Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis

Article abstract of DOI:10.1021/jm400898x

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-β (DAGLβ), a principal biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (2-substituted)-piperidyl-1,2,3-TUs that selectively inactivate DAGLβ in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts. We also describe an expanded repertoire of DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,2,3-TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic pathways.

Full text of DOI:10.1021/jm400898x

Article
pubs.acs.org/jmc
Development and Optimization of Piperidyl-1,2,3-Triazole Ureas as
Selective Chemical Probes of Endocannabinoid Biosynthesis
Ku-Lung Hsu,*,†,‡,§ Katsunori Tsuboi,†,‡,§ Landon R. Whitby,†,‡ Anna E. Speers,†,‡ Holly Pugh,‡
Jordon Inloes,†,‡ and Benjamin F. Cravatt*,†,‡
†The Skaggs Institute for Chemical Biology and ‡Department of Chemical Physiology, The Scripps Research Institute, SR107 10550
North Torrey Pines Road, La Jolla, California 92037, United States.
S
* Supporting Information
ABSTRACT: We have previously shown that 1,2,3-triazole
ureas (1,2,3-TUs) act as versatile class of irreversible serine
hydrolase inhibitors that can be tuned to create selective
probes for diverse members of this large enzyme class,
including diacylglycerol lipase-β (DAGLβ), a principal
biosynthetic enzyme for the endocannabinoid 2-arachidonoyl-
glycerol (2-AG). Here, we provide a detailed account of the
discovery, synthesis, and structure−activity relationship (SAR)
of (2-substituted)-piperidyl-1,2,3-TUs that selectively inacti-
vate DAGLβ in living systems. Key to success was the use of
activity-based protein profiling (ABPP) with broad-spectrum
and tailored activity-based probes to guide our medicinal
chemistry efforts. We also describe an expanded repertoire of
DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass
spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,2,3-
TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic
pathways.
endocannabinoid−eicosanoid network involved in inflamma-
tory responses.13
INTRODUCTION
■
Serine hydrolases (SHs) represent one of the largest and most
diverse enzyme families in Nature. The 200+ human members
of this enzyme class catalyze the hydrolysis of small-molecule
transmitters, lipids, peptides, and proteins1,2 and have emerged
as therapeutic targets for several clinically approved drugs that
treat obesity,3 type 2 diabetes,3,4 and cognitive disorders.5
Despite their pervasive roles in biology, most mammalian SHs
remain poorly characterized with respect to their biochemical
and physiological functions. The development of selective
inhibitors to probe the function of individual SHs in living
systems would be of great value, but this goal has only been
accomplished for a limited number of SH targets.6−12
Here, we provide a full account of the discovery, synthesis,
structure−activity relationship (SAR), and inhibitory activities
of (2-substituted)-Pip-1,2,3-TUs that culminated in the
discovery of the DAGLβ-selective inhibitor 11 (KT109) and
the dual DAGLα/β inhibitor 27 (KT172).13 We also describe
the synthesis of alkyne and biotin-coupled 1,2,3-TUs and their
use as tailored activity-based probes for DAGL enzymes.
Finally, we show that structurally related (2-phenyl)-Pip-1,2,3-
TUs do not inhibit DAGLβ but maintain potency against the
off-target enzyme ABHD6, thus serving as useful negative-
control probes for DAGLβ-related studies, as well as a novel
scaffold for developing in vivo-active inhibitors of ABHD6.18
We have shown that 1,2,3-triazole ureas (1,2,3-TUs) serve as
a versatile scaffold for developing selective inhibitors of SHs.8
1,2,3-TUs inhibit SHs by an irreversible mechanism involving
carbamylation of the active-site serine nucleophile (Supporting
Information Figure 1). We recently reported the development
of potent and selective inhibitors of diacylglycerol lipase-β
(DAGLβ) based on a (2-substituted)-piperidyl (Pip)-1,2,3-TU
scaffold.13,14 DAGLβ and DAGLα are sequence-related SHs
that produce the endocannabinoid, 2-arachidonoylglycerol (2-
AG).15−17 The development of selective, in vivo-active
inhibitors targeting DAGLβ enabled the functional analysis of
this enzyme in peritoneal macrophages where it regulates an
RESULTS
■
Discovery and Characterization of (2-Substituted)-
Pip-1,2,3-TUs as DAGLβ Inhibitors. As described in our
previous studies,13 we found that the 1,4-regioisomer of (2-
substituted)-Pip-1,2,3-TUs, such as 2, show enhanced potency
for DAGLβ compared with the corresponding 2,4-regisomer
(3) as measured by competitive activity-based protein profiling
(ABPP) (Figure 1). ABPP is a chemoproteomic technology
Received: June 17, 2013
© XXXX American Chemical Society
A
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Figure 1. Characterization of (2-benzyl)-Pip-1,2,3-TU isomers as DAGLβ inhibitors. The activity of regio- (2 and 3) and stereo- (2a and b) isomers
was tested against recombinant DAGLβ expressed in HEK293T cells and endogenous ABHD6 in mouse Neuro2A neuroblastoma cell proteome
using a gel-based competitive ABPP assay. Compounds were preincubated with HEK293T-DAGLβ membrane (0.3 mg/mL) or Neuro2A (1 mg/
mL) proteomes for 30 min at 37 °C prior to treatment with the activity-based probes 38 (HT-01)13 or fluorophosphonate-rhodamine (FP-Rh),
respectively (1 μM probe, 30 min), and analysis by gel-based ABPP, where compound activity was measured by reductions in probe-labeling of target
SHs. This gel and subsequent fluorescent gels are shown in gray scale.
Scheme 1. General Synthesis of (2-Benzyl)-Pip-1,2,3-TUs
Scheme 2. Synthesis of (2-Benzyl)-Pip-heterocyclic Urea Derivatives
that utilizes small-molecule probes containing: (1) a reactive
group that binds and covalently modifies the active sites of a
large number of enzymes that share conserved mechanistic
and/or structural features, and (2) a reporter tag, such as a
fluorophore or biotin, to facilitate detection, enrichment, and
identification of probe-labeled enzymes by SDS-PAGE (gel-
based ABPP19) and/or quantitative mass spectrometry-based
proteomic methods (ABPP-SILAC8,13). The regioisomers of
(2-substituted)-Pip-1,2,3-TUs arise due to acylation at the N1
or N2 position of the triazole ring during triphosgene-mediated
urea formation (Scheme 1). Here, we wanted to determine
whether enantiomers of 2 show differences in DAGLβ potency
and selectivity. We resolved racemic 2 into its enantiomers (2a
and b) using chiral-HPLC and found that 2a showed ∼100-fold
greater potency than 2b against DAGLβ (Figure 1). We also
observed a similar increase in activity against the off-target
ABHD6, demonstrating that 2a provides a similar overall
selectivity profile as the racemate. We therefore concluded that
the racemate is suitable for biological studies, given that the
active enantiomer displayed a similar selectivity profile and only
a 2-fold increase in potency over the racemic mixture.
To evaluate whether replacement of the triazole ring with
other nitrogenous heterocycles would affect activity against
DAGLβ, we synthesized the triazole (5), pyrazole (8), and
imidazole (9) derivatives of the 2-piperidyl-heterocyclic urea
scaffold (Scheme 2). We synthesized compound 5 by direct
B
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Table 1. Comparison of Activity of (2-Benzyl)-Pip-heterocyclic Urea Derivatives
Scheme 3. Synthesis of (2-Benzyl)-Pip-1,2,3-TUs with 4-Substituted Triazoles
coupling of 4-(4-(trifluoromethoxy)phenyl)-triazole to 2-
benzylpiperidine and purified the 1,4-regioisomer by prepara-
tive TLC. The heterocyclic derivatives were synthesized by
triphosgene-mediated coupling of 2-benzylpiperidine with
either 4-bromopyrazole or 4-bromoimidazole followed by
Suzuki coupling with (4-(trifluoromethoxy)phenyl)boronic
acid to generate 8 or 9 as single regioisomers. The activities
of compounds 5, 8, and 9 against recombinant DAGLβ and
other SHs in the mouse brain proteome demonstrated that the
triazole (5) showed potent activity against DAGLβ and also
inhibited other SHs at higher concentrations, whereas the
pyrazole (8) and imidazole (9) derivatives were largely inactive
as determined by gel-based ABPP (Table 1). These results
indicate that the triazole ring is important for reactivity of (2-
benzyl)-Pip-1,2,3-TUs with DAGLβ.
Modifications to the Triazole Ring Enhance Potency
and Selectivity of DAGLβ Inhibitors. We next evaluated
substitutions to the 4-position of the triazole ring of DAGLβ
inhibitors. 4-Phenyl-triazoles were synthesized from commer-
cially available alkynes by copper-catalyzed azide−alkyne
cycloaddition reaction20 (CuACC or click chemistry), purified
using silica gel chromatography, and coupled to 2-benzyl
piperidine to yield compounds 10−13 (Scheme 3). Further
coupling of compounds 12 and 13 with phenylboronic acid
afforded the biphenyl derivatives 14 and 15. Compound 19 was
synthesized using commercially available 4-bromo-1,2,3-triazole
coupled to 2-benzyl piperidine under standard conditions to
generate a TU intermediate that was then treated with 4-
(piperidine-1-carbonyl)-phenylboronic acid in the presence of
Pd(II) and K2CO3 to provide compound 19 (Scheme 3).
Competitive ABPP revealed that compounds 5, 10, and 19
were equipotent against DAGLβ (Figure 2A), but 5 and 19
showed better selectivity compared to 10 (Figure 2B,C). We
next synthesized the biphenyl compound 11 and found that it
maintained potency against DAGLβ and exhibited improved
selectivity compared to 5 and 19 (Figure 2A and Table 1). We
also compared the activity of isomers of compound 11 that
varied in the position of the distal phenyl ring and found that
substitutions at the 3- (14) or 2-position (15) reduced potency
against DAGLβ (Figure 2A) or increased off-target activity
(Figure 2 and Table 1).
Compound 11 still showed potent activity against ABHD6,
as well as low-level cross-reactivity with lipoprotein-associated
phospholipase A2 (PLA2G7) at higher concentrations (Figure
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Figure 2. Potency and selectivity of (2-benzyl)-Pip-1,2,3-TU inhibitors of DAGLβ. (A) Inhibitory activity of compounds (0.1 or 10 μM) against
recombinant DAGLβ expressed by transient transfection in HEK293T cells (DAGLβ-HEK293T lysates) or recombinant mouse ABHD11 (50 nM)
as determined by gel-based competitive ABPP using 38 or FP-Rh, respectively. (B,C) Selectivity of compounds (10 or 1 μM) against mouse brain
SHs as measured by gel-based competitive ABPP using FP-Rh (B) or 38 (C). The gel-based ABPP assays were performed as described in Figure 1.
Assignment of SH enzyme activities in competitive ABPP gels are based on gel migration patterns consistent with past studies.13,21
Scheme 4. Synthesis of Biphenyl Derivatives of (2-Benzyl)-Pip-1,2,3-TUs
2C). To improve the selectivity of compound 11 further, we
synthesized derivatives with various substituents added to the
distal phenyl ring (Scheme 4). All compounds were synthesized
using 2 as a common intermediate for subsequent Suzuki
coupling reactions. We tested derivatives with a trifluoromethyl
substituent at para- (24), meta- (25), and ortho-positions (26)
and found that 26 showed the best potency and selectivity
(Figure 3 and Table 2). Substitutions at the para-position of
compounds (20 and 24) increased PLA2G7 off-target activity
with the exception of larger polar groups (21 and 23).
However, these larger polar substitutions also reduced potency
against DAGLβ (Figure 3 and Table 2), and we therefore
focused on smaller substituents and found that addition of a
hydroxymethyl group at the ortho- (29) but not meta-position
(22) improved potency against DAGLβ (Figure 3). Replace-
ment of the hydroxymethyl with a methoxy substituent (27)
eliminated FAAH as an off-target and attenuated PLA2G7 off-
target activity while largely retaining potency against DAGLβ
(Figure 3 and Table 2). Low-level cross-reactivity with
monoacylglycerol lipase (MGLL) was, however, observed
with compound 27 when tested at 10 μM (Figure 3B).
These results demonstrate that ortho-substitutions of the
biphenyl-triazole ring with small, moderately polar groups
generate DAGLβ inhibitors with excellent activity and
selectivity.
In summary, our SAR studies designated compounds 11 and
27 as potent and selective DAGLβ inhibitors. Both compounds
inhibited DAGLβ in vitro with IC50 values of 50−80 nM as
measured by gel-based ABPP and LC-MS substrate assays.13
Compounds 11 and 27 showed good selectivity with minimal
and complementary cross-reactivity against other SHs with only
a single shared off-target, ABHD6 (Figure 2B and 3B). Further
studies showed that, while compound 27 was equipotent
against DAGLα and DAGLβ, 11 displayed enhanced potency
for DAGLβ over DAGLα (∼60-fold selectivity) as measured by
gel-based ABPP,13 designating these compounds as dual
DAGLα/β and DAGLβ-selective inhibitors, respectively. Both
compounds displayed excellent potency and selectivity in situ,
with near-complete inactivation of DAGLβ (and ABHD6) in
Neuro2A cells at concentrations <50 nM (in situ IC50 values of
11−14 nM) and negligible cross-reactivity with the 45+
additional SHs detected in this cell line by ABPP-SILAC.13
Finally, both compounds inactivated DAGLβ in peritoneal
macrophages from mice (1−5 mg kg−1 of compound, ip).13
A Clickable Analogue of Compound 27 Confirms
Proteome-Wide Selectivity for DAGLβ/ABHD6. Our
previous competitive ABPP results (both gel- and MS-based
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Figure 3. Potency and selectivity of biphenyl derivatives of (2-benzyl)-Pip-1,2,3-TUs. (A) Inhibitory activity of compounds (0.1 or 10 μM) against
recombinant DAGLβ expressed by transient transfection in HEK293T cells (DAGLβ-HEK293T lysates) or recombinant mouse ABHD11 (50 nM)
as determined by gel-based competitive ABPP using 38 or FP-Rh, respectively. (B,C) The selectivity of compounds (10 or 1 μM) against mouse
brain SHs was measured using gel-based competitive ABPP with FP-Rh (B) or 38 (C). The gel-based ABPP assays were performed as described in
Figure 1. SH activities in gels were assigned as described in Figure 2
Table 2. Activity of Biphenyl Derivatives of 2-Benzyl Pip-1,2,3-TUs
analyses)13 showed that the DAGL inhibitors 11 and 27 exhibit
excellent selectivity across the SH class but did not address the
possibility that these inhibitors may react with other proteins in
the proteome. To determine proteome-wide selectivity, we
synthesized analogues of 27 (32 and 33; Figure 4A) that bear
an alkyne group to serve as a latent affinity handle suitable for
modification by reporter tags using copper-catalyzed azide−
alkyne cycloaddition chemistry22 (click chemistry). First, we
confirmed that both 32 and 33 retain good inhibitory activity
against DAGLβ and ABHD6 as measured by gel-based
competitive ABPP in Neuro2A proteomes (Figure 4B). Next,
we treated Neuro2A cells with varying concentrations of 32 or
33 for 1 h. Cells were then lysed and the membrane proteomes
conjugated by click chemistry with an azide-Rh tag,23 separated
by SDS-PAGE, and probe-labeled proteins visualized by in-gel
fluorescence scanning (Figure 4C). This analysis identified two
major protein targets of ∼70 and 35 kDa, matching the
molecular weights of DAGLβ and ABHD6, respectively, that
could be detected at concentrations of 32 or 33 as low as 10
nM (Figure 4C). Good selectivity for DAGLβ and ABHD6 was
maintained up to ∼600 nM of the probes, at which point, a
handful of additional probe-labeled proteins were detected.
Considering that the parent inhibitors 11 and 27 exhibit in situ
activities in the 25−50 nM range,13 these data argue that both
inhibitors maintain good proteome-wide specificity at concen-
trations required to inhibit DAGLβ and ABHD6 in cells.
Acyclic Phenethyl-1,2,3-TUs as a Scaffold for Creating
DAGL-Tailored Activity-Based Probes. To determine
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Figure 4. Structure and activity of clickable analogues of compound 27. (A) Chemical structures of clickable probes 32 and 33. (B) In vitro potency
of clickable probes against DAGLβ and ABHD6 in Neuro2A proteome as measured by gel-based competitive ABPP using 38. Neuro2A lysates (1
mg/mL) were incubated with the indicated concentration of compounds (30 min, 37 °C), followed by labeling with 1 μM 38 (30 min, 37 °C). (C)
Click chemistry ABPP of Neuro2A cells treated in situ with 32 and 33. Neuro2A cells were treated with the indicated concentrations of compound
(1 h, 37 °C), lysed and labeled proteins in the membrane fraction were visualized by click chemistry reaction with azide-Rh followed by SDS-PAGE
and in-gel fluorescence scanning. Protein bands corresponding to DAGLβ and ABHD6 are labeled. SH activities in gels were assigned as described in
Figure 2
Scheme 5. Synthesis of Acyclic Phenethyl 1,2,3-TUs
whether modifications to the carbamoylating portion of (2-
substituted)-Pip-1,2,3-TUs would affect potency and selectivity
toward DAGLβ, we synthesized acyclic phenethyl-derivatives
following the procedure outlined in Scheme 5. Reductive
amination of 2-phenylethanamine with aldehydes resulted in
secondary amines that were subsequently coupled with 4-(4-
(trifluoromethoxy)phenyl)-triazole to yield compounds 35−37
(Scheme 5). The acyclic phenethyl-1,2,3-TUs still retained
potent activity against recombinant DAGLβ (Figure 5A) with
reasonable selectivity against other SHs in the mouse brain
proteome as judged by gel-based ABPP (Figure 5B). Depend-
ing on the compound, off-target activity was observed for a
subset of SHs, including FAAH, KIAA1363, PLA2G7, ABHD6,
and an additional unidentified FP-reactive protein (∼80 kDa)
(Figure 5C and Table 3). These studies demonstrate that
acyclic phenethyl-1,2,3-TUs serve as a suitable scaffold to
incorporate diverse substituents, including a bulky Boc-group
(compound 37), into the DAGLβ inhibitor scaffold.
We found that 37 served as a versatile intermediate for the
design of DAGL-tailored activity-based probes. Initially, we
substituted a BODIPY-dye in place of the Boc-group of 37,
resulting in the development of the activity-based probe, 38
(Figure 6A), that could detect native DAGL activity in gel-
based formats.13 Compound 38 also provided a complementary
probe for other SHs, including PLA2G7, that are difficult to
visualize in gel-based assays with the more broad-spectrum
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Figure 5. Potency and selectivity of acyclic phenethyl-1,2,3-TUs. (A) Inhibitory activity of compounds (0.1 μM) against recombinant DAGLβ
expressed by transient transfection in HEK293T cells (DAGLβ-HEK293T lysates) as determined by gel-based competitive ABPP using 38. (B,C)
Selectivity of compounds against mouse brain SHs as measured by gel-based competitive ABPP using FP-Rh (B) or 38 (C). For the gel-based ABPP
assays, proteomes were incubated with compound (0.1, 1, or 10 μM) for 30 min at 37 °C followed by reaction with fluorescent ABPP probes (38 or
FP-Rh, 1 μM, 30 min, 37 °C). Fluorescent gel images are shown in gray scale. SH activities in gels were assigned as described in Figure 2
Table 3. Activity of Acyclic Phenethyl-1,2,3-TUs
agent FP-Rh.21 Here, we wanted to determine the full spectrum
of SHs targeted by compound 38 in Neuro2A proteomes using
the quantitative mass-spectrometry (MS)-based proteomic
method ABPP-SILAC.7,8,13 In brief, light and heavy Neuro2A
proteomes (membrane and soluble fractions) were treated with
DMSO or 38 (20 μM, 30 min), respectively, followed by
enrichment of SHs using FP-biotin (10 μM, 2 h). As shown in
Figure 6B, we observed substantial blockade of FP-biotin
labeling for ABHD6, DAGLβ, DDHD2, and PAFAH2,
identifying these SHs as principal targets of 38. The MS-
based results helped to assign identities to compound 38-
labeled SH targets observed in our gel-based analysis, indicating
that the ∼80−90 kDa doublet observed in both Neuro2A
(Figure 6C) and mouse brain proteomes with 38 (Figures 2C,
3C, and 5C) is likely the poorly characterized phospholipase,
DDHD2.24,25 Collectively, these results underscore the value of
compound 38 as an activity-based probe for the focused
visualization of low-abundance SHs in mammalian proteomes.
Expansion of DAGL-Tailored Probes to Include Biotin
and Alkyne Derivatives of Compound 38. We next
synthesized biotin and alkyne analogues of compound 38 that
can be used to directly enrich probe-labeled targets. We
incorporated alkyne (39) and biotin (40) functional groups in
the 37 precursor using simple amine coupling reactions. Both
39 and 40 showed moderate potency against recombinant
DAGLα and DAGLβ with IC50 values in the range of 0.4−2
μM range (Figure 7). Next, we compared the specificity of 40
with its more broad-spectrum counterpart, FP-biotin26 using
ABPP-SILAC, where probe-treated (heavy) Neuro2A pro-
teomes were compared to DMSO-treated (light) Neuro2A
proteomes for SH activities enriched by probes (heavy/light
ratios >20). More than 45 SH activities were enriched by FP-
biotin (Table 4), which matches previous experiments that
inventoried the full-range of SH activities in Neuro2A cells.13 In
contrast, compound 40-enriched samples only contained 16
SHs (Table 4), including all of the established targets of 38:
DAGLβ, ABHD6, DDHD2, and PAFAH2 (Figure 6B). Several
additional SH targets were also enriched by compound 40,
including ABHD11, ESD, and LYPLAL1 (Table 4). That these
enzymes were not identified in competitive ABPP studies with
compound 38 (Figure 6B) suggests they are likely lower
potency targets of compounds 38 and 40 (or exhibit
preferential reactivity with 40).
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Figure 6. Development of the DAGL-tailored activity-based probe, compound 38. (A) 37 served as a precursor for coupling of an amine-reactive
BODIPY fluorophore to generate 38. (B) Competitive ABPP-SILAC of SH activities from Neuro2A cells treated in situ with 20 μM of 38 for 4 h.
Compound 38 inhibited DDHD2, DAGLβ, ABHD6, and PAFAH2 by ≥80% but did not inhibit any of the other detected SHs. Error bars represent
mean SEM of heavy/light ratios for the multiple peptides observed for each enzyme (minimum of three unique peptides per enzyme) in both
soluble and membrane fractions. The ABPP-SILAC experiments confirmed (for DAGLβ and ABHD6) and revealed (for DDHD2) the identity of
probe-labeled bands detected by gel-based ABPP in compound 38-treated Neuro2A membrane proteome ((C) experiment performed as described
in Figure 1). Note that PAFAH2 is a soluble enzyme and its signals are likely too low for detection by compound 38 in gel-based ABPP experiments
of Neuro2A membrane proteome.
Figure 7. Inhibition of recombinant DAGLα and β by 39 and 40. Inhibitory activity of the alkyne (A) and biotin (B) probes, 39 and 40, respectively,
was measured by competitive ABPP using DAGL-HEK293T membrane lysates. Proteomes were preincubated with varying concentrations of 39 or
40 for 30 min at 37 °C prior to treatment with 38 (1 μM, 30 min, 37 °C) and analysis by gel-based ABPP. Both probes blocked 38 labeling of
DAGLα and β with IC50 values ranging from 0.4 to 2 μM.
We next compared the subsets of SHs enriched by
compound 40 and FP-biotin and asked whether low- or high-
abundance targets were preferentially identified by each probe,
where SH abundance was estimated by spectral counting
(Figure 8). Certain high-abundance SHs, such as FASN, are
enriched to an equal degree in both sample sets (ratio of log
spectral counts ∼1; Figure 8). However, other high-abundance
SHs, such as PREP, show much lower signals in 40 versus FP-
biotin data sets (log spectral count ratio ∼0.5; Figure 8).
Several additional medium-high abundance SHs (e.g., LYPLA1
and LYPLA2) were also preferentially enriched in FP-biotin-
experiments. Conversely, some of the lower abundance targets,
like DDHD2, showed substantially higher signals in 40-
compared with FP-biotin-enriched samples (log spectral
count ratio ∼2; Figure 8). Taken together, these results show
that 40 functions as a tailored activity-based probe by providing
both enhanced reactivity with low-abundance SHs (DDHD2,
PLA2G6, PAFAH2) and attenuated labeling of higher
abundance targets (PREP, LYPLA1, and LYPLA2).
Development of a DAGLβ-Inactive Control Probe That
Also Provides a Novel Scaffold for ABHD6 Inhibitors.
The ABHD6 cross-reactivity displayed by compounds 11 and
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a
Table 4. Comparison of SHs Enriched by FP-Biotin or 40
Figure 8. Comparative analysis of 40- and FP-biotin-enriched SHs in
ABPP-SILAC experiments of Neuro2A proteomes. The signals (log
spectral counts) for SH activities that were detected with ratios >20 in
probe/DMSO experiments (denoting selective enrichment in probe-
treated proteomes; see Table 4 for complete list) and enriched in both
FP-biotin and compound 40 data sets are plotted. SHs that show equal
enrichment in both probe data sets have log spectral count ratios ∼1
(denoted by dashed line). SHs that show preferential enrichment by
FP-biotin (red) or 40 (blue) deviate from the slope (log spectral count
ratios < or > 1, respectively). The low-abundance SHs DDHD2,
PLA2G6, and PAFAH2 show preferential enrichment by compound
40.
activity against DAGLβ in comparison with the potent 2-
benzyl-derivative 27 (Figure 3A, Table 2). To further explore 2-
phenyl- and 2-phenethyl-scaffolds, we synthesized 50−52 and
found that both the 2-phenyl (50) and 2-phenethyl (51)
derivatives that lack substituents on the biphenyl-triazole group
still retained some activity against DAGLβ (Figure 9B). In
contrast, addition of a para-methoxy group produced
compound 52 (KT195),13 which showed a slight reduction in
potency for ABHD6 (IC50 value of 10 nM13), but negligible
cross-reactivity with DAGLβ (Figure 9B) or other brain SHs
(Figure 9A), and was thus designated as a suitable negative-
control probe that could be utilized at concentrations similar to
those used for compounds 11 and 27 to selectively inhibit
ABHD6.
a
Light and heavy proteomes were treated with DMSO or probe (10
μM of FP-biotin or 40) for 2 h at room temperature and analyzed by
ABPP-SILAC as described in Supporting Information. SHs shown in
the table were detected with ratio values >20 in probe/DMSO
comparisons (minimum of three unique peptides per protein),
denoting selective enrichment in probe-treated proteomes. We also
performed a control experiment where we heat-denatured heavy
proteomes prior to enrichment with FP-biotin to demonstrate that the
detected SHs are enriched in an activity-dependent manner in ABPP-
SILAC experiments (Supporting Information Table 1).
CONCLUSION
■
In summary, we describe the synthesis and SAR of a series of
(2-substituted)-Pip-1,2,3-TUs that culminated in discovery of
potent, selective, and in vivo-active inhibitors of DAGLβ.13 The
successful development of DAGLβ-selective inhibitors high-
lights the utility of ABPP as not only a universal activity assay
for the SH class of enzymes but also adaptable to create tailored
activity-based probes to facilitate detection of low-abundance
enzymes like DAGLβ, PLA2G7, and DDHD2. Key to the
development of these specialized activity-based probes was the
tolerance that DAGLβ displays for acyclic analogues of the
piperidine agents 11 and 27, which permitted the incorporation
of diverse reporter tags into the DAGLβ inhibitor scaffold.
Utilizing this synthetic strategy, we were able to generate a suite
of fluorescent (38), alkyne (39), and biotin (40) probes that
should find broad utility in both gel- and MS-based ABPP
studies.
27 proved difficult to eliminate with medicinal chemistry. We
therefore addressed this issue by synthesizing ABHD6-selective
inhibitors that function as negative-control probes for our
biological studies. Four separate piperidine analogues were
coupled to a 4-bromophenyl-triazole to generate TU
intermediates that were then further coupled with substituted
phenylboronic acids to produce compounds 46−52 (Scheme
6). We tested the activities of these derivatives using
competitive ABPP and found that changing the location of
the benzyl group on the piperidine ring from the 2- (27) to 3-
(47) or 4-position (49) increased off-target activity (Figure
9A). After determining that the 2-position was optimal, we
found that substitution of the 2-benzyl- for a 2-phenyl- (46) or
2-phenethyl-piperidine group (48) dramatically attenuated
A major finding in our SAR studies was that substitution of
mono- for biphenyl triazoles enhanced the selectivity of (2-
benzyl)-Pip-1,2,3-TUs for DAGLβ, leading to the development
of the optimized DAGLβ inhibitors, 11 and 27. We went on to
design clickable analogues (32 and 33) of optimized DAGLβ
inhibitors, which exhibited good proteome-wide selectivity at
I
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Scheme 6. Synthesis of (2-Substituted)-Pip-1,2,3-TUs as Candidate Negative-Control Probes That Inhibit ABHD6
Figure 9. Inhibitory activity and selectivity of candidate (2-substituted)-Pip-1,2,3-TU negative-control probes that target ABHD6. (A) Selectivity of
control probes against mouse brain SHs as measured by gel-based competitive ABPP using FP-Rh. (B) Inhibitory activity of candidate negative-
control probes against DAGLβ-HEK293T lysates as measured by gel-based competitive ABPP using 38. Previous studies showed concentration-
dependent inhibition of ABHD6 in mouse brain proteomes by 52 (IC50 = 10 nM) as measured by competitive ABPP.13 For the gel-based ABPP
assays, proteomes were incubated with compound at the indicated concentrations for 30 min at 37 °C followed by reaction with fluorescent ABPP
probes (38 or FP-Rh, 1 μM, 30 min, 37 °C). SH activities in gels were assigned as described in Figure 2
concentrations matching those used for the parent inhibitors to
inactivate DAGLβ and ABHD6 in Neuro2A cells. We also
discovered from our SAR studies key structural modifications to
the DAGL inhibitor scaffold that attenuate DAGLβ activity
while maintaining potency against ABHD6. These studies
culminated in the discovery of a (2-phenyl)-Pip-1,2,3-TU,
compound 52, that can serve as a negative-control probe for
DAGLβ-related studies, as well as a novel chemotype for
developing next-generation ABHD6-selective inhibitors for
functional studies in vivo.18
Projecting forward, we believe that 1,2,3-TUs will continue
to serve as a useful scaffold for developing selective probes
targeting endocannabinoid biosynthetic pathways. With a
modest number of analogues, we were able to develop potent
and isoform-selective inhibitors capable of inactivating DAGLβ
in vitro and in vivo. Future work will focus on optimizing
DAGL inhibitors for improved brain penetrance and CNS
activity. Another important goal is the development of DAGLα-
selective inhibitors, which should benefit from the availability of
gel-based ABPP assays using DAGL-tailored probes (38).
Achieving these objectives would create a near-complete set of
selective chemical probes for studying 2-AG-mediated
endocannabinoid pathways in vivo.
EXPERIMENTAL SECTION
■
General Synthetic Methods. All chemicals and reagents that
were commercially available were purchased from Sigma-Aldrich,
Matrix Scientific, BioBlocks, Combi-Blocks, Maybridge, Fisher, Acros,
ChemExper, or Capot Chemicals and used without further
purification. Commercial solvents (predried, oxygen-free formulations)
were passed through activated alumina columns to obtain dry solvents.
Unless noted otherwise, all reactions were performed under a nitrogen
atmosphere using oven-baked glassware. Flash chromatography was
performed using mesh silica gel (230−400). Analytical thin-layer
chromatography (TLC) on glass backed silica gel 60 F254 plates was
used to monitor reactions. pTLC on silica gel 60 F254 plates or flash
chromatography on 40−60 MYM mesh silica gel were used to purify
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1
reactions. H NMR and spectra were recorded in CDCl3 on a Varian
sonication. The total protein concentration in each fraction was
determined using a protein assay kit (Bio-Rad). Samples were stored at
−80 °C until further use.
Mercury-300 spectrometer, a Varian Inova-400, Bruker DRX-500, or a
Bruker DRX-600 spectrometer, and referenced to trimethylsilane
(TMS). Chemical shifts were reported in ppm relative to TMS and J
values were reported in Hz. High resolution mass spectrometry
(HRMS) experiments were performed at The Scripps Research
Institute Mass Spectrometry Core on an Agilent mass spectrometer
using electrospray ionization-time-of-flight (ESI-TOF). The purity of
final compounds were determined to be ≥95% by HPLC analysis
(Agilent 1100 LC/MS).
Gel-Based Competitive ABPP. Gel-based competitive ABPP
experiments were performed following previously described proto-
cols.13,28,29 Proteomes (1 mg/mL) were pretreated with compounds at
indicated concentrations (30 min, 37 °C) followed by labeling with
either FP-rhodamine or 38 (1 μM final concentration) in a 50 μL total
reaction volume. After 30 min or 1 h at 37 °C, the FP-rhodamine- or
38-labeled reactions, respectively, were quenched with SDS-PAGE
loading buffer. After separation by SDS-PAGE (10% acrylamide),
samples were visualized by in-gel fluorescence scanning using a flatbed
fluorescent scanner (Hitachi FMBio IIe). To measure recombinant
DAGLβ activity, proteomes were diluted to 0.3 mg/mL in assay buffer
(50 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM CaCl2, 0.1% v/v TX-
100, 10% v/v DMSO) and subjected to ABPP analysis as described
above.
Cycloaddition Reactions with the Clickable Probes 32 and
33. Click chemistry was performed following previously described
protocols.30 For in vitro experiments, membrane proteomes (1 mg/
mL in PBS) were incubated with DMSO or varying concentrations of
compound 32 or 33 (3 nM to 10 μM) at 37 °C for 30 min prior to
incubation with 38 (1 μM) for 30 min. For in situ experiments,
Neuro2A cells were incubated with DMSO, 32 or 33 (10 nM to 25
μM clickable probes) for 1 h at 37 °C, lysed, and membrane fractions
isolated for click-chemistry. The alkyne-labeled membrane proteomes
(1 mg/mL in PBS) were then incubated with rhodamine-azide (50
μM), followed by TCEP (1 mM), ligand (100 μM), and CuSO4 (1
mM). After 1 h at 25 °C, reactions were analyzed by SDS-PAGE and
in-gel fluorescence scanning.
Competitive ABPP-SILAC. Neuro2A cells were grown for 10
passages in either light or heavy SILAC DMEM medium
supplemented with 10% (v/v) dialyzed FCS and 2 mM L-glutamine.
The light medium was supplemented with 100 μg/mL L-arginine and
100 μg/mL L-lysine. Heavy medium was supplemented with 100 μg/
mL [13C615N4]-L-arginine and 100 μg/mL [13C615N2]-L-lysine. The
heavy cells (in 10 mL medium) were treated with test compound, and
light cells were treated with DMSO for 4 h at 37 °C. Cells were
washed with PBS (2×), harvested, and lysed by sonication in DPBS.
Membrane and soluble proteomes were isolated as described above
and adjusted to a final concentration of 2.0 mg/mL and labeled with
10 μM FP-biotin (500 μL total reaction volume) for 2 h at 25 °C.
After incubation, light and heavy proteomes were mixed in 1:1 ratio,
and excess FP-biotin removed by CHCl3/MeOH extraction. To the
light and heavy mixed proteomes (1 mL volume), 2 mL of CH3OH,
0.5 mL of CHCl3, and 1.5 mL of H2O was added and the mixtures
vortexed and centrifuged at 1400g for 3 min. The top (aqueous) and
bottom (organic) layers were removed, and 600 μL of MeOH was
added to the protein interface, which was subsequently transferred to a
microfuge tube. Next, 150 μL of CHCl3 and 600 μL of H2O were
added, vortexed, and centrifuged as described above. The top and
bottom layers were removed and the protein interface sonicated in 600
μL of MeOH and then centrifuged at 14000 rpm for 5 min to pellet
protein. The MeOH was removed and pellet resuspended in 6 M
urea/25 mM ammonium bicarbonate. Samples were then reduced with
10 mM DTT for 15 min (65 °C) and alkylated with 40 mM
iodoacetamide for 30 min at 25 °C in the dark. Afterward, samples
were treated with SDS (2% final, 5 min) and biotinylated proteins
enriched with avidin beads (50 μL beads; conditions, 1 h, 25 °C, 0.5%
SDS in PBS). The beads were washed three times with 1% SDS in PBS
followed by three washes with PBS. The urea concentration was
reduced to 2 M with 2× volume DPBS. On-bead digestions were
performed for 12 h at 37 °C with sequence-grade modified trypsin
(Promega; 2 μg) in the presence of 2 mM CaCl2. Peptide samples
were acidified to a final concentration of 5% (v/v) formic acid and
stored at −80 °C prior to analysis. LC-MS/MS analysis of ABPP-
SILAC samples were performed as previously described.7,8,13
Hex-5-yn-1-yl (5-(N-Phenethyl-4-(4-(trifluoromethoxy)phenyl)-
1H-1,2,3-triazole-1-carboxamido)pentyl)carbamate (39). A solution
of 5-hexyn-1-ol (10 mg, 0.10 mmol) in CH2Cl2 (2 mL) was treated
with triphosgene (30 mg, 0.10 mmol, 1.0 equiv) and pyridine (8 μL,
0.10 mmol, 1.0 equiv), and the mixture was stirred for 30 min at 4 °C.
The mixture was poured into H2O and extracted with ethyl acetate.
The organic layer was washed with H2O and brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was dissolved
in THF (1 mL), and Boc-deprotected urea 3713 (7 mg, 14 μmol) and
iPr2NEt (10 μL) were added to the solution. After stirring for 1 h at
room temperature, the mixture was poured into H2O and extracted
with ethyl acetate. The organic layer was washed with H2O and brine,
dried over Na2SO4, and concentrated under reduced pressure. pTLC
(ethyl acetate:hexane = 1:3) afforded 39 (8 mg, 98%).
1H NMR (CDCl3, 300 MHz) δ 8.42−7.80 (m, 3H), 7.40−7.10 (m,
7H), 4.70 (br, 1H), 4.15−3.50 (m, 6H), 3.25−2.95 (m, 4H), 2.22 (td,
2H, J = 7.0, 2.6 Hz), 1.95 (t, 1H, J = 2.6 Hz), 1.65−1.20 (m, 10H).
HRMS calculated for C30H35F3N5O4 [M + H]+ 586.2636, found
586.2633.
N-(5-(5-(2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanamido)pentyl)-N-phenethyl-4-(4-(trifluoromethoxy)phenyl)-
1H-1,2,3-triazole-1-carboxamide (40). A solution of the urea 37 (13
mg, 23 μmol) in CH2Cl2 (0.6 mL) was treated with 4N HCl−dioxane
(0.6 mL), and the mixture was stirred for 3 h at room temperature.
The solvent was evaporated and the residue was dissolved in DMF (1
mL). iPr2NEt (12 μL, 69 μmol, 3.0 equiv) and biotin-NHS (8 mg, 23
μmol, 1.0 equiv) was added to the solution, and the mixture was
stirred for 2 h at room temperature. The mixture was poured into H2O
and extracted with ethyl acetate. The organic layer was washed with
H2O and brine, dried over Na2SO4, and concentrated under reduced
pressure. Et2O (1 mL) was added and the mixture was sonicated. The
supernatant was removed, and this procedure was repeated twice to
afford 40 (10 mg, 63%).
1H NMR (CDCl3, 300 MHz) δ 8.43−7.80 (m, 3H), 7.35−7.05 (m,
7H), 6.25−6.03 (m, 2H), 5.30 (s, 1H), 4.48 (m, 1H), 4.29 (m, 1H),
4.00−3.50 (m, 4H), 3.30−2.80 (m, 6H), 2.70 (d, 1H, J = 13.0 Hz),
2.18 (t, J = 7.4 Hz), 1.80−1.20 (m, 10H). HRMS calculated for
C33H41F3N7O4S [M + H]+ 688.2887, found 688.2883.
Materials for Biological Experiments. FP-rhodamine,26 FP-
biotin,26,27 and 3813 were synthesized as previously described.
Neuro2A and HEK293T cells were purchased from ATCC.
Cell Culture and Preparation of Cell Line Proteomes.
Neuro2A and HEK293T cells were grown in DMEM supplemented
with 10% fetal bovine serum at 37 °C with 5% CO2. For in vitro
experiments, cells were grown to 80−90% confluency, washed twice
with cold PBS (pH 7.5), and scraped. Cell pellets were then isolated
by centrifugation at 1400g for 3 min at 4 °C. The pellets were
resuspended in 500 μL of cold PBS (pH 7.5), sonicated, and
centrifuged at 100000g for 45 min to generate soluble and membrane
fractions. Mouse DAGLα and DAGLβ were transiently overexpressed
in HEK293T cells as previously described.13 Total protein
concentration of membrane and soluble fractions was determined
using a protein assay kit (Bio-Rad). Samples were stored at −80 °C
until use.
Preparation of Mouse Brain and Liver Proteomes. Brains and
livers from C57Bl/6 mice were Dounce-homogenized in PBS, pH 7.5,
followed by a low-speed spin (1400g, 5 min) to remove debris. The
supernatant was then centrifuged (100000g, 45 min) to generate the
cytosolic fraction in the supernatant and the membrane fraction as a
pellet. The pellet was washed and resuspended in PBS buffer by
K
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(9) Long, J. Z.; Li, W.; Booker, L.; Burston, J. J.; Kinsey, S. G.;
Schlosburg, J. E.; Pavon, F. J.; Serrano, A. M.; Selley, D. E.; Parsons, L.
H.; Lichtman, A. H.; Cravatt, B. F. Selective blockade of 2-
arachidonoylglycerol hydrolysis produces cannabinoid behavioral
effects. Nature Chem. Biol. 2009, 5, 37−44.
(10) Chang, J. W.; Nomura, D. K.; Cravatt, B. F. A potent and
selective inhibitor of KIAA1363/AADACL1 that impairs prostate
cancer pathogenesis. Chem. Biol. 2011, 18, 476−484.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplemental methods, synthetic procedures and character-
ization of 1,2,3-triazole ureas, HRMS data, chiral separation of
compound 2, and supplemental figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
(11) Ahn, K.; Smith, S. E.; Liimatta, M. B.; Beidler, D.; Sadagopan,
N.; Dudley, D. T.; Young, T.; Wren, P.; Zhang, Y.; Swaney, S.; Van
Becelaere, K.; Blankman, J. L.; Nomura, D. K.; Bhattachar, S. N.; Stiff,
C.; Nomanbhoy, T. K.; Weerapana, E.; Johnson, D. S.; Cravatt, B. F.
Mechanistic and pharmacological characterization of PF-04457845: a
highly potent and selective fatty acid amide hydrolase inhibitor that
reduces inflammatory and noninflammatory pain. J. Pharmacol. Exp.
Ther. 2011, 338, 114−124.
(12) Zuhl, A. M.; Mohr, J. T.; Bachovchin, D. A.; Niessen, S.; Hsu, K.
L.; Berlin, J. M.; Dochnahl, M.; Lopez-Alberca, M. P.; Fu, G. C.;
Cravatt, B. F. Competitive activity-based protein profiling identifies
aza-beta-lactams as a versatile chemotype for serine hydrolase
inhibition. J. Am. Chem. Soc. 2012, 134, 5068−5071.
(13) Hsu, K. L.; Tsuboi, K.; Adibekian, A.; Pugh, H.; Masuda, K.;
Cravatt, B. F. DAGLbeta inhibition perturbs a lipid network involved
in macrophage inflammatory responses. Nature Chem. Biol. 2012, 8,
999−1007.
(14) Hsu, K. L.; Tsuboi, K.; Speers, A. E.; Brown, S. J.; Spicer, T.;
Fernandez-Vega, V.; Ferguson, J.; Cravatt, B. F.; Hodder, P.; Rosen, H.
Optimization and characterization of a triazole urea inhibitor for
diacylglycerol lipase beta (DAGL-β). In Probe Reports from the NIH
Molecular Libraries Program [Internet]; National Center for Bio-
technology Information: Bethesda, MD, 2012; (updated Feb 25
2013).
AUTHOR INFORMATION
■
Corresponding Authors
*For K.L.H.: phone, 858-784-8636; E-mail, kenhsu@scripps.
edu.
*For B.F.C.: phone, 858-784-8633; E-mail, cravatt@scripps.
edu.
Author Contributions
§These authors contributed equally to this work
Notes
The authors declare the following competing financial
interest(s): Dr. Cravatt is a cofounder and scientific advisor
to Abide Therapeutics, a biotechnology company interested in
developing SH inhibitors as therapeutics.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
Grants DA017259 (B.F.C.), DA033760 (B.F.C.), and
MH084512 (B.F.C.), a Hewitt Foundation Postdoctoral
Fellowship (K.L.H.), the Skaggs Institute for Chemical Biology,
and Dainippon Sumitomo Pharma (K.T.)
(15) Bisogno, T.; Howell, F.; Williams, G.; Minassi, A.; Cascio, M.
G.; Ligresti, A.; Matias, I.; Schiano-Moriello, A.; Paul, P.; Williams, E.
J.; Gangadharan, U.; Hobbs, C.; Di Marzo, V.; Doherty, P. Cloning of
the first sn1-DAG lipases points to the spatial and temporal regulation
of endocannabinoid signaling in the brain. J. Cell Biol. 2003, 163, 463−
468.
(16) Gao, Y.; Vasilyev, D. V.; Goncalves, M. B.; Howell, F. V.; Hobbs,
C.; Reisenberg, M.; Shen, R.; Zhang, M. Y.; Strassle, B. W.; Lu, P.;
Mark, L.; Piesla, M. J.; Deng, K.; Kouranova, E. V.; Ring, R. H.;
Whiteside, G. T.; Bates, B.; Walsh, F. S.; Williams, G.; Pangalos, M. N.;
Samad, T. A.; Doherty, P. Loss of Retrograde Endocannabinoid
Signaling and Reduced Adult Neurogenesis in Diacylglycerol Lipase
Knock-out Mice. J. Neurosci. 2010, 30, 2017−2024.
(17) Tanimura, A.; Yamazaki, M.; Hashimotodani, Y.; Uchigashima,
M.; Kawata, S.; Abe, M.; Kita, Y.; Hashimoto, K.; Shimizu, T.;
Watanabe, M.; Sakimura, K.; Kano, M. The endocannabinoid 2-
arachidonoylglycerol produced by diacylglycerol lipase alpha mediates
retrograde suppression of synaptic transmission. Neuron 2010, 65,
320−327.
(18) Hsu, K. L.; Tsuboi, K.; Chang, J. W.; Whitby, L. R.; Speers, A.
E.; Pugh, H.; Cravatt, B. F. Discovery and optimization of piperidyl-
1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of
alpha/beta-hydrolase domain containing 6 (ABHD6). J. Med. Chem.
2013 DOI: 10.1021/jm400899c.
(19) Jessani, N.; Liu, Y.; Humphrey, M.; Cravatt, B. F. Enzyme
activity profiles of the secreted and membrane proteome that depict
cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 10335−
10340.
(20) Kalisiak, J.; Sharpless, K. B.; Fokin, V. V. Efficient Synthesis of 2-
Substituted-1,2,3-triazoles. Org. Lett. 2008, 10, 3171−3174.
(21) Nagano, J. M.; Hsu, K. L.; Whitby, L. R.; Niphakis, M. J.; Speers,
A. E.; Brown, S. J.; Spicer, T.; Fernandez-Vega, V.; Ferguson, J.;
Hodder, P.; Srinivasan, P.; Gonzalez, T. D.; Rosen, H.; Bahnson, B. J.;
Cravatt, B. F. Selective inhibitors and tailored activity probes for
lipoprotein-associated phospholipase A(2). Bioorg. Med. Chem. Lett.
2013, 23, 839−843.
ABBREVIATIONS USED
■
SH, serine hydrolase; Pip-1,2,3-TU, piperidyl-1,2,3-triazole
urea; ABPP, activity-based protein profiling; CuAAC, copper-
catalyzed azide alkyne cycloaddition; SILAC, stable isotope
labeling by amino acids in cell culture; FP-Rh, fluorophosph-
onate-rhodamine
REFERENCES
■
(1) Simon, G. M.; Cravatt, B. F. Activity-based proteomics of enzyme
superfamilies: serine hydrolases as a case study. J. Biol. Chem. 2010,
285, 11051−11055.
(2) Long, J. Z.; Cravatt, B. F. The metabolic serine hydrolases and
their functions in mammalian physiology and disease. Chem. Rev.
2011, 111, 6022−6063.
(3) Henness, S.; Perry, C. M. Orlistat: a review of its use in the
management of obesity. Drugs 2006, 66, 1625−1656.
(4) Thornberry, N. A.; Weber, A. E. Discovery of JANUVIA
(Sitagliptin), a selective dipeptidyl peptidase IV inhibitor for the
treatment of type 2 diabetes. Curr. Top. Med. Chem. 2007, 7, 557−568.
(5) Racchi, M.; Mazzucchelli, M.; Porrello, E.; Lanni, C.; Govoni, S.
Acetylcholinesterase inhibitors: novel activities of old molecules.
Pharmacol. Res. 2004, 50, 441−451.
(6) Bachovchin, D. A.; Cravatt, B. F. The pharmacological landscape
and therapeutic potential of serine hydrolases. Nature Rev. Drug
Discovery 2012, 11, 52−68.
(7) Bachovchin, D. A.; Mohr, J. T.; Speers, A. E.; Wang, C.; Berlin, J.
M.; Spicer, T. P.; Fernandez-Vega, V.; Chase, P.; Hodder, P. S.;
Schurer, S. C.; Nomura, D. K.; Rosen, H.; Fu, G. C.; Cravatt, B. F.
Academic cross-fertilization by public screening yields a remarkable
class of protein phosphatase methylesterase-1 inhibitors. Proc. Natl.
Acad. Sci. U. S. A. 2011, 108, 6811−6816.
(8) Adibekian, A.; Martin, B. R.; Wang, C.; Hsu, K. L.; Bachovchin,
D. A.; Niessen, S.; Hoover, H.; Cravatt, B. F. Click-generated triazole
ureas as ultrapotent in vivo-active serine hydrolase inhibitors. Nature
Chem. Biol. 2011, 7, 469−478.
L
dx.doi.org/10.1021/jm400898x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(22) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed
Regioselective “Ligation” of Azides and Terminal Alkynes. Angew.
Chem., Int. Ed. 2002, 41, 2596−2599.
(23) Speers, A. E.; Cravatt, B. F. Profiling enzyme activities in vivo
using click chemistry methods. Chem. Biol. 2004, 11, 535−546.
(24) Schuurs-Hoeijmakers, J. H.; Geraghty, M. T.; Kamsteeg, E. J.;
Ben-Salem, S.; de Bot, S. T.; Nijhof, B.; van de Vondervoort, I. I. G.
M.; van der Graaf, M.; Nobau, A. C.; Otte-Holler, I.; Vermeer, S.;
Smith, A. C.; Humphreys, P.; Schwartzentruber, J.; Ali, B. R.; Al-
Yahyaee, S. A.; Tariq, S.; Pramathan, T.; Bayoumi, R.; Kremer, H. P.
H.; van de Warrenburg, B. P.; van den Akker, W. M.; Gilissen, C.;
Veltman, J. A.; Janssen, I. M.; Vulto-van Silfhout, A. T.; van der Velde-
Visser, S.; Lefeber, D. J.; Diekstra, A.; Erasmus, C. E.; Willemsen, M.
A.; Vissers, L. E.; Lammens, M.; van Bokhoven, H.; Brunner, H. G.;
Wevers, R. A.; Schenck, A.; Al-Gazali, L.; de Vries, B. B. A.; de
Brouwer, A. P. M. Mutations in DDHD2, encoding an intracellular
phospholipase A(1), cause a recessive form of complex hereditary
spastic paraplegia. Am. J. Hum. Genet. 2012, 91, 1073−1081.
(25) Nakajima, K.; Sonoda, H.; Mizoguchi, T.; Aoki, J.; Arai, H.;
Nagahama, M.; Tagaya, M.; Tani, K. A novel phospholipase A1 with
sequence homology to mammalian Sec23p-interacting protein, p125. J.
Biol. Chem. 2002, 277, 11329−11335.
(26) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein
profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 1999, 96,
14694−14699.
(27) Kidd, D.; Liu, Y.; Cravatt, B. F. Profiling serine hydrolase
activities in complex proteomes. Biochemistry 2001, 40, 4005−4015.
(28) Bachovchin, D. A.; Ji, T.; Li, W.; Simon, G. M.; Blankman, J. L.;
Adibekian, A.; Hoover, H.; Niessen, S.; Cravatt, B. F. Superfamily-wide
portrait of serine hydrolase inhibition achieved by library-versus-library
screening. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20941−20946.
(29) Jessani, N.; Niessen, S.; Wei, B. Q.; Nicolau, M.; Humphrey, M.;
Ji, Y.; Han, W.; Noh, D. Y.; Yates, J. R., III; Jeffrey, S. S.; Cravatt, B. F.
A streamlined platform for high-content functional proteomics of
primary human specimens. Nature Methods 2005, 2, 691−697.
(30) Speers, A. E.; Adam, G. C.; Cravatt, B. F. Activity-Based Protein
Profiling in Vivo Using a Copper(I)-Catalyzed Azide-Alkyne [3 + 2]
Cycloaddition. J. Am. Chem. Soc. 2003, 125, 4686−4687.
M
dx.doi.org/10.1021/jm400898x | J. Med. Chem. XXXX, XXX, XXX−XXX