Competitive activity-based protein profiling identifies Aza-β-lactams as a versatile chemotype for serine hydrolase inhibition

Article abstract of DOI:10.1021/ja300799t

Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC₅₀-30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors.

Full text of DOI:10.1021/ja300799t

Communication
pubs.acs.org/JACS
Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams
as a Versatile Chemotype for Serine Hydrolase Inhibition
Andrea M. Zuhl,†,§ Justin T. Mohr,‡,§ Daniel A. Bachovchin,† Sherry Niessen,† Ku-Lung Hsu,†
Jacob M. Berlin,‡ Maximilian Dochnahl,‡ María P. Lopez-Alberca,‡ Gregory C. Fu,*,‡
́
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
‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
the serine hydrolase class still lacks selective inhibitors that are
suitable for pharmacological studies in living systems.1,2
ABSTRACT: Serine hydrolases are one of the largest and
most diverse enzyme classes in Nature. Most serine
hydrolases lack selective inhibitors, which are valuable
probes for assigning functions to these enzymes. We
recently discovered a set of aza-β-lactams (ABLs) that act
as potent and selective inhibitors of the mammalian serine
hydrolase protein-phosphatase methylesterase-1 (PME-1).
The ABLs inactivate PME-1 by covalent acylation of the
enzyme’s serine nucleophile, suggesting that they could
offer a general scaffold for serine hydrolase inhibitor
discovery. Here, we have tested this hypothesis by
screening ABLs more broadly against cell and tissue
proteomes by competitive activity-based protein profiling
(ABPP), leading to the discovery of lead inhibitors for
several serine hydrolases, including the uncharacterized
enzyme α,β-hydrolase domain-containing 10 (ABHD10).
ABPP-guided medicinal chemistry yielded a compound
ABL303 that potently (IC50 ≈ 30 nM) and selectively
inactivated ABHD10 in vitro and in living cells. A
comparison of optimized inhibitors for PME-1 and
ABHD10 indicates that modest structural changes that
alter steric bulk can tailor the ABL to selectively react with
distinct, distantly related serine hydrolases. Our findings,
taken together, designate the ABL as a versatile reactive
group for creating first-in-class serine hydrolase inhibitors.
To facilitate the discovery of serine hydrolase inhibitors, we
have introduced a high-throughput screening-compatible
variant of the activity-based protein profiling (ABPP)
technology11,12 that measures the reactivity of active site-
directed fluorophosphonate (FP) probes with serine hydrolases
by fluorescence polarization (fluopol).13 We recently applied
fluopol-ABPP to screen the NIH 300,000+ compound library
against the serine hydrolase protein phosphatase methylester-
ase-1 (PME-1), resulting in the discovery of a highly potent
(low nM) and selective class of aza-β-lactam (ABL) inhibitors
of PME-1.14 The lead inhibitor ABL127 (1) was found to
covalently inhibit PME-1 in cells and mice through acylation of
the enzyme’s active-site serine nucleophile.14
Even though ABL127 showed high selectivity for PME-1, we
wondered whether changes to its core structure could generate
inhibitors for other serine hydrolases. We initially tested this
premise by screening a set of ABLs and oxa-β-lactams (OBLs)
(Figure 1A) against the mouse brain membrane proteome by
competitive ABPP. The ABL and OBL compounds were
synthesized by [2+2] cycloaddition chemistry using chiral 4-
pyrrolidinopyridine catalysts.15,16 Mouse brain proteome was
treated with compounds 1−11, followed by a FP-rhodamine
(FP-Rh) probe,17 and the reactions were then quenched and
separated by SDS-PAGE. FP-Rh-labeled proteins were detected
by in-gel fluorescence scanning (Figure 1B). At 10 μM,
compounds 1−11 inhibited several serine hydrolases that,
based on past ABPP studies of the mouse brain,6−8,10 could be
identified as neutral lipases (AADACL1, MAGL, ABHD6),
amidases (FAAH), thioesterases (FASN), and peptidases
(APEH, PREP). Interestingly, several ABLs also inhibited an
as yet unidentified 27 kDa serine hydrolase that did not exhibit
sensitivity to previously screened carbamate5 or triazole urea10
libraries. ABL117 (7) and ABL143 (8) proved to be particularly
potent and selective inhibitors of this 27 kDa hydrolase,
producing near-complete blockade of its FP-Rh labeling when
tested at 1 μM (Figure 1B). Conversely, ABL113 (both
enantiomers 3 and 4) and OBL716 (11) selectively inhibited
APEH. Several ABLs/OBLs inhibited additional serine hydro-
erine hydrolases constitute ∼1% of all proteins encoded by
Smammalian genomes and play important roles in a wide
range of (patho)physiologic processes.1,2 Many serine hydro-
lases, however, remain unannotated with respect to their
natural substrates and functions.1,2 Selective chemical inhibitors
have served as valuable probes for the functional annotation of
serine hydrolases and led to approved drugs for treating
disorders such as obesity, diabetes, microbial infections, and
Alzheimer’s disease.2 Efforts to develop inhibitors for serine
hydrolases have uncovered specialized chemotypes, such as
lactones/lactams,3,4 carbamates,5−8 and ureas,9,10 that inactivate
these enzymes by covalent modification of the conserved serine
nucleophile. In many cases, structural features can be
introduced into the inhibitors to tailor their selectivity for
individual serine hydrolases. Despite these advances, much of
Received: January 24, 2012
Published: March 8, 2012
© 2012 American Chemical Society
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nor have substrates or functions been discovered for the
enzyme. These factors, combined with the very limited
sequence homology (<25%) that ABHD10 shares with other
mammalian serine hydrolases, designated the enzyme as an
attractive target for chemical probe development.
We first confirmed that ABHD10 was inhibited by ABL117
in a COS7 cell proteome transiently overexpressing this
enzyme (Figure 1D). We next evaluated the structure−activity
relationship (SAR) for ABL inhibition of ABHD10 versus other
serine hydrolases by competitive ABPP of proteomes from the
mouse neuronal cell line Neuro2A (Table 1 and Figure S2).
Table 1. SAR for ABL Inhibitors versus ABHD10 and Other
Representative Serine Hydrolases
Since we had already established that the ethyl substituent (R1)
on the stereogenic carbon was an important factor in
promoting ABHD10 inhibition, we sought to explore other
features of the core scaffold, including the carbamate
substitution (R3), the aryl ring substituent (R2), and the
configuration of the stereocenter. Increasing the steric bulk of
the O-alkyl groups (R3) on the carbamates from methyl
(ABL117, 7) to isopropyl (ABL243, 14) increased potency for
ABHD10 approximately 4-fold, but also produced an equivalent
increase in the inhibition of PREP. The PREP cross-reactivity
was reduced by either substituting the m-methyl group on
ABL243 with a fluorine atom (ABL223, 17) or moving this
methyl group to the para position (ABL303, 19). ABL303
stood out as the most potent ABHD10 inhibitor among the
tested ABLs (IC50 = 30 nM) with at least 40-fold selectivity
over ABHD6, PREP, PME-1, and other serine hydrolases
(Table 1 and Figure S2). Finally, as was observed previously for
the PME-1 inhibitor ABL127, the R-enantiomer of ABL303
(19) was found to be substantially more active against
ABHD10 than the S-enantiomer (ent-ABL303, 20).
We next tested whether ABL303 inhibited ABHD10 with
good selectivity in living cells. Gel-based ABPP confirmed
excellent in situ activity for ABL303 in Neuro2A cells, where
the compound inhibited ABHD10 with IC50 = 21 nM (Figure
2B,D). Near-complete inhibition of ABHD10 was maintained
for at least 6 h following a single treatment with ABL303 (250
nM, Figure S3). No detectable off-targets were observed for
ABL303 until much higher concentrations (≥1 μM), and no
significant inhibition of PME-1 was observed even at 10 μM
(Figure 2C, Figure S3). As previously noted,14,19 however, gel-
based ABPP lacks the resolving power to evaluate the full
spectrum of serine hydrolase activities in complex biological
systems. For this purpose, we assessed the cellular activity of
ABL303 using the quantitative mass spectrometry-based
proteomic method ABPP-SILAC.10,14 Neuro-2A cells were
cultured with isotopically light or heavy amino acids and then
treated with DMSO or 100 nM ABL303 for 2 h, respectively.
Figure 1. Structures and competitive ABPP of ABL/OBL compounds.
(A) Structures of ABL/OBL compounds. (B,C) Competitive ABPP
gels of mouse brain membrane (B) and soluble (C, see also Figure S1)
proteomes treated with ABLs/OBLs (30 min) followed by FP-Rh (1
μM, 30 min). Serine hydrolases inhibited by individual ABLs and
OBLs are labeled. (D) Competitive ABPP gel of mock- versus mouse
ABHD10 (mABHD10)-transfected COS-7 cell proteomes treated with
ABL117, followed by FP-Rh (1 μM, 30 min). Fluorescent images
shown in gray scale.
lases found in the soluble brain proteome, including PME-1
(Figure 1C, Figure S1). Taken together, these data indicate that
ABLs/OBLs are capable of inhibiting a diverse set of
mammalian serine hydrolases, including enzymes that here-
tofore have lacked selective chemical probes.
Our attention next turned to determining the identity of the
27 kDa brain serine hydrolase that was inhibited by ABL117
and ABL143 using a competitive ABPP-MudPIT platform.6,7
Briefly, we treated mouse brain proteome with ABL117 (2 μM)
or DMSO control followed by the activity-based probe FP-
biotin and then enriched FP-biotin-labeled enzymes by avidin
chromatography, subjected these enzymes to an on-bead
trypsin digestion, and analyzed the resulting tryptic peptide
mixture by multidimensional liquid chromatography−tandem
mass spectrometry (LC/LC-MS/MS). Comparison of spectral
counts for serine hydrolases in DMSO- versus ABL117-treated
proteomes identified two enzymes that were significantly
inhibited by ABL117: PME-1 and the uncharacterized enzyme
α,β-hydrolase domain-containing 10 (ABHD10) (Table S1).
ABHD10 is a 297 amino acid (aa) protein with a predicted
MW = 33 kDa. Proteomic studies have identified ABHD10 as a
mitochondrial protein18 with a predicted leader sequence and
proteolytic cleavage site at aa’s 46−47. The mature ABHD10
protein would thus have a mass that closely matches the size of
the ABL117-sensitive enzyme in brain proteomes. To our
knowledge, inhibitors of ABHD10 have not yet been described,
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methods, may reveal the natural substrates and biochemical
pathways regulated by ABHD10 in cells. More generally, our
findings, combined with previous work showing minimal cross-
reactivity of a clickable analogue of ABL127 against proteins
outside of the serine hydrolase class,14 designate the ABL as a
privileged scaffold for serine hydrolase inhibitor development
complements other known inhibitor classes, such as carba-
mates,5−8 ureas,9,10 and activated ketones.20,21 That the
inhibitory potential of ABLs was initially uncovered in a screen
of the public NIH compound library performed against an
individual serine hydrolase (PME-1) and then leveraged to
create inhibitors for other enzymes from this class underscores
the potential of integrated organic synthesis, high-throughput
screening, and chemoproteomics to identify versatile chemo-
types for small-molecule probe development.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic schemes, compound characterization data, exper-
imental protocols, additional ABPP profiles, and complete ref 9.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
cravatt@scripps.edu; gcf@mit.edu
■
Figure 2. Competitive ABPP of ABL303 in situ. (A) Structures of ABL
probes. (B,C) Gel-based ABPP of membrane (B) and soluble (C, see
also Figure S3) proteomes from Neuro-2A cells treated with ABL303
(10 − 0.001 μM, 2 h), afforded (D) IC50 = 21 nM for inhibition of
ABHD10 and no significant inhibition of PME-1. Data are presented
as mean values SEM; n = 3/group. (E) ABPP-SILAC analysis of
Neuro-2A cells treated with ABL303 (red) or ABL127 (black) (100
nM, 2 h; heavy samples) versus DMSO (light samples) revealed
selective inhibition of ABHD10 and PME-1, respectively. Data are
reported as mean values SEM of all peptides quantified for each
serine hydrolase.
Author Contributions
§These authors contributed equally.
Notes
The authors declare the following competing financial
interest(s): Drs. Cravatt and Fu are advisors for a company
interested in developing serine hydrolase inhibitors as new
therapeutics.
ACKNOWLEDGMENTS
■
Proteomes were harvested, labeled with FP-biotin, and
combined in a 1:1 ratio for avidin enrichment and LC/LC-
MS/MS analysis on an LTQ-Velos Orbitrap instrument. Serine
hydrolase activities were quantified by measuring the heavy:-
light ratios of the MS1 ions for their tryptic peptides. ABL303-
treated cells showed a near-complete (>95%) and selective loss
in ABHD10 activity among the ∼40 serine hydrolase activities
detected (Figure 2E). For comparison, we assessed the activity
of ABL127, which caused a complete and selective disruption of
PME-1 activity in Neuro2A cells (Figure 2E). Finally, we also
evaluated ABL303 at a higher concentration (250 nM) and in a
second cell line, BW5147-derived mouse T-cell hybridoma
cells. In both of these studies, we again observed complete and
selective inhibition of ABHD10 (Figure S4).
We thank B. Kipper, K. Masuda, D. Milliken, J. Murphy, H.
Pugh, and K. Tsuboi for technical assistance. This work was
supported by National Institutes of Health grants CA132630
and GM090294 (B.F.C.), GM57034 (G.C.F.), and GM086040
(postdoctoral fellowship to J.T.M.); the National Science
Foundation (predoctoral fellowships to A.M.Z.. and D.A.B.);
the California Breast Cancer Research Program (predoctoral
fellowship to D.A.B.); Hewitt Foundation for Medical Research
(postdoctoral fellowship to K.L.H.); the German Academic
Exchange Service (postdoctoral fellowship to M.D.); Spanish
Ministry of Education (FECYT, postdoctoral fellowship to
M.P.L.A.); Abide Therapeutics; and The Skaggs Institute for
Chemical Biology.
In summary, we have described herein the functional
proteomic analysis of a series of ABL/OBL compounds and
found that they show broad reactivity with diverse branches of
the serine hydrolase class, including peptidases, lipases,
thioesterases, and uncharacterized enzymes. Using ABPP-
guided medicinal chemistry, we developed an optimized
inhibitor ABL303 that shows excellent potency and selectivity
for the uncharacterized enzyme ABHD10. A comparison of the
structures of ABL303 and the previously reported PME-1
inhibitor ABL12714 indicates that only minor alterations to
groups appended to the ABL are required to create compounds
with excellent selectivity for individual serine hydrolases. Future
studies with ABL303, in combination with metabolomic
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