Superfamily-wide portrait of inhibition achieved by library-versus-library screening

Daniel A. Bachovchina,1, Tianyang Jia,1, Weiwei Lia,1, Gabriel M. Simona, Jacqueline L. Blankmana, Alexander Adibekiana, Heather Hooverb, Sherry Niessenb, and Benjamin F. Cravatta,2

aThe Skaggs Institute for Chemical Biology, bCenter for Physiological Proteomics, and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Edited by Kevan M. Shokat, University of California, San Francisco, CA, and approved October 4, 2010 (received for review August 5, 2010)

Serine (SHs) are one of the largest and most diverse en- general screening assay in hand, achieving ligand selectivity for zyme classes in mammals. They play fundamental roles in virtually one member of a large family presents a major challenge. all physiological processes and are targeted by drugs to treat dis- Methods are particularly needed to assess selectivity in native bio- eases such as diabetes, obesity, and neurodegenerative disorders. logical systems, where are regulated by posttranslational Despite this, we lack biological understanding for most of the 110þ mechanisms that may alter their activity and ligand affinity (6, 7). predicted mammalian metabolic SHs, in large part because of a Activity-based protein profiling (ABPP) (8) is a chemical dearth of assays to assess their biochemical activities and a lack proteomic technology that addresses some of the aforementioned of selective inhibitors to probe their function in living systems. challenges. ABPP employs -directed chemical probes We show here that the vast majority (>80%) of mammalian meta- that covalently label large numbers of mechanistically related en- bolic SHs can be labeled in proteomes by a single, active site-direc- zymes in native biological systems. ABPP probes have so far ted fluorophosphonate probe. We exploit this universal activity- been developed for more than a dozen families, including based assay in a library-versus-library format to screen 70þ SHs hydrolases (9–14), kinases (15), histone deacetylases (16), and against 140þ structurally diverse carbamates. Lead inhibitors were (17, 18). When performed in a competitive discovered for ∼40% of the screened , including many format, where compounds are assayed for their ability to block poorly characterized SHs. Global profiles identified carbamate in- probe labeling (10, 12, 19), ABPP offers a powerful means to hibitors that discriminate among highly sequence-related SHs discover small-molecule inhibitors of enzymes that is indepen- and, conversely, enzymes that share inhibitor sensitivity profiles dent of their degree of functional annotation. A key advantage despite lacking . These findings indicate that of competitive ABPP is that it permits simultaneous optimization sequence relatedness is not a strong predictor of shared pharma- of the potency and selectivity of inhibitors against numerous cology within the SH superfamily. Finally, we show that lead car- related enzymes directly in their native cellular environment. This bamate inhibitors can be optimized into pharmacological probes strategy has led to the identification of selective inhibitors for that inactivate individual SHs with high specificity in vivo. many enzymes (12, 19–25), including several uncharacterized proteins (22–24). enzymology ∣ mass spectrometry ∣ profiling ∣ proteomics Despite these advantages, competitive ABPP has not yet been demonstrated to be feasible for screening the large majority of major challenge facing biological researchers in the 21st cen- enzymes from an expansive family against a small-molecule Atury is the functional characterization of the large number of library. Here, we address this problem by evaluating the perfor- unannotated products identified by genome sequencing mance of ABPP against the mammalian serine hydrolase (SH) efforts (1). Many proteins partly or completely uncharacterized superfamily. We show that >80% of the 110þ predicted metabolic with respect to their biochemical activities belong to expansive, SHs in mammals are targeted by a single fluorophosphonate (FP) sequence-related families (2). Although such membership can in- activity-based probe. We employ this FP probe to perform a form on the general mechanistic class to which a protein belongs library-versus-library competitive ABPP screen, wherein 72 SHs (e.g., enzyme, receptor, or channel), it is insufficient to predict are assayed against a collection of ∼140 carbamate small mole- specific biochemical and physiological functions, which require cules. From this screen, lead inhibitors were discovered for more knowledge of substrates, ligands, and interacting biomolecules. than 30 SHs, including several uncharacterized enzymes. Impor- On the contrary, membership within a large can tantly, we show that competitive ABPP can be used to identify even present a barrier to achieving these goals by frustrating inhibitors that discriminate among highly sequence-related the implementation of standard genetic and pharmacological hydrolases and direct the rapid optimization of these inhibitors methods to probe protein function. For example, targeted gene into pharmacological probes that selectively inactivate individual disruption of one member of a may result in SHs in living animals. cellular compensation from other family members. Problems are also encountered when attempting to develop Results specific inhibitors and/or ligands for uncharacterized members Global Cell and Tissue Profiling with a FPActivity-Based Probe. Human of large protein families, where at least two major experimental SHs can be divided into two near-equal-sized subfamilies—the issues must be addressed. First, there is an intrinsic difficulty

facing ligand discovery for uncharacterized proteins, which often BIOCHEMISTRY Author contributions: D.A.B., W.L., G.M.S., and B.F.C. designed research; D.A.B., T.J., W.L., lack the functional information required to develop high-quality J.L.B., and H.H. performed research; D.A.B., W.L., and A.A. contributed new reagents/ assays for compound screening. Creative solutions to this pro- analytic tools; D.A.B., W.L., S.N., and B.F.C. analyzed data; and D.A.B. and B.F.C. wrote blem have emerged for specific protein classes, such as G-protein the paper. coupled receptors (GPCRs) (3) and kinases (4, 5), where generic The authors declare no conflict of interest. assays have been developed that exploit conserved functional This article is a PNAS Direct Submission. and/or structural features displayed by members of each protein 1D.A.B., T.J., and W.L. contributed equally to this work. family (e.g., G-protein coupling for GPCRs; ATP-binding sites 2

To whom correspondence should be addressed. E-mail: [email protected]. CHEMISTRY “ ” for kinases). Whether such universal assay parameters exist This article contains supporting information online at www.pnas.org/lookup/suppl/ for other protein superfamilies is unclear. Second, even with a doi:10.1073/pnas.1011663107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1011663107 PNAS ∣ December 7, 2010 ∣ vol. 107 ∣ no. 49 ∣ 20941–20946 Downloaded by guest on September 25, 2021 / class of serine proteases (∼125 human mem- cell and tissue proteomes that showed diverse labeling patterns bers) and the metabolic SHs (∼115 human members) (26). with a fluorescent FP (FP-rhodamine) as judged by 1D-SDS- The latter set of enzymes includes a wide range of structurally di- PAGE (Fig. 1A and SI Appendix, Fig. S2). We identified the SH verse peptidases, , , , and . targets of FP probes in these tissues by using the MS platform Although FP probes (SI Appendix,Fig.S1)havebeenshownto ABPP-MudPIT (28). Briefly, proteomes were incubated with a label both serine proteases and metabolic SHs (10, 27, 28), we biotinylated FP probe [FP-biotin (9)], and FP-labeled enzymes elected to focus our analysis on metabolic SHs, because serine pro- were enriched with avidin beads, digested with trypsin, and ana- teases are often produced as inactive precursors (zymogens) and lyzed by multidimensional liquid chromatography (LC)-MS/MS. therefore are difficult to assay in heterologous expression systems. Probe-labeled SHs were defined as those that showed (i) an aver- As has been recently reviewed (26), metabolic SHs play key age of ≥5 spectral counts in FP-treated proteomes and (ii) >5- roles in diverse (patho)physiological processes, and several of fold more spectral counts in probe-treated versus “no probe” con- these enzymes are targeted by clinically approved drugs, includ- trol samples. In total, 101 FP-labeled SHs were identified in ing (AChE) for Alzheimer’s disease (29), mouse cells and tissues (SI Appendix, Table S1), and this number pancreatic lipases for obesity (30), and dipeptidylpeptidase IV for was further increased to 105 by evaluating FP labeling for recom- diabetes (31). Despite the importance of metabolic SHs in binantly expressed versions of SHs that show low signals in tissues mammalian physiology, nearly half of these enzymes remain with- 1 5 out any assigned biochemical activity or (26). Selective ( < average spectral counts < ). Several SHs showed tissue- inhibitors are also lacking for the vast majority (>80%) of mam- restricted patterns of probe labeling consistent with their known malian metabolic SHs, further complicating their functional expression profiles and functions [e.g., pancreatic lipases (PNLIP, characterization. Previous studies have showcased the value of FP PNLIPRP1, and PNLIPRP2) were found exclusively in the pan- probes for ABPP of SHs in proteomes (27, 28, 32) and for the creas; hepatic (LIPC) was most strongly labeled in the discovery of inhibitors for members of this enzyme family (19, liver] (Fig. 1B). The 105 FP-labeled SHs number accounts for 22, 23, 25). Whether competitive ABPP can serve as a universal 82% of the 128 predicted metabolic SHs in mice (Fig. 1C). We assay for SH inhibitor discovery depends, however, on the frac- conclude from these data that a FP activity-based probe can serve tion of mammalian SHs that can be profiled by FP probes. as a near-universal assay to characterize mammalian SHs in pro- We set out to determine the full complement of mammalian teomes. We next sought to adapt this assay for screening a library metabolic SHs targeted by FP probes by using a panel of mouse of small-molecule inhibitors against the SH superfamily.

Fig. 1. Determining the full complement of mammalian metabolic SHs targeted by FP activity-based probes. (A) A panel of mouse tissue proteomes (1 mg of protein per mL) was labeled with FP-rhodamine (2 μM, 45 min) and proteomes analyzed by 1D-SDS-PAGE and in-gel fluorescence scanning. Representative fluorescent gel of FP-rhodamine-labeling events shown in gray scale. (B) Hierarchical cluster analysis of SH activity signals identified in mouse tissues by ABPP-MudPIT. Data are presented as the average spectral counts from three independent experiments normalized for each SH to the tissue containing the most spectral counts for that enzyme. (C) A dendrogram showing all 128 members of the mouse metabolic SH family with branch length depicting se- quence relatedness. This analysis includes two additional human SHs, FAAH2 and PNPLA4, that lack mouse orthologues. SHs that were labeled by FP activity- based probes are shown in red (105 enzymes or 82% of the metabolic SH family). cm, conditioned media; LPS, cells treated with 10 μg∕mL lipopolysaccharide for 24 h; RAW, RAW264.7 mouse macrophage cell line.

20942 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1011663107 Bachovchin et al. Downloaded by guest on September 25, 2021 A Library-Versus-Library Format for Competitive ABPP. A typical for- 50 μM against the 72-member SH panel. A compound was scored mat for competitive ABPP involves incubation of a proteome as active against a given SH if it blocked >75% of FP-rhodamine with a small molecule, followed by labeling of the sample with labeling. A representative profile for the SH FAAH2 is shown in a fluorescent activity-based probe, separating proteins by SDS- Fig. 2C. Primary competitive ABPP data for the other 71 SHs are PAGE, and quantifying the fluorescence intensity of protein shown in SI Appendix, Fig. S4, and a summary of the full library- band(s) on the gel relative to a control (DMSO-treated) proteome versus-library dataset can be accessed at the Web site: http:// (19, 23). Although this type of competitive ABPP experiment can www.scripps.edu/cgi-bin/cravatt/BachovchinJiLi2010. Carbamate be performed in native cell and tissue proteomes, the large differ- hits were identified for 33 SHs (SI Appendix, Table S3), corre- ences in endogenous expression levels of SHs, combined with sponding to ∼46% of the screened enzymes. Certain SHs, such their tendency to cluster in certain mass ranges (25–35 and as the (CESs), showed broad reactivity with 55–65 kDa), means that only a fraction of SHs can be resolved the carbamate library (SI Appendix, Fig. S4). This finding is con- by 1D-SDS-PAGE in native proteomes. Complementary MS- sistent with previous studies designating CESs as common targets based platforms, such as ABPP-MudPIT, have been introduced for a wide range of SH-directed inhibitors (19, 34, 35), which to address these problems (23, 25); however, such LC-MS meth- likely reflects the role that these enzymes play in xenobiotic me- ods are too time consuming to permit screening of a library of tabolism (see SI Appendix for details). We also identified carba- compounds. We therefore adopted a different strategy wherein mate inhibitors for a substantial fraction (36%) of non-CES SHs each mammalian SH is recombinantly expressed (e.g., by transient (Fig. 3A and SI Appendix, Table S3). Notably, several of these transfection in eukaryotic cells) and then mass-resolvable subsets carbamates were found to selectively inactivate a single (non- of these enzymes are combined into groups of up to eight enzymes CES) SH (Fig. 3A and Table 1). We used competitive ABPP to create a multiplexed SH library for inhibitor screening by 1D- to calculate IC50 values for a representative set of these inhibi- SDS-PAGE. Importantly, this library-versus-inhibitor library for- tors, which ranged from 0.008 to 5.3 μM (Table 1, and SI mat for competitive ABPP enabled, with only a handful of excep- Appendix, Fig. S5). Other carbamates showed slightly broader re- tions, screening of enzymes directly in crude cell proteomes (i.e., activity with the SH family, inhibiting a handful of enzymes (three without requiring any enzyme purification). to six) by >75% at 50 μM (Fig. 3A). These results indicate that We selected a total of 72 SHs for analysis, 66 of which were reasonably potent (nanomolar to low micromolar) and selective assayed as recombinant proteins and six of which were more con- lead inhibitors can be obtained for many SHs from a modest-sized veniently obtained from endogenous sources (e.g., secreted SHs collection of carbamate small molecules. that could be accessed from the conditioned media of cell lines) Several sequence-related clades of enzymes exist within the SH (SI Appendix, Table S2). These enzymes were selected to cover family (Fig. 1C), and we wondered whether these enzymes would most branches of the metabolic SH family (SI Appendix, Fig. S3). show equivalent inhibitor sensitivity profiles or, alternatively, if Only six enzymes required an additional, single purification step carbamates could discriminate among homologous enzymes. in order to visualize a band by FP-rhodamine labeling. Labeling Our data strongly support the latter conclusion, because several with FP-rhodamine confirmed the activity of recombinant SHs carbamates were identified that selectively inactivate one mem- and maintenance of their gel-resolved signals in multiplexed ber of a pair of nearest-sequence neighbor enzymes, including groups (Fig. 2A). We estimated that this assay format would per- FAAH-1/FAAH-2, AADACL1/AADAC, and PLA2G7/PAFAH2 mit the screening of more than 100 compounds against the entire (Fig. 3 B–D). Interestingly, we also found cases where the most 72-enzyme panel within the time frame of 4–6 wk. Given this level potent “off target” activity was not a homologous, but rather a very of throughput, we elected to screen a targeted library of candi- distantly related SH. For instance, WWL38 inhibited AADACL1 date inhibitors on the basis of the carbamate group, which cova- and ACHE with IC50 values of 4.8 and 13.3 μM, respectively, while lently reacts with the conserved serine nucleophile of SHs to form showing no activity (IC50 > 100 μM) against AADAC (the near- hydrolytically stable enzyme adducts (Fig. 2B). Carbamates have est-sequence neighbor to AADACL1) (SI Appendix, Fig. S5). been developed that show excellent selectivity for individual SHs Such data indicate that sequence homology is not a particularly and have proven valuable as research tools (22, 23, 25, 33) and strong predictor of shared inhibitor sensitivity profiles among therapeutic drugs (29). SHs, thus underscoring the importance of proteomic methods like We synthesized a structurally diverse set of ∼140 carbamates ABPP that can uncover unanticipated pharmacological cross- (see SI Appendix for details) and screened these compounds at points within enzyme superfamilies.

Fig. 2. A library-versus-library format for competi- tive ABPP. (A) SHs were expressed individually and assayed for activity in crude cell lysates by treatment with FP-Rh and analysis by gel-based ABPP. Gel-resol- vable SHs were combined and screened for inhibition by the carbamate library; a representative example is shown for the carbamate URB597, which is a known inhibitor of FAAH (33). (B) General structure of a carbamate library and mechanism of SH inactivation by carbamates. (C) Representative example of the primary competitive ABPP screening data for the en- zyme FAAH2 expressed by transient transfection in 293T cells. A mock-transfected proteome and FP- BIOCHEMISTRY rhodamine signals from an endogenously expressed SH are shown for comparison. Unless otherwise indi- cated, each number on the horizontal axis refers to a carbamate (lacking the WWL prefix to conserve space). From this analysis, several hits were identified, including WWL44, which selectively inhibited FAAH2 relative to other SHs with an IC50 value of 1.7 μM (Table 1). See SI Appendix, Fig. S4 for a complete CHEMISTRY set primary competitive ABPP data from our library- versus-library screen.

Bachovchin et al. PNAS ∣ December 7, 2010 ∣ vol. 107 ∣ no. 49 ∣ 20943 Downloaded by guest on September 25, 2021 Fig. 3. Identification and characterization of lead inhibitors for SHs (A) Hierarchical cluster analysis of carbamate inhibition profiles for a representative subset of SHs. From this analysis, compounds that inactivate several SHs (e.g., WWL98 and WWL202) can be readily discriminated from those that show high selectivity for individual SHs (listed in Table 1 and B–D). (B–D) Concentration-dependent inhibition profiles for carbamates that show high selectivity for one member of a pair of sequence-related enzymes. (B) FAAH-1 versus FAAH-2, (C) AADAC versus AADACL1, and (D) PLA2G7 versus PAFAH2. See SI Appendix, Fig. S5 for more expanded concentration-dependent inhibition curves used to generate the reported IC50 values.

Optimization of Carbamate Hits to Create in Vivo Pharmacological ample, to the six-membered piperidine ring analogue WWL215 Probes. Although some highly potent inhibitors were identified (Table 2), which inhibited several additional SHs, including directly from the carbamate library (e.g., WWL65, which was a FAAH-1, FAAH-2, and PLA2G7 (Fig. 4A and SI Appendix, sub-10-nM inhibitor of FAAH), most of the carbamate hits exhib- Fig. S4)]. Because only a limited number of azepane analogues ited inhibitory activities in the range of high-nanomolar to low- are commercially available, we postulated that exchanging this micromolar. We therefore asked whether these moderate- ring system with a monosubstituted piperidine ring might offer potency leads could be efficiently optimized into selective and a more facile medicinal-chemistry strategy for improving potency, efficacious inhibitors for in vivo pharmacological studies. To ad- while, at the same time, preserving selectivity for ABHD11. From dress this question, we selected WWL151 as a case study, which this effort, we identified the 2-methyl substituted piperidyl carba- selectively inhibited the uncharacterized SH ABHD11 with an mate WWL219 (Table 2), which showed markedly improved μ IC50 value of 5.3 M (Table 1). The most unusual feature of activity against ABHD11 (IC50 ¼ 160 nM) but still inhibited WWL151 compared to other members of the carbamate library the SH FAAH (Fig. 4 A and B). Extension of this 2-substitution was the seven-membered azepane ring, which appeared to grant to an ethyl group to give WWL222 (Table 2) maintained potency this compound high selectivity for ABHD11 [compared, for ex- against ABHD11 (IC50 ¼ 170 nM) (Fig. 4C) while eliminating activity against other SHs (Fig. 4 A and B). Table 1. Selective lead inhibitors (and their SH targets) identified by An attractive feature of carbamates is that these agents typically library-versus-library competitive ABPP (see SI Appendix, Fig. S5 for show excellent pharmacological activity in vivo, including the concentration-dependent inhibition curves used to generate ability to penetrate the nervous system (25, 33, 36), where their reported IC50 values) SH targets may play important roles in regulating neurochemical signaling pathways (37, 38). Consistent with this precedent, μ Hydrolase Compound Structure IC50, M we found that administration of WWL222 to mice (10 mg∕kg, AADACL1 WWL57 0.36 i.p., 4 h) completely inactivated brain ABHD11 as judged by competitive ABPP-MudPIT (Fig. 4D). Remarkably, none of the other ∼50 brain SHs detected in this proteomic analysis were ACHE WWL52 3.5 inhibited by WWL222. These data demonstrate that WWL222 acts as a selective and efficacious inhibitor of ABHD11 in vivo. To confirm that other carbamate hits also showed in vivo activity, ABHD6 WWL123 0.43 we treated mice with the ABHD6 inhibitor WWL123 (Table 1; 5–20 mg∕kg, i.p., 4 h). Competitive ABPP profiles of brain tissue ABHD11 WWL151 5.3 from WWL123-treated animals revealed selective inactivation of ABHD6 (SI Appendix, Fig. S6).

CEL WWL92 4.1 Discussion Complete genome sequences promise to radically change the field of molecular pharmacology. Systematic efforts to subclone FAAH WWL65 0.008 and express the full complement of protein-coding sequences from mouse and human genomes are underway and should pro- FAAH2 WWL44 1.7 vide straightforward access to “expression-ready” constructs for these proteins (39, 40). The principal remaining challenge is then PLA2G7 WWL153 0.29 to develop general functional assays for mammalian proteins, a problem that is compounded by the fact that many of these pro- teins are uncharacterized with respect to biochemical and cellular PNPLA8 WWL210 2.9 activity. Here, we have shown that competitive ABPP offers a near-universal assay platform for the SH superfamily, which re- presents one of the largest and most diverse enzyme classes in

20944 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1011663107 Bachovchin et al. Downloaded by guest on September 25, 2021 Table 2. Structures of a carbamate sublibrary targeting ABHD11, rapid way to screen the SH superfamily against hundreds of in- including the optimized inhibitor WWL222 hibitors. Although this platform is not compatible with screening of thousands of compounds, we should emphasize that the output is comparable to such higher-throughput screens in terms of aggregate data points (>11;000 for our library-versus-library analysis) and information content. Because competitive ABPP Compound n R1 R2 tests each compound against numerous (70þ) SHs in parallel,

WWL151 3 H 4-NO2-Ph a family-wide portrait of pharmacological activity is generated WWL209 3 H Ph for each inhibitor that immediately informs on its potency and WWL210 3 H 2-Cl-Ph selectivity. We used this combined information to identify useful WWL211 3 H 2-OMe, 4-NO2-Ph lead carbamate inhibitors for several SHs, including enzymes, such as ABHD11, CEL, and PNPLA8, for which no other selec- WWL214 3 H tive inhibitors have yet been described. In the case of ABHD11, we furthermore showed how structure-activity relationship data 4 from the initial competitive ABPP screen could guide the rapid WWL215 2 H -NO2-Ph optimization of a low-micromolar hit into a selective and effica- WWL216 1 H 4-NO2-Ph cious inhibitor, WWL222, that is suitable for in vivo pharmaco- WWL219 2 2-Me 4-NO2-Ph WWL220 2 4-Me 4-NO2-Ph logical studies. Like many other SH inhibitors, our carbamate hits WWL222 2 2-Et 4-NO2-Ph showed some cross-reactivity with members of the CES subfam- WWL223 2 2;6-ðMeÞ2 4-NO2-Ph ily. This cross-reactivity should not, however, hinder the use of carbamates to characterize SHs in a wide range of biological 4 WWL225 2 -NO2-Ph systems, because CESs are mostly restricted in their expression to the liver (see Fig. 1B and discussion in the SI Appendix). WWL226 2 3-Me 4-NO2-Ph WWL227 2 2-CH2OH 4-NO2-Ph ABHD11 is a poorly characterized SH that exhibits a broad tissue distribution, including high expression in the brain and 4 WWL228 2 -NO2-Ph heart (Fig. 1B). Proteomic studies have identified ABHD11 as a

WWL229 2 4-NO2-Ph mitochondrial protein (41), although its actual biochemical func- tion, including its endogenous substrates and products, remains WWL230 2 4-NO2-Ph unknown. The ABHD11 gene is located in a region of chromo- some 7 (7q11.23) that is hemizygously deleted in Williams–Beu- 4 WWL231 2 -NO2-Ph ren syndrome, a rare genetic disease with symptoms that include

WWL232 2 4-NO2-Ph vascular stenosis, mental retardation, and excessive sociability (42). Whether ABHD11 plays a role in Williams–Beuren syn- drome remains unclear. The inhibitor WWL222 should assist future investigations of ABHD11’s relevance to symptoms ob- mammals. We determined that more than 80% of the predicted served in Williams–Beuren syndrome, as well as to elucidate metabolic SHs in mice can be profiled in cell and tissue pro- the enzyme’s endogenous biochemical and cellular functions. teomes with a single FP activity-based probe. SHs that were Projecting forward, it is worthwhile to consider the grand not detected by ABPP in this study could represent enzymes that question—how long might it take to generate selective and in show highly restricted tissue distributions [e.g., searches of the vivo-active inhibitors for every member of the SH family by using BioGPS database (http://biogps.gnf.org/) suggest that AADACL2 a near-universal, proteomic assay like competitive ABPP? and AADACL4 are exclusively expressed in mouse skin and re- Although our discovery of lead inhibitors for ∼46% of the tina, respectively, two tissues that were not profiled in this study] screened SHs (∼36% of the non-CES enzymes) is encouraging, or an inability to recognize and react with the FP probe. We also we also note that several of these leads are not yet selective en- cannot exclude the possibility that some of the predicted SH ough for use as pharmacological probes. It is possible that such are in fact pseudogenes that do not produce a functional multitarget carbamates can serve as medicinal-chemistry starting protein product. These factors, taken together, suggest that more points for generating selective inhibitors of individual SHs [as has extensive tissue profiling, perhaps with structural analogues been accomplished for multitarget kinase inhibitors (7) and as we of the prototype FP probe (10), should further enhance ABPP have previously shown for WWL98, which led to the development coverage of the metabolic SH superfamily. of the selective (MGLL) inhibitor The library-versus-library format for competitive ABPP pre- JZL184 (25)]. We also anticipate that some multitarget carba- sented herein offers a technically straightforward and reasonably mates may show greater selectivity for individual SHs when tested

Fig. 4. Development of a selective and in vivo-active inhi- bitor of ABHD11. (A) Competitive ABPP signals for WWL151 and structural analogues (5 μM) against ABHD11 and the common off-targets for this compound scaffold— FAAH, MGLL, ABHD6, and PNPLA8. (B) Cluster analysis of the competitive ABPP data shown in A, designating BIOCHEMISTRY WWL222 as a potent and selective ABHD11 inhibitor. (C) Concentration-dependent inhibition curve for WWL222 against ABHD11. From this curve, an IC50 value of 170 nM was calculated. Data are presented as means standard error of the mean (SEM); n ¼ 3∕group. (D) ABPP-MudPIT analysis of SHs from the brain proteomes of mice treated with vehicle or WWL222 (10 mg kg−1, i.p., 4 h); Among the ∼50 SHs detected in this analysis, only ABHD11 was inhibited by WWL222 (*p < 0.02). Data are CHEMISTRY presented as means SEM; n ¼ 3∕group.

Bachovchin et al. PNAS ∣ December 7, 2010 ∣ vol. 107 ∣ no. 49 ∣ 20945 Downloaded by guest on September 25, 2021 at lower concentrations. As an initial assessment of this postulate, tigations of small molecule–protein interactions. The resulting we measured IC50 values of 0.05, 1.57, and 2.75 μM for WWL110 pharmacopeia should greatly enhance our understanding of pro- versus BCHE, ABHD2, and CEL, respectively (SI Appendix, tein function in mammalian physiology and disease. Fig. S5), indicating that this agent is a relatively selective inhibitor of BCHE but also potent enough to act as a lead for inhibitors of Materials and Methods ABHD2 and CEL. Beyond this line of future research, achieving Global Mouse Cell and Tissue Profiling with FP Probes. See the SI Appendix for complete pharmacological coverage of the SH family will likely details. require screening either a much-expanded carbamate library or additional structural classes of compounds. Carbamates are quite Expression of SH Library. See the SI Appendix for details. straightforward from a synthetic perspective, and it is certainly possible to consider producing a larger (1;000þ-member) library Synthesis of Carbamate Library. See the SI Appendix for details. of these agents. One could also create additional small-molecule ∼3–6 libraries on the basis of other chemotypes that mechanistically Primary Screening of Carbamate Library by Gel-Based ABPP. Typically, gel- resolvable SHs were combined into a single sample (25 μL) and incubated inhibit SHs, such as phosphonates (43), electrophilic ketones with DMSO or a carbamate (50 μM) for 45 min at 25 °C. FP-rhodamine (19), and lactones (30) or lactams (44). As these compound (2 μM) was then added for an additional 45 min at 25 °C. The reactions were libraries grow in size, they will eventually exceed the capacity of gel-based competitive ABPP. We have recently introduced quenched, separated by SDS-PAGE, and visualized by in-gel fluorescence scanning. IC50 values for select compounds were determined as described one potential solution to this problem, a fluorescence polarization in the SI Appendix. platform for ABPP that is compatible with ultrahigh-throughput screening (24). ABPP-MudPIT Analysis of SHs Inhibited by Carbamates in Vivo. See the Finally, we anticipate that the knowledge gained from our SI Appendix for details. competitive ABPP studies with SHs can apply to genome-wide pharmacological studies of other enzyme families for which activ- ACKNOWLEDGMENTS. We thank David Milliken, Brent Martin, Sarah Tully, ity-based probes have been developed, including kinases (15), and Andrea Zuhl for technical assistance. This work was supported by the oxidoreductases (17, 18), and additional classes of hydrolases National Institutes of Health (DA025285, GM090294, DA026161), the (12–14). In this way, future medicinal-chemistry pursuits may take Deutscher Akademischer Austausch Dienst (Postdoctoral Fellowship to on a decidedly proteomic flavor, such that projects focused on a A.A.), the National Science Foundation (Predoctoral Fellowship to D.A.B.), single enzyme give way to more target-agnostic, family-wide inves- Activx Biosciences, and The Skaggs Institute for Chemical Biology

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