical representation of the percent inhibition of Tdp1 by 1, progesterone, and methyl-p-toluene sulfonate. Each point represents the
mean ( SEM of three independent experiments.

7126 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Dexheimer et al.
amine groups were quenched with an injection of 1 M ethanol-
amine (pH 8.0). A reference surface was prepared in the same
manner excluding Tdp1. Inhibitors were diluted in running
buffer [10 mM sodium phosphate (pH 7.5), 150 mM NaCl,
and 1% DMSO (v/v)] and injected at 20 μL/min at 25 ꢀC.
A solution of 50 mM NaOH was used to regenerate the surface
after each injection. Each cycle of inhibitor injection was
followed by a running buffer cycle for referencing purposes.
A DMSO calibration curve was included to correct for refractive
index mismatches between the running buffer and inhibitor
dilution series.
derived peptide [PDB accession code 1NOP29] was obtained
from the Protein Data Bank.37 Before the docking procedure,
the X-ray structure was manipulated with the program Maestro
€
(Schrodinger, Inc., Portland, OR). The water molecules and the
cocrystallized DNA-peptide substrate were deleted from the
structure. Hydrogen atoms were assigned, active site residues
(K265 and K495) were neutralized, and a series of minimizations
were performed using the OPLS2005 force field. The lowest
energy conformation of each inhibitor was located by using the
Monte Carlo stochastic dynamics (MCMM) conformational
search routine in Macromodel (version 9.6). In each case, all
rotatable bonds were selected, 50 000 conformations were gen-
erated, and unique conformations within 2 kcal/mol of the
Molecular Docking. The X-ray crystal structure of human
Tdp1 in complex with vanadate, DNA, and a human Top1-
Figure 4. Structure-activity relationship of analogues of 1. (A) Chemical structures of the analogues tested. (B) Representative gel
demonstrating dose-dependent inhibition of Tdp1 by 6 and 7. (C) Graphical representation of the percent inhibition of Tdp1 by 1-7. Each
point represents the mean ( SEM of three independent experiments.
Figure 5. The processing of both single-stranded and duplex substrates by Tdp1 is inhibited by 1. (A) Representative gels showing the
inhibition of Tdp1 activity by 1 using single-stranded, blunt end duplex or 50-overhang duplex substrates. (B) Graphical representation of the
percent inhibition of Tdp1 by 1 using the three different DNA substrates shown in part A. Each point represents the mean ( SEM of three
independent experiments.

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7127
Figure 6. Differential binding of 1 and 7 to Tdp1 using surface plasmon resonance (SPR). SPR sensorgrams show drug interactions with Tdp1.
Ten successive 2-fold dilutions from 100 μM were tested for each inhibitor.
lowest energy conformation were retained. The initial geome-
tries of the structures were optimized using the OPLS 2005 force
field performing 1000 steps of conjugate minimization. Docking
€
was performed using the program Glide (version 5.0, Schrodin-
ger Inc., New York, NY) with Extra Precision mode running on
a Silicon Graphics workstation. The inhibitors were docked into
˚
a 16 A cavity, which was defined around the Tdp1 active site.
Free Energy of Binding Calculation. Theoretical free energy of
binding (FEB) was calculated utilizing the eMBrAcE procedure
€
developed by Schrodinger Inc. as part of the MacroModel
package.38 For each inhibitor, the energies of the enzy-
me-inhibitor complex (Ecomplex), the free enzyme (Eenzyme),
and the free inhibitor (Einhibitor) were all subjected to energy
minimization using the OPLS 2001 force field with a dielectric
constant of 1.0. A conjugate gradient minimization protocol
with default values was used in all minimization. eMBrAcE
minimization calculations were performed using an interaction
mode. The energy difference is then calculated using the equa-
tion: ΔE = Ecomplex - Eenzyme - Einhibitor
Results
Figure 7. Use of the SCAN1 Tdp1 mutant (H493R) to assess the
ability of 1 to inhibit the binding of Tdp1 to DNA. (A) Schematic
representation of covalent intermediate accumulation of the H493R
SCAN1 mutant. (B and C) Representative denaturing DNA-
PAGE gel and protein-SDS-PAGE gel, respectively. Both show
inhibition of Tdp1-DNA complex formation by 1. (D) Graphical
representation of percent Tdp1 binding to DNA as shown in parts B
and C. Each point and bar represent the mean ( SEM of three
independent experiments.
Identification and Confirmation of 4-Pregnen-21-ol-3,20-
dione-21-(4-bromobenzenesufonate) (NSC 88915) as a Tdp1
Inhibitor. We recently implemented a high-throughput elec-
trochemiluminescence assay to identify small molecule in-
hibitors of Tdp1.31,33 In screening the 1981 compounds from
the “diversity set” of the NCI-Developmental Therapeutics
Program, we identified a C21-substituted progesterone deri-
vative, 1 (Figure 1A), which exhibited inhibitory activity
against Tdp1 at low micromolar concentrations.31 The in-
hibitory activity was confirmed using a gel-based assay,
which utilizes a 50-[32P]-labeled single-stranded oligonucleo-
tide comprising a 30-phosphotyrosine (N14Y). Recombinant
Tdp1 hydrolyzes the phosphodiester linkage between the
tyrosine and the 30-end of this DNA substrate, resulting in
the generation of an oligonucleotide with a free 30-phosphate
end (N14P) (Figure 1B,C).13 Through the analysis of con-
centration versus percentage inhibition curves, we estimated
the IC50 value for 1 as 7.7 ( 0.8 μM. (Figure 1D).
100 μM, Figure 2). These data suggest that the inhibitory
action of 1 on Tdp1 is independent of DNA interactions.
Importance of Both the Steroid and the Phenyl Sulfonyl
Ester Moieties. To establish the relative contribution of
specific substructures toward the overall Tdp1 inhibitory
activity, we deconstructed 1 into two available fragments.
Progesterone and methyl-p-toluene sulfonate were used to
mimic the steroid and phenylsulfonyl ester moieties, respec-
tively (see structures in Figure 3A). While progesterone was
weakly active, methyl-p-toluene sulfonate remained inactive
even at 1 mM, failing to give an IC50 value (Figure 3A,B).
Testosterone was also assayed, yet showed activity similar to
that of progesterone (data not shown). Thus, both the
combination of the steroid and phenylsulfonyl ester frag-
ments is essential for the activity of 1 against Tdp1.
To evaluate the selectivity of 1 for Tdp1, we coun-
terscreened 1 against two different enzymes that interact
and process DNA. No evidence for inhibition of the DNA
repair enzyme, apurinic/apyrimidinic endonuclease (APE1),
or human Top1 was achieved in the presence of 1 (up to

7128 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Dexheimer et al.
Figure 8. Molecular docking of inhibitors into the active site of Tdp1. (A) Overlay of Tdp1 substrate and the best potential docking pose of
1 within the active site of Tdp1. The surface of the protein is shown in gray with the active site residues (H263, K265, H493, and K495)
highlighted in pink. The Tdp1 substrate is colored by atom type: carbon in white, nitrogen in blue, oxygen in red, phosphorus in orange, and
vanadate in black. The inhibitor is colored by atom type: carbon in green, oxygen in red, sulfur in yellow, and bromine in brown. (B) Linear
correlation between the experimental activities (log 1/IC ) attained from the Tdp1 gel-based assay and the calculated free energy of binding from
50
docking for 1-7 (see Figure 4).
The importance of the phenylsulfonyl ester moiety became
more evident following structure-activity relationship stu-
dies. Initial replacement of the p-bromide in 1 by various
substituents (R = H, CH3, NO2, F, and Cl; see 2-6 in Figure
4A) resulted in minimal changes in the Tdp1 inhibition (<3-
fold change in IC50 values, Figure 4B,C). More interestingly,
the mesylate analogue (7) (Figure 4A) was minimally active
(Figure 4B,C). These results indicate that Tdp1 inhibition is
dependent on the presence of the phenyl ring attached via
sulfonyl ester bond at the C21-position of these steroid
derivatives.
Inhibition of Tdp1 Processing of Both Single- and Double-
Stranded DNA Substrates. As reported previously, Tdp1
can utilize both single- and double-stranded DNA sub-
strates.39,40 Thus, in addition to the single-stranded
N14Y substrate (Figures 1 and 3), we assayed the activity
of 1 using double-stranded DNA substrates bearing a
blunt or 22-base recessed 30-phosphotyrosine terminus
(Figure 5A). As demonstrated in Figure 5A,B, Tdp1 inhibi-
tion was similar with both the single- and double-stranded
DNA substrates. These findings provide additional evidence
that the steroid derivatives do not exert their Tdp1 inhibitory
effects through binding or intercalating with the DNA
substrate.
Direct Biophysical Analysis of Inhibitor Binding to Recom-
binant Tdp1 Using Surface Plasmon Resonance. To better
understand the Tdp1-inhibitor interactions and measure
the affinity of 1 to Tdp1, we used surface plasmon resonance
(SPR). Tdp1 was amine-coupled to a sensor chip, and the
drug binding was determined in a Biacore 2000. In Figure 6
(left), the increase in resonance units from the initial baseline
represents the binding of 1 to the surface-bound Tdp1. The
plateau corresponds to the steady-state phase of the
Tdp1-inhibitor interaction, while the decrease in response
units represents the dissociation or reversibility of the inter-
action. No interaction with Tdp1 was detected for the weakly

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7129
active inhibitor 7 (Figure 6, right). These experiments de-
monstrate direct binding of 1 to Tdp1.
repair inhibitors also have the potential to be used as mono-
therapies given that most cancer cells are deficient in one or
more of the DNA damage response pathways, making them
hyperdependent upon the remaining functional pathways (for
reviews, see refs 44, 33, and 45). Thus, inactivation of these
compensatory repair pathways may lead to an increase of
endogenous tumor-specific DNA lesions and selective killing
of cancer cells, while normal cells would remain resistant
owing to the redundancy in their DNA repair systems. The
most noteworthy examples of this concept are the PARP
inhibitors, which have shown significant therapeutic potential
in BRCA1- and BRCA2-deficient tumors46,47 and in combi-
nation with gemcitabine48 and Top1 inhbititors.49
Cellular sensitivity or resistance to Top1 inhibitors is likely
determined by the loss or increased activity of specific DNA
repair pathways, respectively.50 Thus, a viable therapeutic
approach to enhance the pharmacological action of Top1
inhibitors is inactivation of the pathways involved in the
repair of drug-induced Top1-mediated DNA damage.
Top1-DNA covalent complexes or the presumed cytotoxic
lesions generated by the Top1 inhibitors are the primary
substrates for Tdp1.14,15 Tdp1 has also been shown to process
30-phosphoglycolates, although less efficiently than 30-phos-
photyrosylmoieties.16 Thus, Tdp1 inhibitorsare envisioned to
enhance the antipoliferative effects of Top1 inhibitors as well
as ionizing radiation. The observation that Tdp1 knockout
mice are hypersensitive to camptothecins and bleomycin
provides proofofprinciplefor suchstrategies.23,24 Inaddition,
the viability and mild phenotype of Tdp1 knockout mice
are indicative of a lack of severe side effects when applying
Tdp1 inhibitors.23,24 However, an alternative checkpoint-
mediated nucleolytic pathway involving at least three endo-
nuclease complexes [yMus81/yMms4 (hMus81/hEme1),
yMre11/yRad50/yXrs2 (hMre11/hRad50/hNbs1), yRad1/
yRad10 (hXPF/hERCC1)] has also been demonstrated
through genetic analysis in yeast cells.40,51-54 Although re-
dundancy exists in the repair of Top1-DNA lesions, Tdp1
inhibitors have the potential to provide an increased selectiv-
ity and a broader therapeutic window for Top1 inhibitors
given that defects in one or more DNA damage repair path-
ways are a commoncharacteristic ofcancer cells, aspreviously
mentioned. Thus, in an effort to extend this new paradigm of
targeting DNA repair for anticancer therapy, we have in-
itiated a search for selective inhibitors of Tdp1.31,32
To date, only a limited number of Tdp1 inhibitor chemo-
types have been reported.30-33 Here, we describe the identifi-
cation, synthesis, and in vitro biochemical evaluation of a new
chemical family of steroidal Tdp1 inhibitors, which were
identified in a high-throughput screen.31 The initial positive
hit, 1, inhibits Tdp1 activityatlow micromolar concentrations
(Figure 1). We also demonstrate the selectivity of 1 for Tdp1
by counterscreening against other DNA processing enzymes
(Figure 2). Initially, we explored the structure-activity rela-
tionship of 1 by asking whether parsing the compound into
two fragments, which were related components of the larger
molecule (i.e., progesterone and methyl-p-toluene sulfonate;
see Figure 3), maintained Tdp1 inhibitory activity. As shown
in Figure 3, we found that the composite structure was much
more active than the isolated fragments. Another key struc-
tural modification we examined was the incorporation of
functional groups at the para-position of the phenyl ring.
Our results demonstrate that a wide variety of substituents
were tolerated at this position without seriously reducing the
activity against Tdp1, whereas complete elimination of the
Mechanistic Insights of Tdp1 Inhibition by the Steroid
Derivatives Using the SCAN1 Mutant. In human Tdp1
cleavage reactions two active site histidines (H263 and H493)
perform two successive nucleophilic attacks to hydrolyze the
phosphotyrosyl Top1-DNA bond. The first step involves
an enzyme-substrate intermediate between H263 and the
30-DNA end.19,28,41 The second histidine, H493, is critical
for hydrolysis of this Tdp1-DNA covalent intermediate
(Figure 7A). The homozygous H493R mutation associated
with the recessive neurodegenerative disease SCAN1 has been
shown to preferentially disrupt the second step of the Tdp1
reaction [i.e., the hydrolysis of the phosphohistidine (H263)
intermediate] resulting in an accumulation of the Tdp1-DNA
covalent intermediate (Figure 7A).19,23,42 We took advantage of
the catalytic properties of the Tdp1 H493R mutant to elucidate
the mode of inhibition by 1. In the absence of the Tdp1
inhibitor, the H493R mutant accumulates Tdp1-DNA com-
plexes, as demonstrated by the appearance of DNA-labeled
material in the wells of DNA sequencing gels (Figure 7B) and as
a DNA-labeled slowly migrating band in protein SDS-PAGE
gels (Figure 7C).32 In the presence of 1, a concentration-
dependent decrease of the Tdp1-DNA complexes was ob-
served in both DNA sequencing and protein SDS-PAGE gels
(Figure 7D). Inhibition in the formation of Tdp1-DNA
covalent intermediate indicates that 1 interferes with the bind-
ing of the DNA substrate to the Tdp1 active site (Figure 7A).
Molecular Docking of Steroid Derivatives into the Active
Site of Tdp1 and the Correlation of Binding Free Energies with
Inhibitory Activities. To investigate the binding mode of the
steroid derivatives to Tdp1 at the molecular level, we per-
formed docking analysis using the high-resolution structure
of Tdp1, cocrystallized with a peptide-vanadate-DNA
substrate mimic (PDB accession code 1NOP).29 After con-
struction of a molecular model from 1NOP (see Experimen-
tal Section), the eight steroid derivatives were docked into
the substrate binding pocket of Tdp1. Figure 8A shows a
stereoview representation of 1 and the tyrosyl-vanadate-
DNA substrate mimic overlapped within the active site of
Tdp1. On the basis of the model, the inhibitor appears to
mimic the substrate-Tdp1 interaction. The vanadate parti-
cipating in the 30-phosphotyrosyl linkage of the Tdp1 sub-
strate is mimicked by the sulfonyl ester moiety of 1, while the
steroid and phenyl bromide moieties of the inhibitor are
substituted for the DNA and tyrosyl portions of the Tdp1
substrate, respectively. In addition, Figure 8B provides
evidence for a linear relationship between the calculated free
energy of binding (docking) and the IC50 values for 1-7
(correlation coefficient R2 of 0.89).
Discussion
Cells have developed multiple pathways to repair damaged
DNA. Dysregulation of these pathways is a primary cause of
genomic instability and carcinogenesis; yet, when functioning
properly, their action can reduce the effectiveness of conven-
tional DNA-damage-based anticancer therapies. For exam-
ple, increased DNA repair has been shown to be a clinically
important mechanism of resistance to platinum-based thera-
pies (for review, see ref 43). Accordingly, the pharmacological
inhibition of specific DNA repair proteins has emerged as a
promising strategy to both combat the resistance and potenti-
ate the cytotoxicity of current anticancer treatments. DNA

7130 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Dexheimer et al.
para-substituent resulted in a moderate decrease in activity
(∼3-fold) (Figure 4). More interestingly, we found a signifi-
cant reduction in the activity of the mesylate analogue (7),
which suggests that the phenyl ring portion is important to the
activity of this series of compounds. Overall, the p-bromide
compound (1) or the initial hit from the screen was determined
to be most active.
Cellular studies have been performed with compound 1 in
combination with CPT in HCT116 cells. Compound 1 ex-
hibited limited activity by itself (up to 25 μM), and only an
additive effect was observed with camptothecin under these
conditions (data not shown). In addition, we did not observe
an enhancement in the amount of CPT-induced Top1-DNA
cleavage complexes by 1 using the immunocomplex of enzyme
(ICE) bioassay (data not shown). It is likely that limited
cytotoxicity and lack of detectable Tdp1 inhibition by 1 in a
cellular environment may be due to limited drug uptake and
possibly “off target” effects. Thus, compound 1 represents a
lead molecule for development of next generation agents
against Tdp1 in vivo.
Acknowledgment. The wild-type Tdp1 and SCAN1 mu-
tant Tdp1 constructs were generous gifts from Drs. J. J.
Champoux (University of Washington) and Dr. H. Nash
(Laboratory of Molecular Biology, National Institute of
Mental Health, NIH), respectively. This project has been
funded in whole or in part with federal funds from the
National Cancer Institute, National Institutes of Health,
under Contract HHSN261200800001E. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services nor does
mention of trade names, commercial products, or organiza-
tions imply endorsement by the U.S. Government.
Supporting Information Available: HPLC chromatograms
and high-resolution mass spectral data of compounds 1-6. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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