Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance

Article abstract of DOI:10.1021/ja2066972

Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC₅₀ = 21 nM) and in situ (IC₅₀ = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance.

Full text of DOI:10.1021/ja2066972

ARTICLE
pubs.acs.org/JACS
Potent and Selective Inhibitors of Glutathione S-Transferase Omega 1
That Impair Cancer Drug Resistance
Katsunori Tsuboi,† Daniel A. Bachovchin,† Anna E. Speers,† Timothy P. Spicer,§ Virneliz Fernandez-Vega,§
Peter Hodder,§ Hugh Rosen,†,‡ and Benjamin F. Cravatt*,†
†The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, and ‡The Scripps Research Institute Molecular
Screening Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
§The Molecular Screening Center, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
S Supporting Information
b
ABSTRACT: Glutathione S-transferases (GSTs) are a super-
family of enzymes that conjugate glutathione to a wide variety of
both exogenous and endogenous compounds for biotransfor-
mation and/or removal. Glutathione S-tranferase omega 1
(GSTO1) is highly expressed in human cancer cells, where it
has been suggested to play a role in detoxification of chemother-
apeutic agents. Selective inhibitors of GSTO1 are, however,
required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we
performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1
and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of
selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates
GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened
sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance.
’ INTRODUCTION
enzymatic activity is sensitive to generic thiol-alkylating agents
(e.g., N-ethylmaleimide),8 sulfonate esters (SEs),2 and halo-
acetamides.9,10 A limited number of substrate assays have been
developed, but these are not well-suited for HTS due to poor
turnover rates and/or reliance on UV absorbance at short wave-
lengths (305 nm), where many small-molecules exhibit intrinsic
absorbance.11 We previously showed, however, that GSTO1
activity could be assayed by fluopol-ABPP with a rhodamine-
tagged SE activity-based probe.5 Fluopol-ABPP, when performed
in a competitive format, is compatible with HTS of compound
libraries, whereby enzyme inhibition is assessed by the ability of
compounds to out-compete probe labeling of the target enzyme
(resulting in reduced fluopol signal). When used in the context of
a complex proteome, competitive ABPP also offers a means to
assess inhibitor selectivity against a wide range of probe-reactive
enzymes. Here, we apply fluopol-ABPP to screen a 300K+ small-
molecule library, made available through the NIH Molecular
Libraries Probe Production Centers Network (MLPCN), for
GSTO1 inhibitors. Lead hits were optimized using competitive
ABPP in cancer cell proteomes, resulting in the generation of an
agent KT53 that is a highly potent, selective, and cell-active
inhibitor of GSTO1. KT53 sensitizes cancer cells to the cytotoxic
effects of cisplatin, providing the first pharmacologic evidence
that GSTO1 contributes to chemotherapy resistance in cancer.
Glutathione S-transferases (GSTs) are a large and diverse class
of enzymes that conjugate glutathione to a variety of both exogenous
and endogenous compounds for biotransformation and/or removal.1
Using activity-based protein profiling (ABPP), we discovered
that glutathione S-tranferase omega 1 (GSTO1) is overexpressed
in human cancer cell lines that show enhanced aggressiveness,2
and other studies have implicated GSTO1 in chemotherapeutic
resistance.3,4
Despite its potential role in cancer, few inhibitors have been
described for GSTO1. In our original report of a fluorescence
polarization (fluopol)-ABPP platform for high-throughput
screening (HTS),5 we identified lead GSTO1 inhibitors from a
2000-compound library, but the potency, selectivity, and biolo-
gical activity of these compounds were not extensively examined.
More recently, Son et al.6 reported that the commercially available
fluorescent protein tag, CellTracker Green (5-chloromethylfluo-
rescein diacetate, Invitrogen), inhibits GSTO1 with good potency
(IC50 = 51 nM) and selectivity. While this probe may be useful
for certain applications, it readily undergoes hydrolysis by endogen-
ous esterases, rendering the compound membrane-impermeable.
We therefore sought to develop an improved inhibitor that showed
the requisite combination of potency, selectivity, and cellular activity
for assessing the function of GSTO1 in cancer.
Among GSTs, GSTO1 is unusual in that, rather than having a
catalytic serine or tyrosine in the active site, GSTO1 utilizes a
hyperreactive catalytic cysteine nucleophile.7 As a consequence,
Received: July 18, 2011
Published: September 07, 2011
r
2011 American Chemical Society
16605
dx.doi.org/10.1021/ja2066972 J. Am. Chem. Soc. 2011, 133, 16605–16616
|

Journal of the American Chemical Society
ARTICLE
’ MATERIALS AND METHODS
quenched with 2ꢀ SDS-PAGE loading buffer, separated by SDS-PAGE,
and visualized by in-gel fluorescent scanning and analyzed as described
for recombinant GSTO1 competitive ABPP. When available, data from
multiple experiments were averaged (typical stdev = (3%). IC50 values
(KT53, KT59) were determined from doseꢁresponse curves from three
trials at each inhibitor concentration (0.1ꢁ250 nM) using Prism soft-
ware (GraphPad).
Competitive ABPP of GSTO1 in Situ. MDA-MB-435 cells were
grown to 90% confluency in RPMI medium (5 mL total volume; sup-
plemented with 10% fetal calf serum [FCS]) and treated with 0.01 nM to
1 μM inhibitor (5 μL of a 1000ꢀ stock in DMSO) for 1 h at 37 °C. Cells
were harvested, washed four times with 10 mL of DPBS, and homo-
genized by sonication in DPBS. The soluble fraction was isolated by
centrifugation (100 000g, 45 min), and the protein concentration was
adjusted to 1 mg/mL with DPBS. SE-Rh (1 μL of 50ꢀ stock in DMSO)
was added to a final concentration of 10 μM in 50 μL total reaction
volume. The reaction was incubated for 1 h at 25 °C, quenched with
2ꢀ SDS-PAGE loading buffer, separated by SDS-PAGE, and visualized
by in-gel fluorescent scanning and analyzed as described for recombi-
nant GSTO1 competitive ABPP. When available, data from multiple
experiments were averaged (typical stdev = (3%). IC50 values for KT53
and KT59 were determined from doseꢁresponse curves from three trials
at each inhibitor concentration (0.01ꢁ1000 nM) using Prism software
(GraphPad).
CC-ABPP of GSTO1 in Vitro. MDA-MB-435 soluble proteome
(1 mg/mL in DPBS) was treated with 1 nM to 10 μM KT59 (1 μL of
50ꢀ stock in DMSO) for 30 min at 25 °C. For competition with KT53,
soluble proteome was first preincubated with KT53 (0.1ꢁ10 μM, 30 min)
prior to addition of KT59 (equal molar concentration, 30 min). Each
sample was reacted with rhodamine-azide (Rh-N3; 50 μM) under click
chemistry reaction conditions [1 mM TCEP, 100 μM TBTA ligand,
1 mM Cu(II)sulfate].13 The reaction was incubated for 1 h at 25 °C,
quenched with 2ꢀ SDS-PAGE loading buffer, separated by SDS-PAGE,
and visualized by in-gel fluorescent scanning. The percent labeling was
determined by measuring the integrated optical density of the GSTO1
band relative to the integrated optical density of the GSTO1 band at the
highest test compound dose (where saturated labeling is achieved).
CC-ABPP of GSTO1 in Situ. MDA-MB-435 cells were grown to
90% confluency in RPMI medium (5 mL total volume; supplemented
with 10% FCS) and treated with 16ꢁ1000 nM KT59 (1 μL of 1000ꢀ
stock in DMSO) for 6 h at 37 °C. Cells were harvested, washed four
times with 10 mL of DPBS, and homogenized by sonication in DPBS.
The soluble fraction was isolated by centrifugation (100 000g, 45 min),
and the protein concentration was adjusted to 1 mg/mL with DPBS.
Each sample was reacted with Rh-N3 (50 μM) under CC reaction
conditions and analyzed as described for CC-ABPP of GSTO1 in vitro.
Gel Filtration Analysis of Inhibition Mechanism. Recombi-
nant GSTO1 (250 nM) in DPBS was incubated with DMSO or inhibitor
(1 μM) for 30 min at 25 °C, and each reaction was split into two frac-
tions. One fraction was reacted directly with CA-Rh, and the other was
passaged over a Sephadex G-25 M column (GE Healthcare) and then
reacted with CA-Rh at a final concentration of 5 μM in 50 μL total
reaction volume. The reaction was incubated for 1 h at 25 °C, quenched
with 2ꢀ SDS-PAGE loading buffer, separated by SDS-PAGE, visualized
by in-gel fluorescent scanning, and analyzed as described for recombi-
nant GSTO1 competitive ABPP. Following in-gel scanning, the gel was
stained with Coomassie for protein load comparison.
Materials. (phenyl) SE-Rh was synthesized as described previously;12
the synthesis of CA-Rh is detailed in the Supporting Information. All
chemical reagents and solvents were obtained from Sigma-Aldrich or
ThermoFisher unless otherwise indicated. All cell culture media and
supplements were obtained from CellGro and Omega Scientific.
Recombinant Protein Expression and Purification. GSTO1
(human isoform) was obtained as an expressed sequence tag from
Invitrogen (Carlsbad, CA) and subconed into pTrcHisB (Invitrogen).
Point mutants were generated using the Quikchange Site-Directed
Mutagenesis Kit (Stratagene). The constructs were expressed in BL21-
(DE3) E. coli and purified as described.5
Fluopol-ABPP HTS Assay. The fluopol-ABPP assay was per-
formed at the Scripps Research Institute Molecular Screening Center
(SRIMSC) in Jupiter, FL, using robotic handlers. Briefly, 4.0 μL of Assay
Buffer (0.01% Pluronic detergent, 50 mM Tris HCl pH 8.0, 150 mM
NaCl, 1 mM dithiothreitol) containing recombinant GSTO1 (1.25 μM)
was dispensed into 1536-well microtiter plates. Next, test compound (30
nL in DMSO) or DMSO alone (0.59% final concentration) was added
to the appropriate wells, giving 5.96 μM final concentration, and
incubated for 30 min at 25 °C. The assay was started by dispensing
SE-Rh probe (1.0 μL of 375 nM in Assay Buffer) to all wells, giving a final
concentration of 75 nM. Plates were centrifuged and incubated for 20 h
at 37 °C. Prior to reading, plates were equilibrated at room temperature
for 10 min. Fluorescence polarization was read for 30 s for each polariza-
tion plane (parallel and perpendicular) on a Viewlux microplate reader
(PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a
BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). The
well fluorescence polarization value (mP) was obtained via the PerkinElmer
Viewlux software. Compounds that inhibited GSTO1 greater than 34.81%
(mean + 3 ꢀ standard deviation) were considered active. Assay statistics:
Z0 = 0.80 ( 0.05, S:N = 2.08 ( 0.21, hit rate = 1.06% (3207 compounds).
The top 2374 available compounds were then retested in triplicate
using the same HTS assay conditions and hit cutoff; assay statistics: Z0 =
0.84 ( 0.04, S:N = 3.19 ( 0.14, hit rate = 54% (1286 compounds).
Competitive ABPP of Recombinant GSTO1. Recombinant
GSTO1 (250 nM in 50 μL of Dulbecco’s phosphate buffered saline
[DPBS]) was incubated with 1 μM test compound (1 μL of a 50ꢀ stock
in DMSO) for 30 min at 25 °C followed by reaction with 10 μM SE-Rh
(1 μL of 50ꢀ stock in DMSO) for 1 h at 25 °C. The reaction was
quenched with 2ꢀ SDS-PAGE loading buffer, separated by SDS-PAGE,
and visualized by in-gel fluorescent scanning. The percentage activity
remaining was determined by measuring the integrated optical density of
the GSTO1 band relative to a DMSO-only (no compound) control.
IC50 values (Table 1 compounds) were determined from doseꢁresponse
curves from three trials at each inhibitor concentration (3ꢁ3000 nM)
using Prism software (GraphPad).
Cell Culture and Preparation of MDA-MB-435 Soluble
Proteome. MDA-MB-435 cells were grown to 90% confluency in
RPMI medium supplemented with 10% fetal calf serum (FCS) in a
humidified, 5% CO2 incubator at 37 °C. Cells were washed 3ꢀ with DPBS
and harvested in ice-cold DPBS by scraping. Cell pellets were isolated by
centrifugation (1000g, 3 min), resuspended in ∼4ꢀ (v/v) DPBS, and
lysed by sonication with a probe sonicator (10 ꢀ 3 s pulses, 50% power)
on ice. The soluble fraction was isolated by centrifugation (100 000g; 45
min; supernatant = soluble). Protein concentration was assayed (DC
Protein Bioassay Kit, Bio-Rad), adjusted to 1 mg/mL in DPBS, and
aliquots were frozen at ꢁ80 °C until use.
LCꢁMS/MS Analysis of Inhibition Mechanism. See the
Supporting Information for full details. Briefly, purified GSTO1 (50 μM)
was incubated with inhibitor 1 or 3 (10 μM) for 30 min, followed by reduc-
tion, alkylation, and trypsin digestion. A fraction of each sample was analyzed
by microcapillary LCꢁMS/MS using a nanospray ESI14 source (Agilent
HPLC, ThermoScientific Orbitrap Velos) in data-dependent acquisition
mode. Results were searched using Sequest15 (Cys static mod +57.021,
Competitive ABPP in a Complex Proteome. MDA-MB-435
soluble proteome (1 mg/mL in DPBS) was incubated with 0.1 nM to 10
μM inhibitor (1 μL of a 50ꢀ stock in DMSO) for 30 min at 25 °C. The
proteome was then labeled with either 10 μM SE-Rh or 5 μM CA-Rh
(1 μL of a 50ꢀ stock in DMSO) for 1 h at 25 °C. The reaction was
16606
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Table 1. Top GSTO1 Inhibitor Leads Following Gel-Based
Competitive ABPP Screeninga
Figure 1. Fluopol-ABPP HTS assay for GSTO1 inhibitor discovery.
Screening data for a representative 20 000 compounds of the MLPCN
small molecule library are shown. Compounds that reduced the SE-Rh
fluorescence polarization signal by g34.81% (line) were designated as
hits for GSTO1 (red b).
for 24 h. At the indicated time point (1, 3, 6, 12, and 24 h), an aliquot
(15 μL) was removed and subjected to HPLCꢁMS analysis using an
Agilent 1200 series HPLC interfaced with an Agilent MSD SL controlled
by ChemStation (Agilent) software. Compounds were separated over a
Prep-C18 scalar column (5 μm, 4.6 ꢀ 150 mm, Agilent) using a
waterꢁacetonitrile buffer system (buffer A, 100% water; buffer B, 95%
acetonitrile/5% water, each containing 0.1% trifluoroacetic acid). The
run consisted of 5 min 70% buffer A, followed by a linear, increasing
gradient (30ꢁ80%) of buffer B over 20 min. The eluate was monitored
by UV (254 nm) and ESIꢁMS. The AUC for each species was calculated
(ChemStation) as a percentage of the total species present in the UV
trace.
Cytotoxicity Analysis of Inhibitor Compounds. MDA-MB-
435 cells in RPMI medium (with or without 10% FCS) were dispensed
into a 96-well plate (100 μL, 15E4 cells/well). Next, 0ꢁ50 μM KT53 or
KT59 (10 μL of 11ꢀ stock in medium containing 10% DMSO) was
added to the appropriate wells. Cells were incubated for 48 h at 37 °C,
and cell viability was determined by the WST-1 assay (Roche) according
to manufacturer instructions. CC50 values were determined from doseꢁ
response curves from two biological replicates (n = 6 for each) at each
inhibitor concentration using Prism software (GraphPad).
Cisplatin-Induced Cytotoxicity Studies. MDA-MB-435 cells
in RPMI medium (containing 10% FCS) were dispensed into a 96-well
plate (100 μL, 20E4 cells/well) and incubated overnight at 37 °C. When
cells had reached 60ꢁ70% confluency, 0ꢁ1 μM KT53 (10 μL of an 11ꢀ
stock in medium containing 1% DMSO) was added to the appropriate
wells. Cells were incubated for 1 h at 37 °C prior to addition of 0ꢁ250 μM
cisplatin (10 μL of a 12ꢀ stock in media containing 7.5% DMSO). After
a 12 h (total) incubation, cells were redosed with 0ꢁ1 μM KT53, re-
spectively (10 μL of a 13ꢀ stock in medium containing 1% DMSO) and
incubated at 37 °C for a further 12 h. Cell viability was determined by the
WST-1 assay (Roche) according to manufacturer instructions. The analysis
at 1 μM KT53 includes seven biological replicates (n = 5 ea), at 0.5 μM
includes two biological replicates (n = 5 ea), and at 0.25 and 0.125 μM
includes three biological replicates (n = 5 ea). Statistical analysis was
carried out using an unpaired Student’s t test using Prism Software
(GraphPad); data are presented as the mean ( SEM. For all statistical
tests, significance was established at P < 0.05.
a Observed off-target of both CA-Rh (inhibitor) and SE-Rh (activator);
otherwise, all off-targets listed here and in subsequent tables are off-
target inhibition events observed by CA-Rh labeling competition.
Cys variable mod +274.057 for 1 and +310.148 for 3) and assembled
into protein identifications using DTASelect.16 Sequest results are sum-
marized in Table S2. The area under the curve (AUC) for each modified
peptide was calculated from the extracted ion chromatogram and normal-
ized to the AUC for the corresponding unmodified peptide. Percentage
for each modified peptide is reported as a percentage of the total mod-
ified species observed (sum of all normalized AUCs) in the sample. No
modified peptides were observed in the DMSO-treated sample.
HPLCꢁMS Analysis of Compound Stability. Test compound
(0.1 mg/mL in DPBS containing 1% DMSO) was incubated at 37 °C
Chemical Synthesis. See the Supporting Information.
’ RESULTS AND DISCUSSION
1. Discovery of α-Chloroacetamide (CA) Inhibitors of
GSTO1. Fluopol-ABPP evaluates test compounds for their ability
to block enzyme reaction with a fluorescent activity-based probe.
16607
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
In the absence of a competitive agent, probe binding to the much
larger target enzyme results in slower tumbling of the probe and a
concomitant increase in fluorescence polarization (fluopol). As
such, test compounds are monitored for their ability to block
the increase in fluopol. The fluopol-ABPP technique has been
applied to screenfor inhibitors of numerous probe-reactive enzymes
(http://pubchem.ncbi.nlm.nih.gov/). As described,5 we first ver-
ified that purified, recombinant wild-type GSTO1 (1 μM), but
not a catalytically inactive mutant (C32A), was able to undergo
labeling with a rhodamine-conjugated phenyl sulfonate ester (SE-
Rh; 75 nM) activity-based probe. We then completed a pilot
screen of 2000 compounds from the Molecular Libraries Small
Molecule Repository (MLSMR) validation set, which yielded 38
hit compounds (1.9% hit rate).5 This screen was conducted un-
der kinetically controlled conditions, where an assay time point
(90 min SE-Rh incubation) prior to completion of the labeling
reaction allowed for identification of both reversible and irrever-
sible inhibitors of GSTO1.5 However, given the high hit rate
from the pilot screen, we elected for the full deck screen (302 667
compounds), conducted at The Scripps Research Institute Molec-
ular Screening Center (SRIMSC) branch of the MLPCN, to use
an extended probe incubation time (20 h), where complete in-
hibition of GSTO1 is achieved, to reduce the number of hits and
select for compounds that had a high probability of fully inactivating
GSTO1, likely by an irreversible mechanism. A total of 3207
compounds (1.1%) were active, passing the set threshold of 34.81%
GSTO1 inhibition. The raw data are accessible via PubChem
(BioAssay AIDs 1974 and 2176). A representative subset of the
primary screening data is depicted in Figure 1. The top 2374
active compounds were then retested in triplicate, and 1286 com-
pounds (54%) were confirmed as active.
Because the HTS assay conditions were designed to enrich for
irreversible inhibitors, compounds with potentially reactive func-
tionalities (e.g., α-chloroacetamides, epoxides, and α-aryl chlorides)
featured prominently among the top hits. Initially, we cherry-picked
126 active compounds for secondary gel-based competitive ABPP
screening: 33 epoxides, 30 α-aryl chlorides, and 63 sterically hin-
dered α-chlorocetamides (CAs) (i.e., bearing tertiary nitrogens).
We first assessed whether or not test compounds could inhibit
purified, recombinant GSTO1 (1 μM compound concentration)
Figure 3. Time course of inhibition of GSTO1 in cultured MDA-MB-
435 cells by competitive ABPP with SE-Rh. Compound 1 shows modest
in situ inhibition of GSTO1 at 12 h (65%) and 18 h (25%) following a
single 1 μM dose of compound in serum-containing medium. In com-
parison, compound 3 sustains more than 75% inhibition of GSTO1 for
up to 18 h. GSTO1 indicated with arrow; inhibition calculated relative to
DMSO (no compound) control. Fluorescent image here, and for all
subsequent ABPP figures, shown in gray scale.
Figure 2. CA inhibitors operate by irreversibly acylating the active
site cysteine of GSTO1. (A) Labeling of GSTO1 is not recovered
following gel filtration to remove small molecules (top gel slice;
fluorescent image shown in gray scale). Equal protein recovery shown
in Coomassie-stained bottom gel slice. (B) Proposed modification of
GSTO1 by 1 and 3.
Table 2. Compounds 1 and 3 Label the Active Site Cysteine of GSTO1
compound/control
peptide
1
3
DMSO
R IYSMRFC32aPFAER T
100%
0%
99.4%
0.05%
0.6%
0%
0%
0%
0%
0%
3
3
K KNPFGLVPVLENSQGQLIYESAITCaEYLDEAYPGKK L
3
3
K KLLPDDPYEKACaQKMILELFSKVPSLVGSFIR S
0%
3
3
R LEAMKLNECaVDHTPK L
0%
0.02%
3
3
totalb
100%
100%
a Labeling observed on Cys; active site Cys in italic. b Total modified species observed in the sample as measured by area under the curve of extracted ion
chromatograms normalized to corresponding unmodified peptide; underlined K or R indicates peptide with missed cleavage at that site was also
observed (see Table S2 for Sequest results).
16608
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Scheme 1
Table 3. SAR of Scaffold 1 Analogues
Scheme 2
using a competitive ABPP assay with the SE-Rh probe (Figure S1
and Table S1). The majority of top hits belonged to the CA inhi-
bitor class. Fifty-three of the top compounds (g60% inhibition),
along with an additional 24 cherry-picked HTS CA hits, were
then evaluated for endogenous GSTO1 inhibition in the context
of a complex proteome (1 μM compound concentration; Figure
S2 and Table S1). For this assay, a soluble proteome preparation
of the human melanoma cell line MDA-MB-435, which expresses
high endogenous levels of GSTO1,12 was employed. Of the 77
compounds, 22 (2 epoxides, 3 α-aryl-chlorides, and 17 CAs) showed
high (at least ∼90%) inhibition of the target enzyme.
A third round of gel-based competitive ABPP secondary screen-
ing assessed potency at a lower (100 nM) compound concentra-
tion in the MDA-MB-435 soluble proteome (Figure S3). The top
10 compounds, all CAs with the exception of α-aryl chloride 9,
are shown in Table 1. The majority of the top compounds (1ꢁ7)
exhibited highly similar structural features, which could be broken
down into two subtypes: Scaffold 1 (compound 1) and Scaffold
2 (compounds 2ꢁ7; Table 1). Both structures feature an
R-substituted N-phenyl and an adjacent amide moiety; Scaffold
1 is distinguished by having a hydrogen (H) alpha to the amide
nitrogen, whereas Scaffold 2 has an aromatic/heteroaromatic
(Ar) derivative at that location.
An assessment of selectivity (10 μM compound concentration)
was conducted using both the SE-Rh and cysteine-reactive
CA-Rh17 activity-based probes to characterize potential off-target
inhibition against the more than 30 probe-reactive proteins
visible by gel-based analysis in the MDA-MB-435 soluble pro-
teome (Figure S4). The results, summarized in Table 1, show
that a few of the top leads (e.g., 1, 5) evinced considerable selec-
tivity, having few off-targets (listed by molecular weight of off-
target) at concentrations more than 300-fold above their inhibi-
tory activity for GSTO1.Other inhibitors (e.g., 2ꢁ4, 6ꢁ10)
exhibited some off-target reactivity. However, as a whole, the
selectivity of the Scaffold 1 and 2 classes was deemed sufficient to
allow for medicinal chemistry optimization to remove unwanted
activities. Compounds 8ꢁ10 were deprioritized as potential
leads due to their high off-target rate and reduced inhibition of
GSTO1 at 100 nM in the complex proteome. Thus, our attention
focused on Scaffold 1 and Scaffold 2 compounds for further
investigation.
noting that compound 7 has previously been identified in a
MLPCN campaign by Bittker et al.18 as a potent and selective
cytotoxic agent, capable of inducing death in cells expressing the
oncogenic RAS allele HRASV12, but not cell lines lacking
HRASV12. As a cell-based assay was employed, no specific molec-
ular target was implicated in the cytotoxic effects of 7. It therefore
merits consideration whether one or more of the protein targets
for this compound detected here by competitive ABPP could be
responsible for the observed phenotype.
2. Inhibition Mechanism of Lead Compounds. While it was
surmised that the identified compounds operated by a covalent
mechanism to inactivate GSTO1, this was tested experimentally using
both gel filtration and liquid chromatography-tandem mass spectro-
metry (LCꢁMS/MS). The blockade of CA-Rh probe labeling of
GSTO1 by two representative compounds, 1 and 3, was not reversed
by gel filtration (Figure 2A), indicative of irreversible inhibition.
Further analysis by LCꢁMS/MS revealed that compounds 1
and 3 covalently label the active site cysteine nucleophile, Cys32,2 of
GSTO1 (Table 2). Cys32 was the dominant site of labeling for
compounds 1 and 3, with only a minor fraction (0ꢁ0.6%) of
labeling being observed on nonactive site cysteines. The ob-
served mass shift of the active site peptide (see Sequest analysis,
Table S2) indicates that the reaction occurs via cysteine nucleo-
philic attack to displace the chloride, as expected of this chemo-
type and depicted in Figure 2B.
3. In Situ Activity of Lead GSTO1 Inhibitors. We next sought
to determine whether the lead inhibitors could inactivate
GSTO1 in living cancer cells. Cultured MDA-MB-435 cells were
treated with 1 μM compound for 1 h in medium containing 10%
Before moving on to describe our medicinal chemistry efforts
to improve GSTO1 inhibitor potency and selectivity, it is worth
16609
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Figure 4. Competitive ABPP of Scaffold 1 (A and B) and Scaffold 2 (C and D) analogues in vitro. (A and C) Assessment of GSTO1 inhibition potency
(100 nM compound) in MDA-MB-435 soluble proteome with SE-Rh ABPP probe. (B and D) Assessment of selectivity (5 μM compound) in MDA-
MB-435 soluble proteome with CA-Rh ABPP probe. GSTO1 (black) and off-target bands (maroon) indicated with arrows. Gels imaged at 60% intensity
with the following exceptions: top blue box (B and D), 50% intensity; bottom blue box (B and D), 70% intensity to enhance visualization of off-target
bands. See Figure S6 for full gel images of (A) and (C) and 5 μM profile with SE-Rh.
serum, after which cells were harvested and their soluble proteomes
analyzed by gel-based ABPP with SE-Rh (Figure S5). As sum-
marized in Table 1, compounds 1ꢁ4 completely inhibited GSTO1
activity in cancer cells. For further characterization of in situ activity,
we tested compounds 1 and 3 over an extended time course. The
inhibitory activity of 1 was significantly decreased after 18 h
(Figure 3, 25% inhibition). In contrast, compound 3 showed a
longer duration of activity, maintaining >75% GSTO1 inhibition
at 18 h. However, 3 also exhibited moderate off-target reactivity
and reduced potency as compared to 1 (Table 1), necessitating
optimization of both scaffold types.
4. Optimization of Lead GSTO1 Inhibitors. 4.1. Structureꢁ
Activity Relationships (SAR) and Chemistry. While compound 1
was the only representative member of Scaffold 1 in the screening
library, there were a number of Scaffold 2 analogues, allowing a
preliminary assessment of SAR. A halogen at the 3- or 4-position of
the phenyl ring (R) conferred potency (e.g., 2ꢁ5, Table 1), but
was not tolerated at the 2-position (M04, L08, Table S1). The
aromatic position was amenable to a diversity of ring structures
(3-pyridine for 2 and 3, 4-methoxyphenyl for 4ꢁ6, and thio-
phene for 7, Table 1). Likewise, the amide appeared to be a
potentially structurally flexible position, allowing both cyclohex-
ylamides (2ꢁ6) and phenylethylamides (7). With this informa-
tion in mind, we next initiated an ABPP-guided medicinal chemistry
program to improve the potency, selectivity, and in situ activity of
lead CA inhibitors.
of Scaffold 1 analogues, whereby fluoro- or iodo-nitrobenzenes
A were reacted with diaminoalkanes B to afford amines C, which
were subsequently acylated to give amides D. Alternatively, D
intermediates were synthesized by alkylation of anilines E with
bromides F. N-Alkylation of D was accomplished upon incuba-
tion with chloroacetyl chloride to afford probes G. Synthesis of
Scaffold 2 analogues (H) was accomplished by one-pot Ugi re-
action as depicted in Scheme 2.
4.2. SAR Studies of Scaffold 1 GSTO1 Inhibitors. Consistent
with the SAR determined from HTS library compounds, in vitro
gel-based competitive ABPP of Scaffold 1 analogs (Table 3 and
Figure 4A) revealed that a 3- or 4-substituted electron-with-
drawing nitro group (KT48 and 1, respectively) conferred
potency versus 2-substitution (KT49) at the R position of the
N-phenyl. A halogen could be substituted for nitro at the 4-position
(KT19) with preservation of potency, whereas 4-substituted
methyl (KT16) and methoxymethyl (KT15) were not tolerated
(Table 3 and Figure 4A). Replacing the trifluoroacetamide group
at R1 with other alkyl (KT8, KT45, KT3) or aromatic (KT9)
amides led to, in some cases dramatic, reduction in the in vitro
potency, as did replacement of the acetamide with a straight chain
alkyl group (KT2; Table 3 and Figure 4A). KT2 also exhibited a
higher number of off-targets (Table 3 and Figure 4B), possibly due
to its increased hydrophobicity and/or decreased structural rigidity.
Interestingly, it was noted that KT3 provided more sustained
in situ inhibition versus 1 (80% versus 65%, respectively; Table 3
and Figure 5), despite its comparatively weak in vitro inhibition.
The synthesis of compounds investigated in this study is sum-
marized in Schemes 1 and 2. Scheme 1 was utilized for synthesis
16610
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Table 4. SAR of Scaffold 2 Analogues
Figure 5. Inhibition of GSTO1 in cultured MDA-MB-435 cells. Fol-
lowing a 12 h in situ incubation (1 μM compound; medium containing
10% serum), cells were harvested and soluble proteome isolated and
profiled by gel-based ABPP with SE-Rh. GSTO1 indicated with arrow.
We were able to improve the in vitro potency by adding a
methylene to the alkyl linker connecting the CA with the amide
(KT28; Table 3 and Figure 4A). The sustained in situ inhibition
afforded by KT3 and KT28 indicates that the trifluoroacetamide
moiety, while conferring potency in vitro, may limit cellular stability.
As compared to starting compound 1, KT28 was the most opti-
mized compound based on Scaffold 1, with somewhat dimin-
ished in vitro potency (90% versus g98% inhibition at 100 nM)
but enhanced in situ potency (80% versus 65% inhibition, 1 μM
compound concentration, sustained 12 h) (Table 3).
4.3. SAR Studies of Scaffold 2 GSTO1 Inhibitors. On the basis
of the SAR analysis of library compounds and Scaffold 1, for
investigation of Scaffold 2, we initially chose to fix R as 4-fluoro
for investigation of the aromatic and amide moieties (Table 4). In
addition to the cyclohexylamide and phenylethylamide groups
exhibited by several top-10 compounds at R2 (2ꢁ6 and 7, re-
spectively), benzylamide (KT30) conferred similar potency but
without improvement in selectivity (Table 4 and Figure 4C,D).
Other N-cyclic (KT42), alkoxy (KT44), and aromatic (KT31)
amides were not tolerated, with GSTO1 inhibition at 100 nM
dropping to zero (Table 3 and Figure 4C). Of all of the analogous
Ar derivatives tested, 5-thiazole (KT34), 2-furan (KT35), phen-
yl (KT40), 4-pyridine (KT37), 3-pyridine (3), and 2-pyridine
(KT33), the most potent in vitro derivatives were the 2- and
4-pyridines (Table 4 and Figure 4C). The 2-pyridine analogue
(KT33) was the most selective, having no visible off-targets at
5 μM concentration in the MDA-MB-435 soluble proteome
(Table 4 and Figure 4D). The 2- and 4-pyridine derivatives KT33
and KT37 also exhibited very good in situ potency, inhibiting
GSTO1 activity by at least 90% after 12 h (1 μM compound
concentration; Table 4 and Figure 5).
and selectivity (Table 4 and Figure 4D), having no off-targets at
5 μM compound concentration in the MDA-MB-435 soluble
proteome. Compounds KT53 and KT54 represent a significant
optimization of the Scaffold 2 inhibitors, combining the best
properties of the top HTS compounds (Table 1), including
excellent potency at 100 nM compound concentration (100%
inhibition; Figure 4C), sustained potency for 12 h in situ (95%;
Figure 5), and high selectivity, having no observed off-targets at 5
μM compound concentration (Figure 4D). KT53 and KT54
were also more potent and equally selective as compared to the
optimized Scaffold 1 compound KT28; as such, we elected to
proceed with in depth characterization of KT53.
5. Inhibitory Profiles of KT53 against GSTO1. Dose re-
sponse curves for in vitro and in situ inhibition of GSTO1 by
KT53 (Figure 6A and B) gave IC50 values of 21 and 35 nM, re-
spectively. Time course experiments revealed that KT53 potently
inhibited more than 90% of GSTO1 activity for at least 18 h
(Figure 7), designating the compound as suitable for extended
pharmacological studies of GSTO1 in cells.
6. Click Chemistry (CC)-ABPP Characterization of KT53. In
addition to competitive ABPP assays for analysis of inhibitor
selectivity, we also directly assessed inhibitor-reactive proteins using
click chemistry (CC)-ABPP.13,19 In this approach, an alkyne ana-
logue is synthesized for the compound of interest and added to
both proteomes and living cells, after which CC conjugation to a
fluorescent azide reporter tag reveals inhibitor-labeled proteins.
With the aromatic and amide moieties thus optimized, we briefly
revisited the question of N-phenyl substitution with com-
pounds KT53-KT55. The 3-fluoro derivatives KT53 and
KT54 offered the best in situ inhibition (Table 4 and Figure 5)
16611
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Scheme 3
Scheme 4
Figure 6. IC50 curves for GSTO1 inhibition by KT53 and KT59 in vitro
(A, C) and in situ (B, D) generated by competitive ABPP with SE-Rh
following a 6 h in situ incubation. IC50 values were determined by fitting
a three-parameter doseꢁresponse curve (GraphPad Prism); n = 3, error
is (SEM. Representative gel slice of GSTO1 band shown below
each curve.
the compound was synthesized from ethyl 2-pyridylacetate (I) as
outlined in Scheme 4. Briefly, following α-bromination of I and
reaction with 3-fluoroaniline, the imine J was obtained in a mix-
ture of unreacted 3-fluoroaniline reagent and brominated inter-
mediate K. Conversion of K to J was achieved upon treatment of the
mixture with acetic anhydride in pyridine. Reduction of J with
sodium borohydride gave intermediate L, which was hydrolyzed and
then condensed with 5-hexyn-1-amine to afford the alkynyated
product M. Treatment of M with chloroacetyl chloride gave KT60.
Both analogues had selectivity profiles similar to that of
KT53; however, when tested for GSTO1 inhibition at 100 nM
compound concentration, the potency of KT59 (g98%) more
closely matched that of KT53 (100%), with KT60 providing
only 80% inhibition of GSTO1 (Figure S7). Indeed, KT59 ex-
hibited almost identical doseꢁresponse profiles in vitro and in situ
as compared to KT53 (Figure 6C and D versus A and B). As such,
KT59 was deemed a more appropriate analogue for comparison to
KT53 for selectivity analysis.
The full in vitro doseꢁresponse competitive ABPP experi-
ment with the SE-Rh probe is shown in Figure 8A for KT53
(panel 1) and KT59 (panel 2). Again, both compounds are
observed to have highly similar inhibition profiles. In the third
panel of Figure 8A, instead of competition with SE-Rh, KT59-
labeled proteins are visualized directly following CC conjugation
with a rhodamine-azide (RhꢁN3) reporter tag. At higher (300
nM to 10 μM) concentrations, some concentration-dependent,
nonspecific labeling by KT59 (not competed by pretreatment
with KT53, Figure 8B) of predominantly abundant (e.g., see
Coomassie-stained gel, Figure S8) proteins is observed; however,
at lower (<300 nM) concentrations, GSTO1 is the principal target
labeled by KT59. Importantly, the 30 nM compound concentra-
tion is sufficient to achieve near-complete inhibition of GSTO1
with negligible cross-reactivity with other proteins.
Figure 7. Time course for in situ inhibiton of GSTO1 by KT53 (1 μM)
analyzed by competitive ABPP with SE-Rh. KT53 completely inhibited
GSTO1 activity for up to 6 h and showed at least 90% inhibition up to 18
h. After 2 days, the compound still retained 60% inhibition of the target
enzyme relative to DMSO (no compound) control.
This strategy can reveal additional covalent targets for small-mole-
cules that are either (1) not reactive with competitive ABPP probes
or (2) labeled by the small-molecule to only a minor extent (and
thus not visibly depleted in reactivity by competitive ABPP). We
designed two alkyne analogues for KT53, KT59, and KT60, and
synthesis was completed as outlined in Schemes 3 and 4, respec-
tively. KT59, bearing an alkyne in place of fluorine at the meta
position of the N-phenyl, was synthesized via four-component
Ugi reaction (Scheme 3) as described for other Scaffold 2
derivatives (Scheme 2). For KT60, an aliphatic alkyne was sub-
stituted for cyclohexyl as the right-hand amide substituent, and
KT59 also inhibited GSTO1 in living cells with equivalent
potency and selectivity as compared to KT53. The competitive
ABPP (SE-Rh probe) and CC-ABPP profiles generated from
in situ treatment of MDA-MB-435 cells (6 h time point) are
shown in Figure 9 (panels 1 and 2). Similar to the in vitro
16612
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Figure 8. In vitro competitive ABPP and CC-ABPP profiles of KT53 and KT59 in MDA-MB-435 soluble proteome. (A) Competitive ABPP for KT53
(panel 1) and KT59 (panel 2) with the SE-Rh probe shows selective inhibition of GSTO1. To directly assess the degree of off-target reactivity, proteins
labeled by KT59, which bears a terminal alkyne handle, are functionalized via CC with an azide-conjugated fluorescent reporter tag Rh-N3 (panel 3).
Near-complete inhibition of GSTO1 is observed at 30 nM or greater concentrations of KT59, while labeling of off-targets is minimal until higher
inhibitor concentrations (300 nM or greater). (B) Competitive CC-ABPP experiment where proteome is first incubated with KT53 followed by reaction
with KT59 and CC conjugation with Rh-N3. Besides GSTO1, none of the KT59-labeled bands (e.g., 45ꢁ70 kDa region) are competed by preincubation
with KT53, indicating that the latter are nonspecific targets of KT59.
Figure 9. In situ competitive ABPP and CC-ABPP profiles of KT53 and KT59 in the MDA-MB-435 soluble proteome. Competitive ABPP for KT53
(panel 1) and KT59 (panel 2) with the SE-Rh probe shows selective inhibition of GSTO1 following a 6 h in situ incubation. To directly assess the degree
of off-target reactivity, proteins labeled by KT59 are functionalized via CC with the fluorescent reporter tag Rh-N3 (panel 3). Near-complete inhibition
of GSTO1 is observed at 63 nM or greater concentrations of KT53 and KT59, while labeling of off-targets is minimal until higher inhibitor
concentrations (1 μM).
proteome labeling experiments (Figure 8), a concentration range
of KT59 was identified (∼63ꢁ250 nM) where near-complete
inhibition of GSTO1 was achieved with negligible off-target
reactivity (as assessed by CC-ABPP in Figure 9, panel 3). Even
at higher concentrations (0.5ꢁ1 μM), KT59 did not show
substantial reactivity with other proteins as judged by both
competitive ABPP and CC-ABPP (Figure 9, panels 2 and 3).
Given the highly similar structures, profiles, and potencies of KT59
and KT53, these data strongly suggest that KT53, like KT59, can
inhibit GSTO1 to near-completion in cancer cells without showing
substantial reactivity with other proteins.
7. Stability of KT53. During the course of evaluating KT53,
we observed that the compound was not stable if stored as a
DMSO stock at room temperature. Analysis by HPLC revealed
that KT53 (Figure 10A, retention time 16.8 min) readily trans-
formed into an inactive compound (Figure 10A, retention time
9.1 min). Plotting a time course of inactivation under conditions
relevant to in situ treatment (DPBS, 37 °C) reveals that the
16613
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
Figure 10. KT53 (A) and KT59 (B), but not compound 3 (C), are unstable in DPBS at 37 °C. A 24 h time course monitored by HPLC-MS and UV
shows that KT53 and KT59 decompose to the corresponding pyridinium salt via intramolecular attack of the pyridine nitrogen on the CA. The
conversion is over 50% complete at 3 h and ∼85% complete at 6 h. This phenomenon is only observed with compounds bearing a 2-substituted pyridine,
where the nitrogen is aligned for nucleophilic attack of the α position of the CA (e.g., compound 3 is stable under the same conditions; (C)).
majority of active KT53 is converted quite rapidly, within 6 h
(Figure 10A, inset). The decomposition product was observed
to have a unit mass of 368, which is consistent with intramo-
lecular nucleophilic attack of the pyridyl nitrogen to displace
the chloride, giving the pyridinium salt (Figure 10A). This
phenomenon was observed for all 2-pyridyl derivatives, such
as KT59 (Figure 10B), KT33, KT54, and KT55 (data not
shown). In contrast, compound 3, which cannot adopt a confor-
mation wherein the 3-pyridyl nitrogen is in position to attack the
CA at the α position, was stable under the same conditions for up
to 24 h (Figure 10C).
Although this property requires careful storage of KT53
(in DMSO solution, the compound remains active if stored at
ꢁ80 °C for several months, data not shown), the ability of KT53
to self-inactivate may actually provide a fortuitous advantage in
pharmacology experiments by decreasing off-target effects, as the
compound could react rapidly with GSTO1 (within 1 h, Figure 7)
and then self-deactivate (∼85% within 6 h), thus limiting the op-
portunity for reaction with other proteins. Additionally, the com-
paratively rapid inactivation versus sustained GSTO1 inhibition
(90% after 18 h, Figure 7) suggests that the enzymeꢁinhibitor
complex is highly stable.
Figure 11. Effects of GSTO1 inhibition on cisplatin-induced cytotoxi-
city. Cultured MDA-MB-435 cells were treated with KT53 (1 μM) or
DMSO for 1 h prior to administration of cisplatin (0ꢁ250 μM). After 12
h, cells were readministered KT53 (1 μM). After 24 h total cisplatin
incubation time, cell viability was determined using a WST-1 assay. Data
shown are average ( SEM of seven biological replicates (n = 5 ea); ***P
< 0.0005; **P < 0.005; *P < 0.05. Statistical analysis: unpaired Student’s t
test (GraphPad Prism).
8. Effect of GSTO1 Inhibition on Cisplatin-Induced Cyto-
toxicity. KT53 and KT59 showed minimal cytotoxicity in MDA-
MB-435 cells, with CC50’s > 25 μM (Figure S9) that are well
above the dose required to inhibit GSTO1 in situ (Figure 9). These
results suggest that inhibiting GSTO1 is not, on its own, deleterious
to cancer cell survival. However, it has recently been reported
that HeLa cells overexpressing GSTO1 show enhanced survival
upon exposure to the cytotoxic agent cisplatin as compared to
nontranfected cells expressing endogenous levels of GSTO1 and
cells transfected with GSTO1-siRNA.3 We therefore asked whether
pharmacological inhibition of GSTO1 also impacted cancer cell
viability in the presence of other cytotoxic agents.
Cultured MDA-MB-435 cells treated with or without KT53
(1μMdosedatt= 0 and 12 h) were exposed to cisplatin (0ꢁ250 μM,
dosed at t = 1 h) for 24 h, and cell viability was determined using
16614
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
the WST-1 Reagent (Roche). KT53-treated cells were found to
show significantly greater sensitivity to cisplatin-induced cytotoxicity
as compared to control (DMSO-treated) cells (Figure 11). The
experiment was also repeated at lower doses of KT53 (0.5, 0.25,
and 0.125 μM), with less dramatic, although still significant,
increases in cell death observed relative to DMSO treated cells
(Figure S10). It is possible that these lower concentrations
of KT53 may not provide sufficient inhibition of GSTO1 over
the extended 24 h time course of the experiment (KT53 dosed
twice, once at time zero and again at 12 h, with a 12 h in situ
incubation providing 90%, 80%, and70%inhibition at 0.5, 0.25, and
0.125 μM, respectively, versus 95% for 1 μM; see Figure S11) to
see as pronounced of a response. These results thus provide the
first pharmacologic evidence in support of the hypothesis that
GSTO1 plays an important role in chemotherapy resistance in
cancer cells.
(RNAi) knockdown of GSTO1 sensitizes cancer cells to cisplatin
cytotoxicity,3 point to a function for this enzyme in cancer and
drug resistance. Future studies will be required to more broadly
evaluate this premise and include: (1) determining the metabo-
lomic and proteomic effects of inhibiting GSTO1 on cancer cell
biochemistry, (2) assessing the impact of GSTO1 inhibition and
RNAi knockdown on cancer cell sensitivity to other chemother-
apeutic agents both in vitro and in vivo, and (3) understanding
the cellular mechanism for GSTO1-mediated pro-survival effects.
If these studies continue to support a role for GSTO1 in cancer,
suitably optimized inhibitors of this enzyme might one day
find value as drugs to enhance the activity of conventional
chemotherapies. We also note with interest that GSTO1 was
recently identified as a target of the anticancer agent piperlongu-
mine, which might indicate an additional role for this enzyme in
modulating reactive-oxygen species in tumor cells.20 The output
of our study, which reports a large-scale screen that yielded a very
high hit rate for GSTO1 inhibitors (1.1% in primary screen), as
well as the optimization of a subset of these compounds into
selective agents with good cellular activity, provides both (1)
excellent starting points for expanded efforts to develop addi-
tional classes of GSTO1 inhibitors, and (2) pharmacological probes
suitable for exploring the function of this enzyme in cancer and other
(patho)physiologic processes.
’ CONCLUSION
GSTO1 belongs to an ancient class of GST enzymes that differ
from other members of this superfamily in possessing an active
site cysteine nucleophile. This residue grants GSTO1 a range of
thioltransferase and reductase activities, although the physiologic
substrates and functions of this enzyme remain enigmatic. GSTO1-
knockdown or overexpression studies point to a role for this enzyme
in chemotherapy resistance in cancer cells, where it appears to block
cisplatin-induced apoptosis by stimulating pro-survival kinase
pathways.3,4 The impact of pharmacologically inactivating GSTO1
on cancer cell survival has, however, not yet been examined due
in large part to a lack of selective and cell-active inhibitors. Here,
we report the development of a series of potent and selective α-
chloroacetamide inhibitors of GSTO1 that irreversibly inactivate
this enzyme by covalent modification of the enzyme’s active site
cysteine. These inhibitors, which emerged from a fluopol-ABPP
screen of the 300 000+ MLPCN small molecule library, inactivate
GSTO1 in cancer cells with excellent potency (IC50 ∼30ꢁ40 nM),
selectivity, and duration of action (>12 h). At higher concentra-
tions in vitro (>100 nM), the optimized alkyne inhibitor KT59
showed some evidence of modifying other proteins, but we
should note that these modification events exhibited strong
concentration-dependent increases in signal intensity, even at
the highest inhibitor concentration tested (10 μM), which sug-
gests that they reflect low-percentage labeling of abundant proteins
(a premise that is also supported by our competitive ABPP experi-
ments, where the labeling of these proteins by activity-based probes
was not competed by KT53 at concentrations below 10 μM; see
Figure 8). We therefore do not believe that these additional
protein interactions will interfere with the use of KT59 (and its
nonalkyne analogue KT53) as a selective probe of GSTO1 func-
tion in living systems. We should, however, point out that the
apparent selectivity of KT59 as judged by click chemistry-ABPP
applies only to mechanisms of covalent inhibition, and it remains
possible that KT59 or metabolites of this compound also interact
with additional proteins in a noncovalent manner. Additional
studies will also be required to determine whether KT53 and/or
KT59 can be used to inhibit GSTO1 in vivo.
’ ASSOCIATED CONTENT
S
Supporting Information. LCꢁMS/MS analysis of site of
b
labeling protocol and results; synthetic protocols and analysis of
1, 3, and all KT-series compounds; structures and gel-based
competitive ABPP analysis of HTS hit compounds; additional
gel-based competitive ABPP analysis of Scaffold 1 and 2 com-
pounds; CC50 doseꢁresponse curves for KT53 and KT59; and
complete ref 7. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
cravatt@scripps.edu
’ ACKNOWLEDGMENT
We thank Steven Brown, Jill Ferguson, and Kim Masuda for
technical assistance. This work was supported by National Institutes
of Health grants CA087660 (B.F.C.), MH084512 (H.R.); Dainip-
pon Sumitomo Pharma (K.T.); the National Science Foundation
(predoctoral fellowship to D.A.B.); the California Breast Cancer
Research Program (predoctoral fellowship to D.A.B.); and The
Skaggs Institute for Chemical Biology.
’ REFERENCES
(1) Hayes, J. D.; Flanagan, J. U.; Jowsey, I. R. Annu. Rev. Pharmacol.
Toxicol. 2005, 45, 51.
(2) Adam, G. C.; Burbaum, J.; Kozarich, J. W.; Patricelli, M. P.;
Cravatt, B. F. J. Am. Chem. Soc. 2004, 126, 1363.
(3) Piaggi, S.; Raggi, C.; Corti, A.; Pitzalis, E.; Mascherpa, M. C.;
Saviozzi, M.; Pompella, A.; Casini, A. F. Carcinogenesis 2010, 31, 804.
(4) Yan, X. D.; Pan, L. Y.; Yuan, Y.; Lang, J. H.; Mao, N. J. Proteome
Res. 2007, 6, 772.
Using the optimized inhibitor KT53, we found that inactivat-
ing GSTO1 sensitized cancer cells to cisplatin-induced cell death,
thus supporting a role for this enzyme in chemotherapy resis-
tance. This finding, coupled with previous discoveries that GSTO1
is overexpressed in cancer cells that show enhanced aggressive-
ness and chemotherapy resistance3,4 and that RNA-interference
(5) Bachovchin, D. A.; Brown, S. J.; Rosen, H.; Cravatt, B. F. Nat.
Biotechnol. 2009, 27, 387.
16615
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616

Journal of the American Chemical Society
ARTICLE
(6) Son, J.; Lee, J. J.; Lee, J. S.; Schuller, A.; Chang, Y. T. ACS Chem.
Biol. 2010, 5, 449.
(7) Board, P. G.; et al. J. Biol. Chem. 2000, 275, 24798.
(8) Whitbread, A. K.; Masoumi, A.; Tetlow, N.; Schmuck, E.;
Coggan, M.; Board, P. G. Methods Enzymol. 2005, 401, 78.
(9) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.;
Dillon, M. B.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F.
Nature 2010, 468, 790.
(10) Weerapana, E.; Simon, G. M.; Cravatt, B. F. Nat. Chem. Biol.
2008, 4, 405.
(11) Board, P. G.; Coggan, M.; Cappello, J.; Zhou, H.; Oakley, A. J.;
Anders, M. W. Anal. Biochem. 2008, 374, 25.
(12) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Nat. Biotechnol.
2002, 20, 805.
(13) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535.
(14) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse,
C. M. Science 1989, 246, 64.
(15) Eng, J.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom.
1994, 5, 976.
(16) Tabb, D. L.; McDonald, W. H.; Yates, J. R., III. J. Proteome Res.
2002, 1, 21.
(17) Bachovchin, D. A.; Zuhl, A. M.; Speers, A. E.; Wolfe, M. R.;
Weerapana, E.; Brown, S. J.; Rosen, H.; Cravatt, B. F. J. Med. Chem. 2011,
54, 5229.
(18) Bittker, J. A.; Weiwer, M.; Shimada, K.; Yang, W. S.; MacPherson,
L.; Dandapani, S.; Munoz, B.; Palmer, M.; Stockwell, B. R.; Schreiber, S. L.
Probe Reports from the NIH Molecular Libraries Program, 2010.
(19) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003,
125, 4686.
(20) Raj, L.; Ide, T.; Gurkar, A. U.; Foley, M.; Schenone, M.; Li, X.;
Tolliday, N. J.; Golub, T. R.; Carr, S. A.; Shamji, A. F.; Stern, A. M.;
Mandinova, A.; Schreiber, S. L.; Lee, S. W. Nature 2011, 475, 231.
16616
dx.doi.org/10.1021/ja2066972 |J. Am. Chem. Soc. 2011, 133, 16605–16616