Discovery and optimization of sulfonyl acrylonitriles as selective, covalent inhibitors of protein phosphatase methylesterase-1

Article abstract of DOI:10.1021/jm200502u

The serine hydrolase protein phosphatase methylesterase-1 (PME-1) regulates the methylesterification state of protein phosphatase 2A (PP2A) and has been implicated in cancer and Alzheimer's disease. We recently reported a fluorescence polarization-activit

Full text of DOI:10.1021/jm200502u

ARTICLE
pubs.acs.org/jmc
Discovery and Optimization of Sulfonyl Acrylonitriles as Selective,
Covalent Inhibitors of Protein Phosphatase Methylesterase-1
Daniel A. Bachovchin,†,§ Andrea M. Zuhl,†,§ Anna E. Speers,† Monique R. Wolfe,† Eranthie Weerapana,†
Steven J. Brown,‡ Hugh Rosen,‡ and Benjamin F. Cravatt*,†
†The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037, United States
S Supporting Information
b
ABSTRACT: The serine hydrolase protein phosphatase methylesterase-1
(PME-1) regulates the methylesterification state of protein phosphatase 2A
(PP2A) and has been implicated in cancer and Alzheimer’s disease. We
recently reported a fluorescence polarization-activity-based protein profiling
(fluopol-ABPP) high-throughput screen for PME-1 that uncovered a
remarkably potent and selective class of aza-β-lactam (ABL) PME-1
inhibitors. Here, we describe a distinct set of sulfonyl acrylonitrile inhibitors
that also emerged from this screen. The optimized compound, 28
(AMZ30), selectively inactivates PME-1 and reduces the demethylated
form of PP2A in living cells. Considering that 28 is structurally unrelated to
ABL inhibitors of PME-1, these agents, together, provide a valuable set of
pharmacological probes to study the role of methylation in regulating PP2A
function. We furthermore observed that several serine hydrolases were
sensitive to analogues of 28, suggesting that more extensive structural exploration of the sulfonyl acrylonitrile chemotype may result
in useful inhibitors for other members of this large enzyme class.
’ INTRODUCTION
polarization-activity-based protein profiling (fluopol-ABPP), a
broadly applicable HTS platform for inhibitor discovery where the
ability of compounds to block fluorescent activity-based probe
labeling of proteins is monitored by fluorescence polarization.21
We recently utilized fluopol-ABPP to screen the 300000þ member
NIH small-molecule library against PME-1, leading to the discovery
of a highly potent (IC50 value of ∼10 nM) and selective aza-
β-lactam PME-1 inhibitor, 1 (Figure 1A; ABL127, NIH Probe
ML174).22,23 Generally speaking, however, it is desirable to generate
at least two structurally unrelated classes of inhibitors for an enzyme
target like PME-1 because their shared pharmacological activities can
then be assigned with confidence to disruption of a common protein
target (versus unrelated, compound-specific mechanisms). With this
goal in mind, we report here a set of sulfonyl acrylonitrile inhibitors
that display high selectivity for PME-1.
The serine/threonine protein phosphatase 2A (PP2A) is
involved in a wide range of cellular processes, including DNA
damage repair, cell cycle checkpoint regulation, and the suppres-
sion of pro-oncogenic signaling pathways.1ꢀ3 The targeting of
PP2A to its many substrates in vivo is critical for proper function
and involves dynamic complex assembly with one of several
regulatory (B) subunits and posttranslational modifications,1,2
including C-terminal methylesterification of the catalytic (C)
subunit (referred to hereafter as “methylation”).4ꢀ7 PP2A
methylation is controlled by a methyltransferase (leucine carboxyl-
methyltransferase-1 or LCMT1)8 and methylesterase (protein
phosphatase methylesterase-1 or PME-1),9 which install and
remove this modification, respectively. The impact of methyla-
tion on PP2A function remains largely unknown, but it is thought
to modulate B subunit interactions and substrate specificity.10ꢀ13
Studies have implicated reduced PP2A methylation in cancer14
and neurodegenerative disease,15 indicating that PME-1 could be
an attractive therapeutic target.
’ RESULTS
HTS by Fluopol-ABPP Identifies a Lead Sulfonyl Acryloni-
trile PME-1 Inhibitor. From a 16000 compound Maybridge library
screened by fluopol-ABPP, we identified sulfonyl acrylonitrile 2
Until recently, PME-1 inhibitors had not been reported,
possibly because typical substrate assays for PME-1 were in-
compatible with high-throughput screening (HTS).16ꢀ18 As a
serine hydrolase (SH), PME-1 reacts with fluorophosphonate
(FP) activity-based probes,19,20 making it amenable to fluorescence
Received: April 25, 2011
Published: June 03, 2011
r
2011 American Chemical Society
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Table 1. Structural Exploration of the Sulfonyl Acrylonitrile
Core
Figure 1. Characterization of lead sulfonyl acrylonitrile PME-1 inhibi-
tor 2. (A) Structures of 1 and 2. (B) Evaluation of 2 by gel-based
competitive ABPP with FP-Rh (2 μM) in the soluble proteome (1 mg/
mL protein) of HEK 293T cells. (C) IC50 curve of 2 in the soluble
proteome (1 mg/mL protein) from MDA-MB-231 cells as determined
by gel-based competitive ABPP (IC50 = 10.8 μM). Data are presented as
mean values ( SEM; n = 3/group. (D) Pretreatment of HEK 293T
proteomes with 2 (50 μM, 10 min) before addition of purified PME-1
(500 nM, 3 h) partially inhibits PP2A demethylation as determined by
Western blotting.
a Off-targets are defined as SHs with >50% inhibition at 50 μM.
selectivity. For instance, the bis-sulfonyl compound 16 did not
inhibit PME-1 but exhibited strong activity against another SH,
prolyl endopeptidase (PREP; Supporting Information Figure
S2), and the bis-nitrile compound 17 was 2-fold less potent
against PME-1 but had increased activity against a 40 kDa SH
(Supporting Information Figure S2 and Scheme 3).
(MB51) as a lead PME-1 inhibitor that showed limited off-target
activity against other SHs as judged by gel-based competitive
ABPP assays21,24 with an FP-rhodamine (FP-Rh) probe
We next focused our efforts on modifying the more distal
portions of 2, which led to the discovery that electron-with-
drawing groups on the meta or para positions of the sulfonamide
phenyl ring (18ꢀ21) improved potency (Table 2; Supporting
Information Figure S3). The addition of these electron-with-
drawing groups only increased potency to a point, however, as
the strongly electron-withdrawing compound 22 exhibited no
activity against PME-1. We next altered the size and electronics
of the vinyl sulfone moiety and found that the larger tert-butyl
(23, IC50 = 6.8 μM) and electron-withdrawing 4-F-phenyl
groups (25, IC50 = 4.8 μM) improved potency relative to 2. In
addition to showing improved potency, both 23 and 25 were also
more selective than 2, inhibiting only PME-1 in soluble pro-
teomes (Supporting Information Figure S3).
We finally combined the aforementioned features to create 28
(AMZ30; Table 2), a compound that contains 3-NO2 substituted
sulfonamide and 4-F-phenyl sulfonyl groups and showed sub-
stantially improved inhibition of PME-1 (IC50 value of 600 nM)
with more than 100-fold selectivity relative to other SHs in
human cell lysates (Figure 2A,B). We believe that 28 (and other
acrylonitriles) likely inhibits PME-1 by a covalent, irreversible
mechanism because recovery of enzyme activity was not observed
(Figure 1A,B). 2 showed modest potency for PME-1 (IC50
=
10.8 μM; Figure 1C) and cross-reacted with two additional SHs
in human cell proteomes: acyl-peptide hydrolase (APEH) and
an unidentified 30 kDa SH (Figure 1B; Supporting Information
Figure S1). Despite its limited potency, 2 (50 μM) partially
blocked the PP2A-demethylating activity of PME-1 in cell
extracts (Figure1D). We therefore set out to improve the potency
and cellular activity of 2.
Optimization of the Sulfonyl Acrylonitrile Scaffold for
PME-1 Inhibition. We first synthesized a series of analogues with
perturbations of the sulfonyl acrylonitrile core of 2 (Table 1), which
we suspected might be the key pharmacophore responsible for
PME-1 inhibition. We then screened these compounds by compe-
titive ABPP against PME-1 and other SHs in the mouse brain
soluble proteome, which contains high levels of the PME-1
protein.22 We observed that reduction (Scheme 1) of either the
olefin (3) or the nitrile (4) produced inactive compounds. The
sulfonamide portion of 2 was also critical for activity as analogues
with a free pyrrole group (7), a phenyl group (8), or an electron-
withdrawing para-nitrophenyl group (9), prepared according to
Scheme 2, were all inactive. The presence of mixed electron-
withdrawing groups proved important for both activity and
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Scheme 1. Reduction of Lead Compound 2a
a Reagents and conditions: (i) NaBH4, MeOH, 0 °C, 1 h; (ii) LAH, Et2O, 0 °C, 30 min.
Scheme 2. Synthesis of Compounds 7ꢀ14a
a Reagents and conditions: (i) Et3N, EtOH, mol. sieves, reflux overnight.
Scheme 3. Synthesis of Compounds 6ꢀ28a
a Reagents and conditions: (i) Et3N; DMF, overnight; (ii) NaH, DMF, 0
°C, overnight.
Figure 2. Optimized sulfonyl acrylonitrile 28 selectively inhibits PME-1
in vitro. (A) Evaluation of 28 by gel-based competitive ABPP with FP-
Rh (2 μM) in the soluble proteome (1 mg/mL protein) of HEK 293T
cells reveals selective PME-1 inhibition with (B) an IC50 value of 600
nM. Data are presented as mean values ( SEM; n = 3/group. (C) PME-
1 (500 nM) was incubated with DMSO or 28 (50 μM), and each
reaction was split into two fractions. One fraction was directly labeled
with FP-Rh (left panels), and the other was subjected to gel filtration to
remove free inhibitor and then reacted with FP-Rh (right panels) to
determine the reversibility of inhibition.
Table 2. Optimization of the Sulfonyl Acrylonitrile Scaffold
for Selective PME-1 Inhibitiona
compd
R1
3-CN
R2
Me
PME-1 IC50 (μM)
off-targetsb
olefin or nitrile group in 28 serves as the point of covalent
attachment to PME-1, that 16, which lacks a nitrile group, retains
inhibitory activity against other SHs (PREP) leads us to speculate
that covalent bond formation through 1,4-Michael addition to
the olefin is a likely mechanism for inhibition.
18
19
20
21
22
23
24
25
26
27
28
2.5
3.0
4-NO2
4-Cl
Me
Me
3
APEH
PREP
4-CN
2,3,4,5,6-(F)5
H
Me
4
Me
>20
6.8
Sulfonyl Acrylonitrile 28 Inhibits PME-1 in Situ. We next
asked whether 28 could selectively inhibit PME-1 in living cells.
Incubation of HEK 293T cells with a concentration range of 28
generated an in situ IC50 value for PME-1 inhibition of 3.5 μM as
determined by gel-based ABPP (Figure 3B) without any ob-
served cross-reactivity with other SHs even at 100 μM com-
pound (Figure 3A). We sought to verify the in situ selectivity of
28 by employing the higher resolution, MS-based competitive
SILAC-ABPP technology,22,25 which confirmed complete in
situ inhibition of PME-1 and no activity against 35 other SHs
detected in HEK 293T cells (Figure 3C). Although these
competitive ABPP studies demonstrated that 28 is selective for
PME-1 among SHs in HEK 293T cells, they did not address the
possibility that 28 interacts with other nucleophilic residues on
proteins. Indeed, activated olefins, like the sulfonyl acrylonitriles,
have been shown to react with catalytic cysteine and threonine
residues in enzymes.26 To account for this possibility, we
incubated soluble proteomes harvested from 28-treated HEK
293T cells with chloroacetamide-Rh (CA-Rh) or sulfonate ester-
Rh (SE-Rh) activity-based probes, which react with activated
t-Bu
Ph
H
>20
4.8
H
4-F-Ph
t-Bu
4-F-Ph
4-F-Ph
3-CN
3-CN
3-NO2
3.4
0.64
0.60
a See Supporting Information Figure S3 for gel-based competitive ABPP
data upon which the values in this table are based. b Off-targets are
defined as SHs with >50% inhibition at 50 μM.
after gel filtration chromatography (Figure 2C). However, we
were unable to identify adducts between 28 and the active-site
serine nucleophile of PME-1 (or adducts with any other serine or
cysteine residue in the protein) by mass spectrometry (MS). We
speculate that the putative covalent bond between 28 and PME-
1’s serine nucleophile, while stable in the context of the folded
PME-1 protein, may be broken or eliminated to regenerate the
unmodified enzyme following the protein denaturation steps
required for MS analysis. While we do not yet know whether the
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Figure 3. 28 selectively blocks PME-1 activity and reduces levels of demethylated PP2A in cells. (A) Gel-based competitive ABPP with FP-Rh (2 μM) of
the soluble proteomes from HEK 293T cells treated with 28 (0.1ꢀ100 μM, 1 h) shows selective PME-1 inhibition with (B) an IC50 value of 3.5 μM. (C)
Isotopically “light” and “heavy” HEK 293T cells were treated with 28 (20 μM) or DMSO, respectively, for 1 h. Proteomes were combined in a 1:1 total
protein ratio (0.5 mg each), analyzed by ABPP-MudPIT, and SH activities were quantified by comparing intensities of light and heavy peptide mass
peaks. Data are presented as mean values ( SEM for all quantifiable peptides from each SH. (D) HEK 293T cells stably overexpressing PME-1 were
treated with 28 or 27 (20 μM, 1 h). Compound-treated cells exhibit significant reductions in demethylated PP2A and significant increases in methylated
PP2A, as evident from a representative Western blot (left) and quantified data from replicate experiments (right). (E) Unmodified HEK 293T
cells treated with either 28 (20 μM) or 1 (500 nM) for 1 h showed significant reductions in the level of demethylated PP2A. For (D) and (E), *p < 0.05,
**p < 0.01, and ***p < 0.001 for compound-treated versus DMSO-treated cells. Data are presented as mean values ( SEM; n = 3/group.
cysteines and other nucleophilic residues in proteins.27 28, even
at 100 μM, did not alter the intensity for any CA-Rh or SE-
Rh-labeled proteins relative to their signals in DMSO-treated
samples (Supporting Information Figure S4), indicating that 28
is not a generally thiol-reactive compound.
PME-1 with >100-fold selectivity relative to other SHs in
human cells. 28 (designated as NIH Probe ML136), like the
previously reported PME-1 inhibitor 1, significantly reduced
the levels of demethylated PP2A and, in cells with elevated
levels of PME-1 activity, also increased the levels of methy-
lated PP2A. While 28 is less potent as a PME-1 inhibitor
compared to 1, the fact that these compounds are structurally
unrelated should make them useful as paired pharmacological
probes to study the role that methylation plays in regulating
PP2A function, particularly its impact on determining associa-
tion with specific B subunits. To this point, it remains possible
that 28 and 1 will differentially affect the integrity of PP2A
complexes. Indeed, recent crystallographic studies have shown
that PME-1 can stably interact with the C subunit of PP2A,17
and the ABL and sulfonyl acrylonitrile classes of PME-1 in-
hibitors could affect these proteinꢀprotein interactions in dif-
ferent ways.
Projecting forward, we were also intrigued to find that
several analogues of 28 inhibit SHs in addition to PME-1,
including APEH, PREP, and as-of-yet unidentified 30 kDa and
40 kDa enzymes, in human cell proteomes. These results
suggest that further structural exploration of the sulfonyl
acrylonitrile class of compounds and, in particular, tuning
the electronics of their activated olefin, has the potential to
generate selective inhibitors for additional enzymes from the
SH class. In this way, the sulfonyl acrylonitrile could take
position alongside other classes of covalent SH inhibitors,
such as the carbamate,20,28 lactone/lactam,22,29 and activated
ketones,24 as a versatile chemotype for expanding our phar-
macological coverage of the vast number of SHs present in
eukaryotic and prokaryotic organisms.
Finally, we investigated the impact of 28 on the methylation
state of PP2A. We have shown previously that the effects of
PME-1 inhibition are most pronounced in cell lines over-
expressing PME-1.22 Therefore, we initially treated HEK
293T cells stably overexpressing PME-1 with 28 (20 μM)
and, for comparison, the structurally related PME-1 inhibitor 27
(20 μM; Table 1), and observed that both inhibitors caused an
∼80% reduction in the levels of demethylated PP2A (Figure 3D).
28 and 27 treatment also significantly increased the methylated
form of PP2A (Figure 3D). These changes in PP2A methylation
state were similar in magnitude to those observed previously with
the structurally unrelated inhibitor 1.22 28 (20 μM) and 1 (500
nM) also decreased the demethylated form of PP2A to a similar
degree in untransfected HEK 293T cells expressing basal levels of
PME-1 (Figure 3E).
’ CONCLUSIONS
In summary, we have developed a class of sulfonyl acryloni-
triles that selectively inhibit the SH PME-1. Essential to the
success of this effort were: (1) a fluopol-ABPP HTS assay that
enabled the identification of a lead compound with only a modest
affinity for PME-1, and (2) the coupling of medicinal chemistry
with gel-based competitive ABPP for the parallel refinement of
compound potency and selectivity directly in native proteomes.
The optimized sulfonyl acrylonitrile compound, 28, inhibited
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’ EXPERIMENTAL SECTION
(E)-2-(Methylsulfonyl)-3-(1H-pyrrol-2-yl)acrylonitrile (7). Yield:
18%. 1H NMR (400 MHz) δ 9.64 (br s, 1H), 7.89 (s, 1H), 7.31ꢀ7.29
(m, 1H), 7.06ꢀ7.02 (m, 1H), 6.50ꢀ6.48 (m, 1H), 3.16 (s, 3H). HR-MS
m/z calcd for C8H8N2O2S (M ꢀ H): 195.0234. Found: 195.0240.
(E)-2-(Methylsulfonyl)-3-phenylacrylonitrile (8). Yield: 43%. 1H
NMR (400 MHz) δ 8.14 (s, 1H), 7.96ꢀ7.95 (m, 2H), 7.66ꢀ7.61 (m,
1H), 7.58ꢀ7.53 (m, 2H), 3.22 (s, 3H). ESI-MS m/z calcd for C10H9NO2S
(M þ H): 208.0. Found: 208.0.
Synthetic Methods. Chemistry. General Procedures. All reac-
tions were carried out in flame-dried glassware under a nitrogen atmo-
sphere with dry solvents, using standard anhydrous techniques. Dry N,
N-dimethylformamide (DMF), methanol (MeOH), and diethyl ether
were obtained by passing the previously degassed solvents through
activated alumina columns. Absolute ethanol (EtOH) was purchased
from Pharmco-AAPER and used without further purification. All
reagents were purchased at the highest commercial quality and used
without further purification. Reactions were monitored by analytical
thin-layer chromatography (TLC) on precoated, glass backed silica gel
60 F254 plates. All reported yields are unoptimized. Reactions were
purified either by PTLC, also on silica gel 60 F254 plates or by flash
chromatography on 40ꢀ60 MYM mesh silica gel as specified. The purity
of tested compounds (>95%) was verified by HPLC using a 12 min
gradient elution of increasing concentrations of CH3CN in water
(0ꢀ100%) with UV detection at 254 nm on a Gemini C18 50 mm ꢁ
4.60 mm, 5 μm column. NMR spectra were recorded on Bruker DRX-
600, DRX-500, AMX-400, and Inova-400 instruments and calibrated
using residual chloroform as an internal reference. The following
abbreviations were used to explain multiplicities: s = singlet, br s =
broad singlet, d = doublet, m = multiplet, and all J values are reported in
Hz. High resolution mass spectra (HRMS) were recorded on an Agilent
LC/MSD TOF mass spectrometer by electrospray ionization time-of-
flight reflectron experiments. Low resolution mass spectra were re-
corded on an Agilent LC/MSD ESI mass spectrometer or an Agilent
5973N GC/MS.
2-(Methylsulfonyl)-3-(1-(phenylsulfonyl)-1H-pyrrol-2-yl)propane-
nitrile (3). MB51 (5 mg, 0.015 mmol, 1 equiv) was diluted in methanol
(150 μL) and cooled to 0 °C under N2 in a flame-dried half-dram vial.
NaBH4 (140 μL, 5 mg/mL in methanol, 1.2 equiv) was then added and
the reaction mixture allowed to stir and warm to room temperature over
1 h, at which point it was concentrated in vacuo. PTLC (SiO2, 250 μm,
70% EtOAc/Hx) afforded 5 mg (0.015 mmol, quant) of the desired
product. 1H NMR (400 MHz) δ 7.80ꢀ7.78 (m, 2H), 7.68ꢀ7.63
(m, 1H), 7.57ꢀ7.53 (m, 2H), 7.39ꢀ7.38 (m, 1H), 6.39ꢀ6.38
(m, 1H), 6.32ꢀ6.30 (m, 1H), 4.62 (dd, J = 11.4, 4.2, 1H), 3.58 (dd,
J = 14.6, 4.2, 1H), 3.34 (dd, J = 14.6, 11.4, 1H), 3.20 (s,3H). HR-MS m/z
calcd for C14H14N2O4S2 (M þ Na): 361.0287. Found: 361.0277.
(E)-2-(Methylsulfonyl)-3-(1-(phenylsulfonyl)-1H-pyrrol-2-yl)prop-
2-en-1-amine (4). MB51 (5 mg, 0.015 mmol, 1 equiv) was diluted in
ether (150 μL) and cooled to 0 °C under N2 in a flame-dried half-dram
vial. LAH (18 μL, 1 M in THF, 1.2 equiv) was then added slowly
dropwise and the reaction mixture allowed to stir for 30 min, at which
point it was exposed to air and concentrated under a stream of N2. PTLC
(SiO2, 250 μm, 80% EtOAc/Hx) afforded 0.6 mg (0.002 mmol, 12%) of
the desired product. 1H NMR (500 MHz) δ 7.81ꢀ7.79 (m, 2H),
7.64ꢀ7.61 (m, 1H), 7.55ꢀ7.52 (m, 2H), 7.32ꢀ7.31 (m, 1H),
7.26ꢀ7.24 (m, 1H), 6.22ꢀ6.21 (m, 1H), 6.15ꢀ6.14 (m, 1H), 4.29
(s, 2H), 3.76 (s, 2H), 2.58 (s, 3H). HR-MS m/z calcd for C14H16N2O4S2
(M þ Na): 363.0444. Found: 363.0448.
(E)-2-(Methylsulfonyl)-3-(4-nitrophenyl)acrylonitrile (9). Yield:
13%. 1H NMR (400 MHz) δ 8.41ꢀ8.38 (m, 2H), 8.21 (s, 1H),
8.13ꢀ8.11 (m, 2H), 3.26 (s, 3H). GC-MS m/z calcd for C10H8N2O4S
(Mþ): 252.0. Found: 252.0.
1
2-((1H-Pyrrol-2-yl)methylene)malononitrile (10). Yield: 25%. H
NMR (400 MHz) δ 9.84 (br s, 1H), 7.51 (s, 1H), 7.32ꢀ7.30 (m, 1H),
7.05ꢀ6.99 (m, 1H), 6.49ꢀ6.47 (m, 1H). HR-MS m/z calcd for C8H5N3
(M ꢀ H): 142.0411. Found: 142.0415.
2-(2,2-Bis(methylsulfonyl)vinyl)-1H-pyrrole (11). Yield: 35%. 1H
NMR (400 MHz) δ 8.00 (s, 1H), 7.32ꢀ7.30 (m, 1H), 6.99ꢀ6.97 (m,
1H), 6.45ꢀ6.42 (m, 1H), 3.33 (s, 3H), 3.31 (s, 3H). ESI-MS m/z calcd for
C8H11NO4S2 (M ꢀ H): 249.01. Found: 249.01.
(E)-2-(Phenylsulfonyl)-3-(1H-pyrrol-2-yl)acrylonitrile (12). Yield:
1
59%. H NMR (400 MHz) δ 9.62 (br s, 1H), 7.99ꢀ7.96 (m, 3H),
7.67ꢀ7.65 (m, 1H), 7.61ꢀ7.56 (m, 2H), 7.24ꢀ7.22 (m, 1H),
7.04ꢀ7.02 (m, 1H), 6.46ꢀ6.44 (m, 1H). ESI-MS m/z calcd for
C13H10N2O2S (M ꢀ H): 257.0. Found: 257.0.
(E)-2-((4-Fluorophenyl)sulfonyl)-3-(1H-pyrrol-2-yl)acrylonitrile
1
(13). Yield: 86%. H NMR (400 MHz) δ 9.60 (br s, 1H), 8.01ꢀ7.97
(m, 2H), 7.96 (s, 1H), 7.28ꢀ7.24 (m, 3H), 7.05ꢀ7.02 (m, 1H),
6.47ꢀ6.45 (m, 1H). ESI-MS m/z calcd for C13H9FN2O2S (M ꢀ H):
275.0. Found: 275.0.
(E)-2-(tert-Butylsulfonyl)-3-(1H-pyrrol-2-yl)acrylonitrile (14). Yield:
85%.1H NMR (400 MHz) δ 9.90 (br s, 1H), 7.80 (s, 1H), 7.30ꢀ7.26
(m, 1H), 7.08ꢀ7.05 (m, 1H), 6.48ꢀ6.47 (m, 1H), 1.48 (s, 9H). ESI-MS
m/z calcd for C11H14N2O2S (M ꢀ H): 237.1. Found: 237.0.
General Procedure for the Sulfonation of Pyrroles. To a flame-dried
RBF under nitrogen atmosphere was charged the free pyrrole (1 equiv),
which was subsequently diluted with DMF (0.05 M in pyrrole). Method
A: for the preparation of 19, 20, 22, 23, 25, 27, and 28, TEA (5 equiv)
was then added, followed by the sulfonyl chloride (1.2 equiv). Method
B: for the preparaton of 16ꢀ18, 21, 24, and 26, the reaction mixture was
cooled to 0 °C before the addition of the sulfonyl chloride and then NaH
(5 equiv). For both methods A and B, the reactions allowed to stir
overnight at which point the solvent was removed in vacuo. PTLC
(SiO2, 1000 mm, 50% EA/Hx) afforded the desired products.
2-(2,2-Bis(methylsulfonyl)vinyl)-1-(phenylsulfonyl)-1H-pyrrole (16).
1
Yield: 72%. H NMR (400 MHz) δ 8.85 (s, 1H), 7.96ꢀ7.93 (m, 2H),
7.82ꢀ7.81 (m, 1H), 7.74ꢀ7.73 (m, 1H), 7.70ꢀ7.65 (m, 1H), 7.60ꢀ7.55
(m, 2H), 7.52ꢀ7.50 (m, 1H), 3.33 (s, 3H), 3.02 (s, 3H). ESI-MS m/z calcd
for C14H15NO6S3 (M ꢀ H): 388.01. Found: 388.01.
2-((1-(Phenylsulfonyl)-1H-pyrrol-2-yl)methylene)malononitrile (17).
1
Yield: 74%. H NMR (400 MHz) δ 8.34 (s, 1H), 7.82ꢀ7.79 (m, 2H),
7.76ꢀ7.75 (m, 2H), 7.74ꢀ7.70 (m, 1H), 7.62ꢀ7.58 (m, 2H), 6.58ꢀ6.56
(m, 1H). HR-MS m/z calcd for C14H9N3O2S (M ꢀ H): 282.0343. Found:
282.0337.
General Procedure for the Preparation of Vinyl Aromatics. Pow-
dered 4 Å molecular sieves were charged into a RBF and flame-dried
under vacuum. After replacing the atmosphere with nitrogen, ethanol
(0.15 M in reactants) was added, followed by the carboxaldehyde
(1 equiv), the active methylene compound (1 equiv), and TEA
(4 equiv). The reaction was refluxed for 4 h, cooled to room tempera-
ture, and then quenched with water (0.5 vol equiv). The aqueous layer
was acidified with 1N HCl (0.2 vol equiv) and the product extracted into
DCM (3 ꢁ 2 vol equiv). The combined organic layers were then washed
with water (1 vol equiv), brine (1 vol equiv), dried over MgSO4, and
concentrated in vacuo. Flash chromatography (SiO2, 10ꢀ50% EA/Hx
gradient) afforded the Knoevenagel products.
(E)-3-(2-(2-Cyano-2-(methylsulfonyl)vinyl)-1H-pyrrol-1-ylsulfonyl)
1
benzonitrile (18). Yield: 6%. H NMR (400 MHz) δ 8.63 (s, 1H),
8.15ꢀ8.11 (m, 2H), 7.98ꢀ7.95 (m, 1H), 7.77ꢀ7.74 (m, 3H),
6.55ꢀ6.53 (m, 1H), 3.19 (s, 3H). HR-MS m/z calcd for C15H11N3O4S2
(M ꢀ H): 360.0118. Found: 360.0130.
(E)-2-(Methylsulfonyl)-3-(1-((4-nitrophenyl)sulfonyl)-1H-pyrrol-2-
yl)acrylonitrile (19). Yield: 34%. 1H NMR (500 MHz) δ 8.65 (s, 1H),
8.43ꢀ8.40 (m, 2H), 8.09ꢀ8.06 (m, 2H), 7.78ꢀ7.75 (m, 2H), 6.64ꢀ6.62
(m, 1H), 3.19 (s, 3H). HR-MS m/z calcd for C14H11N3O6S2 (M ꢀ H):
380.0011. Found: 380.0023.
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(E)-3-(1-(4-Chlorophenylsulfonyl)-1H-pyrrol-2-yl)-2-(methyl-
1
sonicated on ice with a probe sonicator, and centrifuged (64000g,
45 min, 4 °C) to provide the soluble fraction as the supernatant and
the membrane fraction as the pellet. For in situ experiments, compounds
were directly added to the cell culture media and cells incubated at 37 °C
for the indicated time before the cells were washed and scraped. The cell
pellets were isolated by centrifugation at 1400g for 3 min, 4 °C,
resuspended in 500 μL of ice-cold PBS (pH 7.5) and sonicated as
described above. Then 250 μL of this total cell extract was saved for
PP2A methylation analysis by Western blotting (described below), and
the remainder was separated into membrane and soluble fractions as
described above. PME-1 was stably overexpressed in HEK 293T cells as
described previously.22 Rat APEH and mouse PREP were transiently
overexpressed in COS-7 cells as described previously.20 Total protein
concentration of each fraction was determined using a protein assay kit
(Bio-Rad). Samples were stored at ꢀ80 °C until use.
sulfonyl)acrylonitrile (20). Yield: 64%. H NMR (400 MHz) δ 8.66
(s, 1H), 7.84ꢀ7.83 (m, 1H), 7.81ꢀ7.80 (m, 1H), 7.75ꢀ7.74 (m, 1H),
7.74ꢀ7.72 (m, 1H), 7.57ꢀ7.56 (m, 1H), 7.55ꢀ7.54 (m, 1H), 6.59ꢀ6.57
(m, 1H), 3.17 (s, 3H). ESI-MS m/z calcd for C14H11ClN2O4S2 (M ꢀ H):
369.0. Found: 369.0.
(E)-4-(2-(2-Cyano-2-(methylsulfonyl)vinyl)-1H-pyrrol-1-ylsulfo-
nyl)benzonitrile (21). Yield: 8%. 1H NMR (400 MHz) δ 8.63 (s, 1H),
8.01ꢀ7.98 (m, 2H), 7.90ꢀ7.87 (m, 2H), 7.76ꢀ7.75 (m, 2H), 6.63ꢀ6.61
(m, 1H), 3.19 (s, 3H). HR-MS m/z calcd for C15H11N3O4S2 (M ꢀ H):
360.0118. Found: 360.0127.
(E)-2-(Methylsulfonyl)-3-(1-((perfluorophenyl)sulfonyl)-1H-pyr-
rol-2-yl)acrylonitrile (22). Yield: 31%. 1H NMR (600 MHz) δ 8.62
(s, 1H), 7.83ꢀ7.82 (m, 1H), 7.75ꢀ7.74 (m, 1H), 6.64ꢀ6.63 (m, 1H),
3.18 (s, 1H). ESI-MS m/z calcd for C14H7F5N2O4S2 (M ꢀ H): 425.0.
Found: 425.0.
Competitive ABPP Assays in Proteomes. For in vitro experi-
ments, proteomes were diluted to 1 mg/mL in PBS (pH 7.5) and
incubated with DMSO or compound for 30 min at 25 °C (25 μL total
reaction volume). FP-rhodamine was then added at a final concentration
of 2 μM. After 45 min, the reactions were quenched with 2ꢁ SDS-PAGE
loading buffer (reducing), separated by SDS-PAGE (10% acrylamide),
and visualized in-gel with a Hitachi FMBio IIe flatbed fluorescence
scanner (MiraiBio). For in situ experiments, soluble proteomes were
prepared from harvested cells treated with DMSO or compound as
indicated and diluted to 1 mg/mL in PBS (25 μL total volume). These
proteomes were then directly labeled with rhodamine probe as follows:
FP-rhodamine (2 μM, 45 min), CA-rhodamine (5 μM, 1 h), or SE-
rhodamine (5 μM, 1 h) at 25 °C, and analyzed as described above.
Determination of IC50 Values. For determination of in vitro IC50
values, compounds were incubated in the indicated soluble proteome
(1 mg/mL, 25 μL total volume) at the specified concentrations
(performed in triplicate) for 45 min at 37 °C. The samples were then
labeled with FP-rhodamine (2 μM) for 45 min, quenched with 2ꢁ
loading buffer (reducing), separated by SDS-PAGE, and visualized by in-
gel fluorescence scanning. For determination of in situ IC50 values, the
soluble fractions from cells treated with compound (1 h, performed in
triplicate using conditions described above) were diluted to 1 mg/mL in
PBS (25 μL total volume) and then labeled with FP-rhodamine and
analyzed as described above. The percentage activity remaining was
determined by measuring the integrated optical intensity of the bands
using ImageJ software. IC50 values were determined from a doseꢀ
response curve generated using Prism software (GraphPad).
Assessing the Reversibility of Inhibition. Purified wild-type
PME-1 (500 nM, 2.6 mL total volume in PBS) was incubated with
DMSO or 28 (50 μM) at 25 °C. After 30 min, 100 μL was removed from
each reaction (fraction A). The remaining 2.5 mL of each reaction was
passaged over a Sephadex G-25 M column (GE Healthcare) and eluted
in a volume of 3.5 mL PBS (fraction B). Then 100 μL of both fractions A
and B were labeled with FP-rhodamine (2 μM, as described above).
After 30 min, the reactions were quenched with 2ꢁ SDS-PAGE loading
buffer, separated by SDS-PAGE, and analyzed by in-gel fluorescence
scanning. Gels were then subjected to Coomassie staining with Instant-
Blue (Expedeon) to verify equivalent protein loading.
(E)-2-(tert-Butylsulfonyl)-3-(1-(phenylsulfonyl)-1H-pyrrol-2-yl)-
1
acrylonitrile (23). Yield: 45%. H NMR (400 MHz) δ 8.59 (s, 1H),
7.90ꢀ7.88 (m, 2H), 7.78ꢀ7.77 (m, 2H), 7.70ꢀ7.67 (m, 1H), 7.60ꢀ7.56
(m, 1H), 6.57ꢀ6.55 (m, 1H), 1.48 (s, 9H). HR-MS m/z calcd for
C17H18N2O4S2 (M þ Na): 401.0606. Found: 401.0611.
(E)-2-(Phenylsulfonyl)-3-(1-(phenylsulfonyl)-1H-pyrrol-2-yl)acryl-
onitrile (24). Yield: 5%. 1H NMR (400 MHz) δ 8.03 (s, 1H), 8.00ꢀ7.98
(m, 2H), 7.69ꢀ7.66 (m, 3H), 7.62ꢀ7.57 (m, 2H), 7.53ꢀ7.51 (m, 2H),
7.26ꢀ7.24 (m, 1H), 7.11ꢀ7.07 (m, 1H), 6.47ꢀ6.45 (m, 1H). HR-MS
m/z calcd for C19H14N2O4S2 (M þ Na): 421.0287. Found: 421.0295.
(E)-2-(4-Fluorophenylsulfonyl)-3-(1-(phenylsulfonyl)-1H-pyrrol-
2-yl)acrylonitrile (25). Yield: quantitative. 1H NMR (400 MHz) δ 8.77
(s, 1H), 8.02ꢀ7.97 (m, 2H), 7.93ꢀ7.92 (m, 1H), 7.91ꢀ7.90 (m, 1H),
7.76ꢀ7.75 (m, 1H), 7.74ꢀ7.70 (m, 1H), 7.63ꢀ7.58 (m, 3H),
7.30ꢀ7.25 (m, 2H), 6.53ꢀ6.51 (m, 1H). HR-MS m/z calcd for
C19H13FN2O4S2 (M ꢀ H): 415.0. Found: 415.0.
(E)-3-(2-(2-(tert-Butylsulfonyl)-2-cyanovinyl)-1H-pyrrol-1-ylsulfonyl)-
1
benzonitrile (26). Yield: 8%. H NMR (400 MHz) δ 8.54 (s, 1H),
8.17ꢀ8.14 (m, 1H), 8.12ꢀ8.11 (m, 1H), 7.97ꢀ7.95 (m, 1H),
7.80ꢀ7.74 (m, 3H), 6.64ꢀ6.62 (m, 1H), 1.48 (s, 9H). HR-MS m/z
calcd for C18H17N3O4S2 (M þ Na): 426.0553. Found: 426.0568.
(E)-3-(2-(2-Cyano-2-(4-fluorophenylsulfonyl)vinyl)-1H-pyrrol-1-
ylsulfonyl)benzonitrile (27). Yield: 70%. 1H NMR (400 MHz) δ 8.76
(s, 1H), 8.20ꢀ8.17 (m, 2H), 8.07ꢀ8.03 (m, 2H), 8.02ꢀ8.00 (m, 1H),
7.83ꢀ7.79 (m, 1H), 7.77ꢀ7.75 (m, 1H), 7.69ꢀ7.68 (m, 1H),
7.36ꢀ7.31 (m, 2H), 6.62ꢀ6.60 (m, 1H). HR-MS m/z calcd for
C20H12FN3O4S2 (M þ Na): 464.0145. Found: 464.0152.
(E)-2-(4-Fluorophenylsulfonyl)-3-(1-(3-nitrophenylsulfonyl)-1H-
pyrrol-2-yl)acrylonitrile (28). Yield: 84%. 1H NMR (500 MHz) δ 8.78
(s, 1H), 8.69ꢀ8.68 (m, 1H), 8.57ꢀ8.55 (m, 1H), 8.28ꢀ8.27 (m, 1H),
8.04ꢀ8.01 (m, 2H), 7.89ꢀ7.86 (m, 1H), 7.77ꢀ7.76 (m, 1H),
7.66ꢀ7.65 (m, 1H), 7.31ꢀ7.28 (m, 2H), 6.60ꢀ6.58 (m, 1H). HR-MS
m/z calcd for C19H12FN3O6S2 (M ꢀ H): 400.0079. Found: 400.0096.
Materials for Biological Experiments. FP-biotin,30,31 FP-
rhodamine,32 SE-rhodamine,33 and 123 were synthesized following pre-
viously described protocols. Demethylated-specific PP2A (clone 4b7),
methylated-specific PP2A (clone 2A10), and total PP2A antibodies were
purchased from Millipore, anti R-tubulin antibodies were purchased from
NeoMarkers, and 2 was purchased from Ryan Scientific. Recombinant
PME-1 was expressed and purified as described previously.22
Cell Culture and Preparation of Human Cell Line Pro-
teomes. MDA-MB-231 cells were grown in L15 media supplemented
with 10% fetal bovine serum at 37 °C in a CO2 free incubator. HEK
293T cells were grown in DMEM with 10% fetal bovine serum at 37 °C
with 5% CO2. For in vitro experiments, cells were grown to 100%
confluency, washed two times with ice cold PBS (pH 7.5), and scraped.
Cell pellets were isolated by centrifugation at 1400g for 3 min at 4 °C.
The pellets were resuspended in 500 μL of ice-cold PBS (pH 7.5),
Isotopic Competitive ABPP-MudPIT. HEK 293T cells were
grown in DMEM SILAC media (ThermoScientific) supplemented with
dialyzed fetal bovine serum (Gemini) and 12C14N-lysine and -arginine
(Sigma) for “light” cells or 13C615N2-lysine and -arginine (Isotec) for
“heavy” cells. Cells were treated as indicated with DMSO or 28 (1 h,
37 °C), washed, harvested, and soluble and membrane proteomes were
isolated as described above. Light and heavy proteome fractions (0.5 mg
each) were combined (1 mL total volume) and labeled with 5 μM of FP-
biotin for 1 h at 25 °C. After incubation, the membrane proteomes were
solubilized with 1% Triton-X and rotated at 4 °C for 1 h. Enrichment
of FP-labeled proteins was achieved as previously described.34 The
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Journal of Medicinal Chemistry
ARTICLE
streptavidin-enriched proteome was washed two times for 3 min with
(1) 1% SDS in PBS, (2) 6 M urea in PBS, (3) PBS (pH 7.5), and finally
resuspended in 200 μL of 8 M urea in 25 mM ammonium bicarbonate.
Samples were then prepared for on-bead digestion by reduction with
10 mM TCEP (Sigma) for 30 min at 25 °C and alkylation with 12 mM
iodoacetamide (Sigma) for 30 min at 25 °C in the dark. Samples were
diluted to 2 M urea with PBS (pH 7.5), and digestions were performed
for 12 h at 37 °C with sequence-grade modified trypsin (Promega; 4 μL
of 0.5 μg/μL) in the presence of 2 mM CaCl2. Last, peptide samples
were acidified with formic acid to a final concentration of 5%.
probed using indicated antibodies following manufacturers’ instructions
and were visualized and quantified using the Odyssey imaging system
(Li-Cor).
’ ASSOCIATED CONTENT
S
Supporting Information. Evaluations of 2 and 16; re-
b
presentative gel-based competitive ABPP profiles for 18, 19, 20,
21, 23, 25, 26, 27, and 28; selectivity profiling of 28 for cross-
reactivity; detailed synthesis of the chloroacetamide (CA)-rho-
damine probe. This material is available free of charge via the
Internet at http://pubs.acs.org.
Digested peptide mixtures were pressure-loaded on a fused silica
loading column (250 μm i.d., 360 μm o.d.) packed with 4 cm of reversed-
phase resin (Aqua C18, 5 μm, 125A, Phenomenex) fitted with a fritted
filter (Upchurch). The loading column was attached in-line to a biphasic
MudPIT capillary column (100 μm i.d., 360 μm o.d., packed with 10 cm
Aqua C18 reversed phase resin followed by 3 cm strong cation exchange
resin (Partisphere, 5 μm, 120A, Whatman) with an in-house pulled tip).
The sample was analyzed by two-dimensional liquid chromatography
(2D-LC) separation in combination with tandem mass spectrometry as
previously described34,35 using an Agilent 1100-series quaternary pump
(manual flow-split system) and LTQ-Orbitrap Velos mass spectrometer
running Xcalibur Software (ThermoScientific). The mass spectrometer
was outfitted with an in-house fabricated nanospray platform. Peptides
were eluted in a five-step MudPIT experiment (using 0%, 10%, 25%,
80%, and 100% salt bumps of 500 mM aqueous ammonium acetate, each
step followed by an increasing gradient of aqueous acetonitrile/0.1%
formic acid) and data were collected in data-dependent acquisition
mode with dynamic exclusion enabled (repeat count of 1, exclusion
duration of 20 s). One full MS1 scan (400ꢀ1800 m/z) was followed by
30 data dependent MS2 scans of the most abundant ions with mono-
isotopic precursor selection enabled. All other parameters were left at
default values. The MS2 spectra data were extracted from the raw file
using RAW Xtractor (version 1.9.7; publically available at http://fields.
scripps.edu/?q=content/download). MS2 spectra data were searched
using the SEQUEST algorithm (Version 3.0)36 against the latest version
of the human IPI database concatenated with the reversed database for
assessment of false-discovery rate.37 SEQUEST searches allowed for
variable oxidation of methionine (þ16), static modification of cysteine
residues (þ57 due to alkylation), and no enzyme specificity. Each data
set was independently searched with light and heavy params files: for the
light search, all other amino acids were left at default masses; for the
heavy search, static modifications on lysine (8.0142) and arginine
(10.0082) were specified. The resulting MS2 spectra matches were
assembled into protein identifications and filtered using DTASelect
(version 2.0.41)38 with the --trypstat option, which applies different
statistical models for the analysis of tryptic, half-tryptic, nontryptic
peptides, and peptides were restricted to fully tryptic using the -y 2
option. DTASelect 2.0 uses a quadratic discriminant analysis to achieve a
user-defined maximum peptide false positive rate; the default parameters
(maximum false positive rate of 5%) was used for the search; however,
the actual false positive rate was much lower (<1%). SILAC ratios were
quantified using in-house software.39 The total proteomic data was
filtered manually for serine hydrolases containing at least two quantifi-
able peptides, and data was globally normalized to a control sample
where both heavy and light cells were treated with DMSO and mixed in a
1:1 ratio to adjust heavy to light peptide abundance to exactly a 1:1 ratio
(correction factor = 1.3 light/heavy). Two serine hydrolases, BCHE and
CES3, displayed ratios >3 light:heavy in the control sample that were
maintained in the inhibitor-treated sample and, for simplicity, were
excluded from analysis.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 858-784-8633. E-mail: cravatt@scripps.edu.
Author Contributions
§These authors contributed equally to this work.
’ ACKNOWLEDGMENT
We thank Tianyang Ji, David Milliken, Kim Masuda, and
Benjamin Kipper for technical assistance. This work was sup-
ported by the National Institutes of Health CA132630 (B.F.C.),
MH084512 (B.F.C., H.R.), the National Science Foundation
(predoctoral fellowships to D.A.B. and A.M.Z.), the California
Breast Cancer Research Program (predoctoral fellowship to D.A.
B.), and The Skaggs Institute for Chemical Biology.
’ ABBREVIATIONS USED
PP2A, protein phosphatase 2A; PME-1, protein phosphatase
methylesterase-1; fluopol, fluorescence polarization; ABPP,
activity-based protein profiling; ABL, aza-β-lactam; LCMT1, leu-
cine carboxylmethyltransferase-1; FP, fluorophosphonate; SH,
serine hydolase; HTS, high-throughput screening; Rh, rhoda-
mine; APEH, acyl-peptide hydrolase; PREP, prolyl endopepti-
dase; SILAC, stable isotope labeling with amino acids in cell
culture; CA, chloroacetamide; SE, sulfonate ester
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