Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes

Article abstract of DOI:10.1021/ja303400u

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

Full text of DOI:10.1021/ja303400u

Communication
pubs.acs.org/JACS
Confirming Target Engagement for Reversible Inhibitors in Vivo by
Kinetically Tuned Activity-Based Probes
Alexander Adibekian,†,⊥,∇ Brent R. Martin,†,#,∇ Jae Won Chang,† Ku-Lung Hsu,† Katsunori Tsuboi,†
Daniel A. Bachovchin,† Anna E. Speers,† Steven J. Brown,‡ Timothy Spicer,‡ Virneliz Fernandez-Vega,‡
Jill Ferguson,‡ Peter S. Hodder,‡,§ Hugh Rosen,†,∥ and Benjamin F. Cravatt*,†
†Skaggs Institute for Chemical Biology and Department of Chemical Physiology and ∥Scripps Research Institute Molecular Screening
Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Molecular Screening Center and §Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter,
Florida 33458, United States
S
* Supporting Information
function in native biological systems,3 can determine target
engagement for enzyme inhibitors in cell and animal models.4
ABSTRACT: The development of small-molecule inhib-
itors for perturbing enzyme function requires assays to
confirm that the inhibitors interact with their enzymatic
targets in vivo. Determining target engagement in vivo can
be particularly challenging for poorly characterized
enzymes that lack known biomarkers (e.g., endogenous
substrates and products) to report on their inhibition.
Here, we describe a competitive activity-based protein
profiling (ABPP) method for measuring the binding of
reversible inhibitors to enzymes in animal models. Key to
the success of this approach is the use of activity-based
probes that show tempered rates of reactivity with
enzymes, such that competition for target engagement
with reversible inhibitors can be measured in vivo. We
apply the competitive ABPP strategy to evaluate a newly
described class of piperazine amide reversible inhibitors for
the serine hydrolases LYPLA1 and LYPLA2, two enzymes
for which selective, in vivo active inhibitors are lacking.
Competitive ABPP identified individual piperazine amides
that selectively inhibit LYPLA1 or LYPLA2 in mice. In
summary, competitive ABPP adapted to operate with
moderately reactive probes can assess the target engage-
ment of reversible inhibitors in animal models to facilitate
the discovery of small-molecule probes for characterizing
enzyme function in vivo.
To date, this approach has been limited to assessing irreversible
inhibitors for multiple reasons. First, many activity-based
probes show poor bioavailability in vivo, which restricts their
utility to the ex vivo analysis of tissue proteomes from inhibitor-
treated animals, an assay protocol that involves considerable
sample processing and dilution that can compromise the
detection of reversible inhibitor−enzyme interactions. Second,
even for activity-based probes that show suitable bioavailability,
managing their rate of reactivity in vivo is difficult, which, if not
properly controlled, can result in the probes “outcompeting”
reversible inhibitors and failing to record their target
interactions. Here, we sought to address these challenges by
creating kinetically tuned activity-based probes that enable
direct competitive profiling of reversible inhibitors in vivo. We
applied this strategy to assess newly described inhibitors for the
serine hydrolases lysophospholipase 1 (LYPLA1) and lyso-
phospholipase 2 (LYPLA2), enzymes that, to date, have lacked
selective and in vivo active inhibitors.
LYPLA1 and LYPLA2 were initially characterized as
phospholipases based on in vitro substrate assays.5 LYPLA1
has since been found also to act as a protein palmitoyl
thioesterase (PPT) that removes palmitate modifications from
cysteine residues on proteins, such as the Ras GTPases.6
LYPLA2 shares 65% sequence identity with LYPLA1, but its
role as a PPT remains unknown. While considerable
biochemical and cellular data support a role for LYPLA1 as a
PPT,7 little is known about the functions of LYPLA1 and
LYPLA2 in vivo. Several inhibitors of LYPLA1 have been
described,7a,8 but none of these agents have been shown to
inhibit LYPLA1 selectively over LYPLA2 or to inhibit LYPLA1
in animal models. This latter goal is further challenged by a lack
of robust substrate/product biomarkers that can be used to
confirm LYPLA1/2 inhibition in vivo.
nzyme inhibitors are valuable chemical probes to perturb
E
biochemical pathways in living systems and in some cases
can be developed into new medicines to treat human disease.
Most enzymes in the human proteome, however, still lack
selective inhibitors.1 Advances in high-throughput screening
(HTS)2 combined with chemoproteomic platforms1 have
begun to accelerate the development of enzyme inhibitors
with excellent potency and selectivity, but determining whether
these inhibitors interact with their targets in living systems is
often challenging, especially for poorly characterized enzymes
lacking endogenous substrate/product biomarkers.
To identify new classes of LYPLA1 and LYPLA2 inhibitors,
we first established enzyme assays compatible with HTS. As
serine hydrolases, LYPLA1 and LYPLA2 react with fluoro-
phosphonate (FP) activity-based probes and thus can be
We and others have previously shown that activity-based
protein profiling (ABPP), a chemoproteomic method that uses
Received: April 9, 2012
Published: June 12, 2012
active site-directed, covalent probes to evaluate enzyme
© 2012 American Chemical Society
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Communication
assayed by a recently described, HTS-compatible competitive
ABPP platform that measures the reactivity of fluorophore-
conjugated FP probes with serine hydrolases by fluorescence
polarization (fluopol).9 In fluopol-ABPP, small molecules
compete with FP probes for binding to serine hydrolases and
are recorded as hits by their ability to reduce fluopol signal. We
expressed and purified human LYPLA1 and LYPLA2 and
confirmed that the wild-type, but not the catalytically dead
serine mutants (S119A and S112A, respectively), reacted with a
rhodamine-conjugated FP probe (FP-Rh) to generate time-
dependent increases in fluopol signal (Figure 1). In
collaboration with the Molecular Libraries Production Centers
Network (MLCPN), we then screened 315 004 compounds for
LYPLA1 or LYPLA2 inhibition. Compounds producing a >30%
reduction of fluopol signal were classified as potential hits
[Figure S1 in the Supporting Information (SI)]. Following a
confirmation screen, 331 and 790 compounds were found to
inhibit LYPLA1 and LYPLA2, respectively. We then manually
filtered these compounds for those with the highest inhibition
and removed nonselective compounds identified as hits in
previous fluopol-ABPP screens of other serine hydrolases
(PME1 and RBBP9).
The remaining 91 LYPLA1, 61 LYPLA2, and 95 dual
LYPLA1/LYPLA2 inhibitors were analyzed by gel-based ABPP
in HEK293T cell proteomes, which express high endogenous
levels of LYPLA1 and LYPLA2. We established assay
parameters compatible with assessing both reversible and
irreversible inhibitors by using a pegylated FP-Rh probe (FP-
peg-Rh), which reacts more slowly with LYPLA1 and LYPLA2
compared to the FP-Rh probe to produce only partial enzyme
labeling under the reaction conditions employed for inhibitor
evaluation (Figure S2). From this analysis, a diverse set of
piperazine amides emerged as attractive inhibitors for LYPLA1
and LYPLA2. The N-(p-methoxyphenyl)piperazine amide 1,
for instance, completely inhibited LYPLA2 when tested at 1
μM, while showing no inhibition of LYPLA1 or other serine
hydrolases detected in the HEK293T proteome (Figure 2A−
C). A complementary profile was observed for the 2-furoyl
piperazine amide 21, which produced near-complete inhibition
of LYPLA1 without showing any cross-reactivity with LYPLA2
or other serine hydrolases (Figure 2A−C).
Figure 1. Screening for LYPLA1 and LYPLA2 inhibitors. Time course
of FP-Rh labeling for LYPLA1 and LYPLA2 as determined by fluopol-
ABPP. No fluopol increase was observed in the absence of enzymes or
with the S119A LYPLA1 and S122A LYPLA2 catalytic mutants.
To better understand the structure−activity relationship
(SAR) of 1 and 21, we purchased and tested the related
Figure 2. Identification of piperazine amides 1 and 21 as potent and selective reversible inhibitors of LYPLA2 and LYPLA1, respectively. (A) Gel-
based competitive ABPP of HTS hits 1 and 21 and structurally related compounds 2−13 and 22−33 tested in a HEK293T proteome. (B) Structures
of profiled compounds. (C) Concentration-dependent blockade of FP-peg-Rh labeling of LYPLA2 and LYPLA1 by 1 and 21, respectively, in a HEK
293T proteome. Fluorescent gels shown in grayscale are representative of at least three independent experiments. (D) Competitive ABPP analysis of
gel filtration experiments to determine the reversibility of LYPLA2 and LYPLA1 inhibition by 1 and 21; also shown is the profile for the irreversible
inhibitor 1,2,3-triazole urea AA26−9. (E) Kinetic values for inhibition of recombinant human LYPLA2 and LYPLA1 by 1 and 21, respectively,
determined using a fluorescent substrate assay. Data represent means SEM (n = 4).
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Communication
compounds 2−13 and 22−33 (Figure 2A,B). Compounds 7, 9,
and 10, all bearing para-substituted phenyl groups, inhibited
LYPLA2 by 70−90%, while ortho-, meta-, or unsubstituted aryl
piperazines did not exhibit inhibitory activity at 1 μM. Both the
2-furoyl piperazine amide and 2,5-substituted anilide groups of
21 were required for maximal and selective inhibition of
LYPLA1, as revealed by the profiles of disubstituted anilides
28−30, which showed moderate cross-inhibition of LYPLA2
(50−70% at the concentration of 1 μM) and the structurally
related amide 26, which did not inhibit LYPLA1 or LYPLA2.
We resynthesized 1 and 21 as described in the SI and
confirmed their structural assignment and inhibitory activity by
1H NMR and competitive ABPP assays, respectively (Figure
S3).
Neither 1 nor 21 possesses any obvious electrophilic groups
for reaction with LYPLA1 or LYPLA2, which led us to
conclude that they were likely acting as reversible inhibitors.
We confirmed the reversibility of enzyme inhibition for 1 and
21 by gel filtration experiments, which showed complete
recovery of FP-peg-Rh labeling of LYPLA1 and LYPLA2 after
two consecutive gel filtrations (Figure 2D). In contrast, no
recovery of FP-peg-Rh labeling was observed for the 1,2,3-
triazole urea irreversible inhibitor AA26−9,4d even after three
gel filtration events (Figure 2D).10 1 and 21 showed good
potency for LYPLA2 and LYPLA1 with IC50 values of 144 and
210 nM, respectively, derived from gel-based competitive ABPP
assays (Figure 2C and Figure S5) and Ki values of 230 and 300
nM, respectively, derived from a fluorogenic substrate assay
using purified enzymes (Figure 2E and Figure S6). We further
verified the activity and selectivity of 1 and 21 using the LC-MS
platform ABPP-SILAC,4a,d which revealed >95% inhibition of
LYPLA2 and LYPLA1, respectively, with no changes in the
activity of ∼25 other serine hydrolases detected in the mouse
BW5147 T-cell hybridoma proteome (Figure S7 and Table S1).
We next asked whether 1 and 21 could also inhibit LYPLA2
and LYPLA1 in living cells. As mentioned previously, it is not
straightforward to answer this question for reversible inhibitors
using standard competitive ABPP protocols, which involve
incubation of living cells with an inhibitor followed by ex vivo
treatment of cell lysates with an activity-based probe. While, in
principle, we could perform competitive ABPP in situ using
alkynylated FP probes,11 we were concerned that the high rates
of reactivity displayed by FP probes with most serine hydrolases
would complicate the analysis of reversible inhibitors.
Consistent with this premise, we found that LYPLA1,
LYPLA2, and several other serine hydrolases were rapidly
inactivated (within 5 min) by an FP-alkyne probe 34 (Figure
3A and Figure S8). We hypothesized that implementing an
alternative probe with more tempered reactivity could facilitate
competitive ABPP in situ under kinetically controlled
conditions. We tested probe 35, which is based on a recently
discovered triazole urea scaffold for serine hydrolase inhib-
itors,4d and found that it reacted much more slowly than FP-
alkyne with LYPLA1, LYPLA2, and most serine hydrolases
(Figure 3A and Figure S8). We next incubated HEK293T and
mouse T cells with 1 and 21 (5 μM) or DMSO for 3 h followed
by a 1 h treatment with probe 35 (50 μM). Cells were
harvested, lysed, and probe-labeled enzymes visualized by click
chemistry12 conjugation with a Rh-azide reporter tag.13 Gel-
based ABPP revealed the selective and near-complete (>95%)
inhibition of LYPLA2 and LYPLA1 in cells treated with 1 and
21, respectively (Figure 3B and Figure S9). The in situ activity
of both inhibitors was further confirmed by ABPP-SILAC,
Figure 3. Competitive ABPP of 1 and 21 in living cells. (A) Time-
dependent competition of FP-Rh labeling by the clickable probes FP-
alkyne 34 and triazole urea 35 in a mouse brain membrane proteome.
(B) Gel-based competitive ABPP of HEK293T cells treated with 1 and
21 (5 μM, 3 h) followed by probe 35 (50 μM, 1 h) to measure in situ
target engagement. (C) ABPP-SILAC analysis of serine hydrolase
activities from inhibitor-treated HEK 293T cells. Bars represent means
SD of light/heavy ratios for the multiple peptides observed for each
enzyme; data are derived from two independent biological replicates.
which revealed selective inhibition of LYPLA2 and LYPLA1 by
1 and 21, respectively, across the 15+ serine hydrolases
detected in this analysis (Figure 3C and Table S1 and Figure
S10).
We finally asked whether competitive ABPP with probe 35
could also be used to determine the inhibition of LYPLA1 and
LYPLA2 in vivo. Mice were administered 1 and 21 (each at 50
mg/kg in PEG300, i.p.) or vehicle and, after 3 h, treated with
probe 35 (100 mg/kg in PEG300, i.p.) for 1 h. Mice were then
sacrificed, and their tissues removed, processed, reacted with
Rh-azide, and analyzed by gel-based ABPP. The gel profiles
confirmed that 1 and 21 selectively and near-completely
(>90%) inhibited LYPLA2 and LYPLA1, respectively, in lung,
heart, and kidney (Figure 4). In contrast, only partial blockade
(∼50%) was observed for LYPLA1 and LYPLA2 in brain,
suggesting limited CNS penetration for the inhibitors. No
detectable inhibition of LYPLA1 or LYPLA2 was observed in
liver, perhaps reflecting rapid metabolism of 1 and 21 in this
organ. These data indicate that competitive ABPP can
quantitatively assess target engagement in vivo across numerous
organs in parallel to reveal cases of tissue-restricted inhibition.
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Present Addresses
⊥Department of Biochemistry, University of Geneva, Sciences
II, 30 Quai Ernest-Ansermet, 1211 Geneva 4, Switzerland
#Department of Chemistry, University of Michigan, 930 North
University Avenue, Ann Arbor, Michigan 48109, United States
Author Contributions
∇A.A. and B.R.M. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank P. Baillargeon and L. Deluca for MLSMR compound
management and D. Davda, D. Milliken, and K. Masuda for
technical assistance. This work was supported by NIH grants
CA087660 (B.F.C.), MH084512 (H.R.), and CA151460
(B.R.M.), the German Academic Exchange Service (postdoc-
toral fellowship to A.A.) California Institute for Regenerative
Medicine (J.W.C.), Hewitt Foundation for Medical Research
(K.L.H.), and the National Science Foundation (D.A.B.).
Figure 4. Evaluating target engagement for 1 and 21 in vivo. (A)
General layout of a competitive ABPP experiment to measure target
engagement for reversible inhibitors in mice. (B) Competitive ABPP
gels of tissue proteomes from mice treated with 1 and 21 (50 mg/kg, 3
h) followed by probe 35 (100 mg/kg, 1 h).
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■
In summary, we have described an advanced strategy for
competitive ABPP that uses kinetically tuned probes to
determine target engagement for reversible inhibitors in living
cells and mice. We applied this approach to identify and
evaluate piperazine amides 1 and 21 as, to our knowledge, the
first selective and in vivo active inhibitors for serine hydrolases
LYPLA2 and LYPLA1, respectively. Future application of 1 and
21 in conjunction with global methods to assess protein
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help to define the endogenous functions of LYPLA1/2. The
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ASSOCIATED CONTENT
* Supporting Information
Additional ABPP profiles, substrate data, compound character-
ization, and experimental protocols. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
cravatt@scripps.edu
■
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