Academic cross-fertilization by public screening yields SPECIAL FEATURE a remarkable class of methylesterase-1 inhibitors

Daniel A. Bachovchina, Justin T. Mohrb, Anna E. Speersa, Chu Wanga, Jacob M. Berlinb, Timothy P. Spicerc, Virneliz Fernandez-Vegac, Peter Chasec, Peter S. Hodderc, Stephan C. Schürerc, Daniel K. Nomuraa, Hugh Rosena,d, Gregory C. Fub,1, and Benjamin F. Cravatta,1

aThe Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; bDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; cLead Identification Division, Molecular Screening Center, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458; and dThe Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved January 13, 2011 (received for review November 24, 2010)

National Institutes of Health (NIH)-sponsored screening centers ulating the binding interaction of the C subunit with various provide academic researchers with a special opportunity to pursue regulatory B subunits (8–10). A predicted outcome of these small-molecule probes for protein targets that are outside the shifts in subunit association is the targeting of PP2A to different current interest of, or beyond the standard technologies employed protein substrates in cells. PME-1 has also been hypothesized by, the pharmaceutical industry. Here, we describe the outcome to stabilize inactive forms of nuclear PP2A (11), and recent struc- of an inhibitor screen for one such target, the protein tural studies have shed light on the physical interactions between phosphatase methylesterase-1 (PME-1), which regulates the PME-1 and the PP2A holoenzyme (12). methylesterification state of 2A (PP2A) and is Notwithstanding the aforementioned models and findings, the implicated in cancer and neurodegeneration. Inhibitors of PME-1 actual functional consequences of perturbing PP2A methylation have not yet been described, which we attribute, at least in part, remain largely unexplored. In yeast, LCMT1 deletion caused CHEMISTRY to a dearth of assays compatible with high-throughput severe growth defects under stress conditions, while PME-1 screening. We show that PME-1 is assayable by fluorescence polar- deletion did not result in an observable cellular phenotype (9). ization-activity-based protein profiling (fluopol-ABPP) and use Disruption of the PME-1 gene in mice, on other hand, caused this platform to screen the 300,000+ member NIH small-molecule early postnatal lethality (13), which has limited the experimental library. This screen identified an unusual class of compounds, opportunities to explore methylation of PP2A in animals. Recent β the aza- -lactams (ABLs), as potent (IC50 values of approximately studies have found that RNA-interference knockdown of PME-1 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not in cancer cells leads to activation of PP2A and corresponding

derive from a commercial vendor but rather an academic contribu- suppression of protumorigenic phosphorylation cascades (14), BIOCHEMISTRY tion to the public library. We show using competitive-ABPP that indicating that PME-1 could be an attractive drug target in on- ABLs are exquisitely selective for PME-1 in living cells and mice, cology. Changes in PP2A methylation have also been implicated where enzyme inactivation leads to substantial reductions in de- in Alzheimer’s disease, where this modification may stimulate methylated PP2A. In summary, we have combined advanced syn- PP2A’s ability to promote neural differentiation (15). thetic and chemoproteomic methods to discover a class of ABL Despite the critical role that PME-1 plays in regulating PP2A inhibitors that can be used to selectively perturb PME-1 activity structure and function, PME-1 inhibitors have not yet been in diverse biological systems. More generally, these results illus- described. This deficiency may be due to a lack of PME-1 activity trate how public screening centers can serve as hubs to create assays that are compatible with high-throughput screening spontaneous collaborative opportunities between synthetic chem- (HTS). Assessment of PME-1 activity typically involves either istry and chemical biology labs interested in creating first-in-class Western blotting with antibodies that recognize specific methyla- pharmacological probes for challenging protein targets. tion states of PP2A (7, 13) or monitoring the release of 3H- methanol from radiolabeled-C subunits (16), but neither assay rotein phosphorylation is a pervasive and dynamic posttran- is easily adapted for HTS. PME-1 is, however, a Pslational protein modification in eukaryotic cells. In mam- and therefore susceptible to labeling by active-site-directed fluor- mals, more than 500 protein kinases catalyze the phosphoryl- ophosphonate (FP) probes (17). We have recently shown that FP ation of serine, threonine, and tyrosine residues on (1). probes can form the basis for a fluorescence polarization-activity- A much more limited number of are responsible for based protein profiling (fluopol-ABPP) assay suitable for HTS reversing these phosphorylation events (2). For instance, protein (18). Here, we apply fluopol-ABPP to screen the 300,000+ Na- phosphatase 2A (PP2A) and PP1 are thought to be responsible tional Institutes of Health (NIH) compound library for PME-1 together for >90% of the total serine/threonine phosphatase inhibitors. From this screen, we identified a set of aza-β-lactam activity in mammalian cells (3). Specificity is imparted on PP2A (ABL) compounds that act as remarkably potent and selective activity by multiple mechanisms, including dynamic interactions between the catalytic subunit (C) and different protein-binding partners (B subunits), as well as a variety of posttranslational Author contributions: D.A.B., J.T.M., H.R., G.C.F., and B.F.C. designed research; D.A.B., J.T.M., J.M.B., T.P.S., V.F.-V., P.C., P.S.H., S.C.S., and D.K.N. performed research; D.A.B., chemical modifications (2, 4). Within the latter category is an J.T.M., C.W., J.M.B., and S.C.S. contributed new reagents/analytic tools; D.A.B., J.T.M., unusual methylesterification event found at the C terminus of A.E.S., C.W., T.P.S., P.S.H., S.C.S., H.R., G.C.F., and B.F.C. analyzed data; and D.A.B., J.T.M., the catalytic subunit of PP2A that is introduced and removed A.E.S., H.R., G.C.F., and B.F.C. wrote the paper. by a specific methyltransferase (leucine carbxoylmethyltransfer- The authors declare no conflict of interest. ase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase This article is a PNAS Direct Submission. methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. “ ” carboxymethylation (hereafter referred to as methylation ) has This article contains supporting information online at www.pnas.org/lookup/suppl/ been proposed to regulate PP2A activity, at least in part, by mod- doi:10.1073/pnas.1015248108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1015248108 PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6811–6816 Downloaded by guest on September 27, 2021 PME-1 inhibitors. We show that these ABLs covalently inactivate ABL103, ABL105, ABL107) that were similar in structure, all PME-1 with high specificity in living cells and animals, where dis- with a branched alkyl group at R1 and with R stereochemistry ruption of this enzyme leads to substantial decreases in demethy- at this position (Fig. 2A). The MLPCN library contained 22 other lated PP2A. ABLs, including the enantiomers of ABL127 (ent-ABL127), ABL103 (ent-ABL103), and ABL105 (ent-ABL105), that were Results all considerably less active toward PME-1 in the primary screen PME-1 Inhibitor Screening by Fluopol-ABPP. Because PME-1 is a (Table S1). Intriguingly, the ABLs did not originate from a com- serine hydrolase that is known to interact with reporter-tagged mercial compound collection but rather were submitted by FP probes (17, 19), we reasoned that this enzyme would be assay- the academic chemistry laboratory of our coauthor Gregory Fu, able by competitive ABPP methods. However, lower-throughput, who generated these compounds as part of an investigation gel-based competitive ABPP screens have not succeeded in iden- into the synthetic utility of chiral 4-pyrrolidinopyridine catalysts tifying lead PME-1 inhibitors (20), indicating the need to survey (23). We further noted that the ABLs are structurally unusual larger compound libraries. We therefore asked whether PME-1 compared to the rest of the MLPCN library, lying a considerable could be assayed using the recently introduced, HTS-compatible distance in a chemical space plot from the typical structures that fluopol-ABPP platform (18). This technique, where compounds populate the compound collection (Fig. 2B). are tested for their ability to block the increase in fluopol signal The ABL hits were next titrated into the soluble proteome of generated by reaction of a fluorescent activity-based probe with a MDA-MB-231 cells, a cell line that endogenously expresses much larger protein target, has enabled inhibitor screening for a PME-1, to assess their potency (Fig. 2C). The two compounds wide range of probe-reactive (http://pubchem.ncbi.nlm bearing cycloalkyl substituents at R1, ABL127 and ABL103, were .nih.gov/). We confirmed that purified, recombinant wild-type extraordinarily potent inhibitors of PME-1, with IC50 values PME-1, but not a mutant PME-1 in which the serine nucleophile of 4.2 and 2.1 nM, respectively (Fig. 2C and Fig. S2). The two was replaced with alanine (S156A), labels with a fluorophospho- compounds with isopropyl substituents, ABL105 and ABL107, nate rhodamine (FP-Rh) (21) probe (Fig. 1B). This reaction exhibited lower IC50 values (92 and 24 nM, respectively) but were generates a strong, time-dependent increase in fluopol signal still good inhibitors of PME-1. Interestingly, a strong preference that is not observed in the absence of enzyme or with the S156A for the R enantiomer of ABLs was observed, as the S enantiomer mutant PME-1 enzyme (Fig. 1C). In collaboration with the Mo- of ABL127 (ent-ABL127) was at least two orders of magnitude lecular Libraries Probe Production Centers Network (MLPCN), less potent at inhibiting PME-1 (Fig. 2 C and D). Indeed, some of we screened 315,002 compounds for PME-1 inhibition using fluo- the apparent activity of the S enantiomer may be due to the small pol-ABPP (see Fig. 1D for a representative subset of the primary amount of residual R enantiomer in the >99% ee sample. screening data). Following a confirmation screen on initial hits, Before proceeding further, we wanted to confirm that ABLs we identified 1,068 compounds as potential PME-1 inhibitors. could inhibit the ability of PME-1 to demethylate PP2A. We As an initial filter, we selected compounds for follow-up studies therefore treated HEK 293T soluble lysates with ABL127 that had <5% hit rates in all other bioassays reported in the Pub- (500 nM, 30 min) or DMSO before adding purified recombinant Chem database, <30% inhibition in three fluopol-ABPP screens PME-1 for an additional hour. In DMSO-treated lysates, we performed on other enzymes (http://pubchem.ncbi.nlm.nih.gov/), observed the expected time-dependent increase in demethylated and >40% inhibition of PME-1 in the confirmation screen. This PP2A and concomitant decrease in methylated PP2A, as deter- filter yielded approximately 300 candidate PME-1 inhibitors. mined by immunoblotting with antibodies that specifically recog- nize either form of the C terminus of PP2A (Fig. 2E). In lysates Discovery of aza-β-lactam (ABL) Inhibitors of PME-1. The approxi- containing ABL127, however, we observed little or no change mately 300 hit compounds were next analyzed by gel-based in the methylation state of PP2A (Fig. 2E), indicating that competitive ABPP (18, 22) in soluble lysates from HEK 293T ABL127 blocks PME-1’s activity on its physiological substrate. cells overexpressing PME-1. This convenient selectivity screen As ABL127 and ABL103 are highly similar structures and assessed in parallel the activity of lead compounds against ap- emerged as virtually indistinguishable in these assays, we selected proximately 25 gel-resolvable, FP-Rh-reactive serine one of these compounds, ABL127, for in-depth characterization. expressed in HEK 293Tcells and rapidly eliminated false-positive and nonselective compounds. Among the compounds that selec- ABLs Covalently Inhibit PME-1. Based on scientific precedent show- tively inhibited PME-1 (Fig. S1) were four ABLs (ABL127, ing that other serine hydrolases can react with and open β-lactam

Fig. 1. A fluopol-ABPP assay for PME-1. (A) Schematic re- presentation of the reversible C-terminal methylation of PP2A introduced by LCMT1 and removed by PME-1. (B) FP-Rh (75 nM) labels purified wild-type PME-1 (1 μM) but not the catalytically dead S156A PME-1 mutant (1 μM). Top panel: Fluorescent gel image shown in gray scale. (C) Time course for fluopol signal generated by reactions of wild-type PME-1 (1 μM) with FP-Rh (75 nM). No signal in- crease is observed in the absence of enzyme or with the S156A PME-1 mutant. The indicated 45-min time point (Z0 ¼ 0.77), prior to reaction completion, was selected for HTS. Data are presented as mean values SD. (D) Screening data for a representative 15,000 compounds of the NIH small-molecule library. Compounds that reduced the FP- Rh fluopol signal by >26.13% were designated as hits for PME-1 (red squares).

6812 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1015248108 Bachovchin et al. Downloaded by guest on September 27, 2021 Fig. 2. Discovery of selective ABL inhibitors of PME-1. (A) SPECIAL FEATURE Four ABLs (ABL127, ABL103, ABL105, and ABL107) identi- fied as active in the MLPCN screen. (B) Chemistry space co- ordinates for compounds (colored red in high occupancy cells; colored blue in low occupancy cells) that were screened against PME-1. The 26 ABLs (shown as enlarged squares; all other compounds are shown as small circles) are located in a sparsely populated region of chemical space. This optimized 6-dimensional BCUTs (31) visualiza- tion of chemistry space for the compound library was gen- erated using the standard 3D hydrogen suppressed descriptors with Diverse Solutions (Diverse Solutions, Tri- pos), with coverage illustrated by compression into an op- timized two-dimensional BCUTs space as described in more detail previously (32). (C) Evaluation of compounds by gel- based competitive ABPP with FP-Rh (2 μM) in the soluble proteome (1 mg∕mL protein) of MDA-MB-231 cells. (D) Complete PME-1 IC50 curves for ABL127 (4.2 nM; 95% con- fidence limits 2.3–7.5 nM) and ent-ABL127 (450 nM; 95% confidence limits 240–850 nM) in the soluble proteome of MDA-MB-231 cells (for IC50 curves of ABL103, ABL105, and ABL107, see Fig. S2). Data are presented as mean values SEM; n ¼ 3∕group. (E) Pretreatment of HEK 293T proteomes with ABL127 (500 nM, 30 min) before addition of purified PME-1 (500 nM, 1 h) blocks PP2A demethylation as determined by Western blotting with antibodies that re- cognize specific methylation states of PP2A.

rings by breaking the bond, resulting in covalent acylation/ utilizes stable-isotope labeling of amino acids in cell culture inhibition (24), we hypothesized that the active-site serine (S156) (SILAC) (26). SILAC involves differential labeling of proteins CHEMISTRY of PME-1 analogously reacted with the ABL ring of ABL127 to with stable isotopes to generate isotopically “light” and “heavy” produce the enzyme-inhibitor adduct shown in Fig. 3A. Consis- samples, which, when pooled and analyzed by MS, yield accurate tent with a covalent mode of inhibition, we observed that block- quantification by comparing intensities of light and heavy peptide ade of FP-Rh labeling of PME-1 by ABL127 was not reversed peaks. SILAC has previously been used to identify enzymes by gel filtration (Fig. 3B). The identity of the expected acylation targets of activity-based probes (27) and small-molecule-binding adduct was confirmed by liquid chromatography-mass spectro- proteins in cell lysates (28). In our competitive ABPP-SILAC metry (LC-MS) analysis of purified, recombinant PME-1 treated experiments, cells grown in light and heavy media were treated

with ABL127 (Fig. 3C). with DMSO or ABL127, respectively, for 1 h. Proteomes were BIOCHEMISTRY then harvested, combined at a 1∶1 total protein ratio, and treated ABL127 Selectively Inhibits PME-1 in Cells. We next investigated with the activity-based probe FP-biotin (29). FP-biotin-labeled whether ABLs could inhibit PME-1 in living cells. We incubated proteins were then enriched with streptavidin-conjugated beads, MDA-MB-231 and HEK 293T cells with a concentration range digested on-bead with , and the resulting peptides analyzed of ABL127 for 1 h, harvested the soluble proteomes, and then by liquid chromatography-high-resolution tandem MS using an reacted these lysates with the FP-Rh probe (Fig. 4A and Fig. S3). LTQ-Orbitrap mass spectrometer. Specifically enriched proteins In both cell lines, we observed highly potent and selective inhibi- were identified and quantified based on analysis of MS2 spectra tion of PME-1 with IC50 values of 11.1 nM and 6.4 nM, respec- and MS1 profiles, respectively. This analysis revealed complete tively (Fig. 4B and Fig. S3). Although these gel-based competitive and selective in situ inhibition of PME-1 by ABL127 with no ABPP studies did not reveal any additional serine hydrolase activity against >50 other serine hydrolases detected in MDA- targets of ABL127 at concentrations under 10 μM, we wanted MB-231 and HEK 293T cells (Fig. 4C and Fig. S4). to verify this selectivity profile by higher-resolution LC-MS/MS We next investigated the impact of ABL127 incubation on the methods. To accomplish this, we employed an advanced version methylation state of PP2A in cells. As expected, in both MDA- of our competitive ABPP-MudPIT technology (20, 25) that MB-231 and HEK 293T cells, we observed significant reductions

Fig. 3. ABLs are covalent inhibitors of PME-1. (A) Proposed mechanism for covalent PME-1 inhibition by ABL127. (B) PME-1 (500 nM) was incubated with DMSO or ABL127 (5 μM), and each reaction was split into two fractions. One fraction was directly labeled with FP-Rh (left panels), and the other was subjected to gel-filtration to remove free inhibitor and then reacted with FP-Rh (right panels) to de- termine the reversibility of inhibition. (C) MS1 traces for the PME-1 peptide not adducted (top panel) or ad- ducted (bottom panel) to ABL127. See SI Materials and Methods for more information on this experiment. Traces were obtained from tryptic digests of purified, recombinant PME-1 (10 μM) treated with ABL127 (50 μM, red trace) or DMSO (black trace).

Bachovchin et al. PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6813 Downloaded by guest on September 27, 2021 in the levels of demethylated PP2A (35% and 80%, respectively; demethylated PP2A for at least 24 h (Fig. 4I). These data, taken Fig. 4 D and E). A trend toward increases in methylated PP2A together, indicate that ABL127 selectively inactivates PME-1 in was also observed in ABL127-treated HEK 293T cells, but this living cells, which in turn causes significant changes in the methy- change did not reach statistical significance (p ¼ 0.12) (Fig. 4 D lation state of PP2A. and E). No difference in methylated PP2A was observed in MDA- MB-231 cells treated with ABL127 (Fig. 4 D and E). These out- A Clickable ABL Confirms Proteome-Wide Selectivity for PME-1. Our comes might be expected if the vast majority of PP2A is consti- competitive ABPP results showed that ABL127 is highly selective tutively methylated under standard cell culture conditions. To for PME-1 among members of the serine hydrolase family, but investigate this hypothesis, we stably overexpressed PME-1 in they did not address the possibility that ABL127 might react with HEK 293T cells (Fig. 4F), which resulted in a dramatic increase other proteins in the proteome. To investigate this possibility, we in demethylated PP2A and a significant decrease in methylated synthesized ABL112, an analog of ABL127 that contains alkyne PP2A relative to a control cell line stably expressing GFP (Fig. 4 groups to serve as latent affinity handles amenable to modifica- G and H). Importantly, treatment of PME-1-transfected cells tion by reporter tags using the copper(I)-catalyzed azide–alkyne with ABL127 for only 1 h reduced the amount of demethylated cycloaddition reaction (“click chemistry”) (30) (Fig. 5A). First, we PP2A back to the level observed in GFP-overexpressing control confirmed that ABL112 retains inhibitory activity for PME-1 by cells (Fig. 4 G and H). These ABL127-treated cells also showed gel-based competitive ABPP in MDA-MB-231 lysates, where it a significant increase in methylated PP2A (Fig. 4 G and H). showed only a threefold reduction in potency (IC50 ¼ 13.8 nM; Time course studies revealed that a single treatment of ABL127 Fig. 5B) compared to the parent inhibitor ABL127 (Fig. S5). resulted in sustained inactivation of PME-1 and reductions in Next, we treated MDA-MB-231 cells with ABL127 (100 nM)

Fig. 4. ABL127 selectively inactivates PME-1 and decreases the demethylated form of PP2A in cells. (A) Gel-based competitive ABPP of the soluble proteomes from MDA-MB-231 cells treated with ABL127 (0.61–10,000 nM; 1 h) reveals selective blockade of PME-1 with (B)anIC50 value of 11.1 nM. (C) Isotopically “light” and “heavy” MDA-MB-231 cells were treated with DMSO or ABL127 (100 nM), respectively, for 1 h. Proteomes were combined in a 1∶1 total protein ratio (0.5 mg each), analyzed by ABPP-MudPIT, and serine hydrolase activities were quantified by comparing intensities of light and heavy peptide peaks (see Fig. S4 for additional SILAC ABPP-MudPIT analyses). Data are presented as mean values SEM for all quantifiable peptides from each serine hydrolase. (D and E) MDA- MB-231 and HEK 293T cells treated with ABL127 (500 nM, 1 h) exhibit significant reductions in demethylated PP2A. (F) Stable overexpression of PME-1 in HEK 293T cells compared to control cells overexpressing GFP. PME-1 is completely inhibited by ABL127 (500 nM, 1 h) in both cell lines. (G and H) Cells overexpressing PME-1 show decreased PP2A methylation, which is reversed by addition of ABL127 (500 nM, 1 h). (I) Time-course for PME-1 inhibition by ABL127 (500 nM) in PME-1-overexpressing HEK 293T cells. For E and I:*p < 0.05,**p < 0.01 for DMSO-treated versus ABL127-treated cells. #p < 0.05,##p < 0.01 for cells overex- pressing GFP versus PME-1. Data are presented as mean values SEM; n ¼ 3∕group.

6814 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1015248108 Bachovchin et al. Downloaded by guest on September 27, 2021 These results demonstrate that the remarkable selectivity dis-

played by ABL127 for PME-1 extends not only across the serine SPECIAL FEATURE hydrolase family but also the greater mammalian proteome.

ABL127 Inactivates PME-1 in Mice. As mentioned earlier, PME-1 (−∕−) mice are not viable (13), which has hindered experimental efforts to characterize this enzyme’s function (and the functional significance of PP2A methylation) in animals. Pharmacological inhibition of PME-1 would thus offer a potentially powerful means to study this enzyme in vivo. With this goal in mind, we asked whether ABL127 could inhibit PME-1 in mice. C57Bl/6 Fig. 5. A clickable ABL reveals proteome-wide selectivity for PME-1. (A) mice were treated with ABL127 (50 mg∕kg, i.p., 2 h) or vehicle, Structure of the ABL alkyne probe ABL112. (B) ABL112 inactivates PME-1 sacrificed, and their brain proteomes assayed for PME-1 activity (IC50 ¼ 13.8 nM; 95% confidence limits 6.5–29 nM) in the MDA-MB-231 solu- by competitive ABPP. Gel-based profiles indicated that brain ble proteome (1 mg∕mL protein) as determined by gel-based competitive PME-1 was inactivated by ABL127 (Fig. 6A), but overlapping ABPP (see Fig. S5). (C) MDA-MB-231 cells were incubated with DMSO or serine hydrolase activities precluded a confident assessment of ABL127 (100 nM, 30 min) followed by ABL112 (10–200 nM, 2 h). Soluble cell 0 5 ∕ “ ” the extent of inactivation. For enhanced resolution of the activity proteomes ( . mg mL) were then subjected to a standard click reaction state of PME-1 and other brain serine hydrolases, we performed using RhN3 (30) and ABL112-labeled proteins were visualized by in-gel fluor- competitive ABPP-MudPIT studies using FP-biotin. These LC- escence scanning. PME-1 was the only protein labeled by ABL112 and com- B peted by ABL127. ABL112 labeled an additional 80 kDa protein that was not MS profiles confirmed complete inactivation of PME-1 (Fig. 6 ) competed by ABL127, suggesting it may be a target for ABL112, but not and no substantial reductions in any of the other approximately ABL127 (see Fig. S5). (D) Mouse brain soluble lysates (1 mg∕mL) were incu- 40 brain serine hydrolases detected in this experiment. We also bated (30 min) with DMSO or ABL127 (100 nM). ABL112 (10 nM) was then observed an approximately 35% reduction in the amount of de- added for an additional 30 minutes before analysis by click chemistry-based methylated PP2A in brain tissue from mice treated with ABL127 ABPP. (Fig. 6 C and D). These results confirm that ABL127 can selec- tively inhibit PME-1 in mice and this inhibition alters the methy- or DMSO for 30 min before adding ABL112 at a range of con- lation state of brain PP2A.

centrations and incubating for another 2 h. Cells were then lysed CHEMISTRY and the soluble proteomes were subjected to a “click” reaction Discussion with an azide-Rh tag (RhN3), separated by SDS/PAGE, and We report herein a class of ABL inhibitors that show remarkable ABL112-labeled proteins were visualized by in-gel fluorescence selectivity for PME-1 and equipotent activity in both cell-free and scanning (Fig. 5C). This analysis identified PME-1 as the only living cell assays. The lead ABL, ABL127 (designated as NIH ABL112-labeled protein that could be competed by pretreatment Probe ML174), is capable of inactivating PME-1 in both human with ABL127. One additional ABL112-reactive protein at ap- cancer cells and mice, suggesting that it should serve as a versatile proximately 80 kDa was also detected, but the labeling of this pharmacological probe for evaluating PME-1 function in a multi- protein was not competed by ABL127 or ent-ABL127 (Fig. S5),

tude of living systems. We found that PME-1 inhibition causes a BIOCHEMISTRY indicating that it is exclusively a target of the clickable probe significant reduction in demethylated PP2A and, in cells with high ABL112 but not ABL127. Consistent with this premise, compe- levels of PME-1 activity, also a concomitant increase in methy- titive ABPP analysis identified an 80 kDa FP-Rh-labeled protein lated PP2A. Future studies with ABL127 should facilitate a more in MDA-MB-231 lysates that was sensitive to inhibition by detailed understanding of the role that methylation plays in ABL112 but not ABL127 (Fig. S5). We performed a similar regulating PP2A function. Will, for instance, alterations in methy- “click” analysis with ABL112 in the soluble proteome of mouse lation state impact the composition and/or stability of specific brain, which again revealed selective labeling of PME-1 (Fig. 5D). PP2A complexes? Structural studies have confirmed that

Fig. 6. ABL127 selectively inhibits PME-1 in vivo. (A) Evaluation of mouse brain soluble proteome from mice treated with vehicle or ABL127 (50 mg kg−1, i.p., 2 h) by gel-based ABPP. In this analysis, proteomes were also treated ex vivo with DMSO or ABL127 (2 μM) before FP-Rh labeling to confirm complete PME-1 inhibition. (B) ABPP-MudPIT analysis of serine hydrolases from brain proteomes of animals treated with vehicle or ABL127. These data confirm selective in- activation of PME-1 in vivo. (C and D) Mice treated with ABL127 exhibited reductions in demethylated PP2A compared with vehicle-treated mice. *p < 0.01, **p < 0.001 for DMSO versus ABL127 groups. Data are presented as mean values SEM; n ¼ 3∕group.

Bachovchin et al. PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6815 Downloaded by guest on September 27, 2021 PME-1 is a component of this complex, where its interactions That the mechanism for PME-1 inhibition involves acylation with the catalytic subunit of PP2A appear to be mediated, at least of the enzyme’s conserved serine nucleophile (Fig. 3) suggests in part, by the PME-1 active site (12). It is thus possible that in- that exploration of a more structurally diverse set of ABLs might hibition of PME-1 will affect PP2A complexes not only through uncover inhibitors for other serine hydrolases. In this way, the altering methylation but also through disrupting physical interac- chemical information gained from a single high-throughput tions between PME-1 and the catalytic subunit of PP2A. Deter- screen may be leveraged to initiate probe development programs mining how such changes in PP2A complexes affect substrate for additional enzyme targets. interactions and the broader phosphoproteome should represent Projecting forward, this research provides an example of how an exciting area of research. On this note, recent studies point public small-molecule screening centers can serve as a portal for to an important role for PME-1 in negatively regulating the tu- spawning academic collaborations between chemical biology and mor-suppressive function of PP2A in cancer cells (14). PME-1 synthetic chemistry labs. By continuing to develop versatile high- inhibitors may thus have utility as anticancer agents. Finally, we throughput screens and combining them with a small-molecule speculate that the net effect of PME-1 inhibition on PP2A methy- library of expanding structural diversity conferred by advanced lation state will be dictated by the expression levels of not only synthetic methodologies, academic biologists and chemists are PME-1 (see Fig. 3G) but also LCMT1. Imbalances in the relative expression of these two enzymes may thus mark specific cellular well-positioned to collaboratively deliver pharmacological probes states where PME-1 inhibitors will produce their most dramatic for a wide range of proteins and pathways in cell biology. pharmacological effects. Materials and Methods There were several keys to the success of our probe develop- PME-1 Protein Expression and Purification. Human recombinant PME-1 was ment effort. First, screening for inhibitors of PME-1 benefited expressed in BL21(DE3) Escherichia coli and purified at approximately from the fluopol-ABPP technology, which circumvented the 5 mg∕L as detailed in SI Materials and Methods. limited throughput of previously described substrate assays for this enzyme. Second, we were fortunate that the NIH compound PME-1 Fluopol-ABPP Assay. See SI Materials and Methods for details. library contained several members of the ABL class of small mo- lecules. These chiral compounds, which represent an academic Competitive ABPP Assays in Proteomes. See SI Materials and Methods for contribution to the NIH library, occupy an unusual portion of details. structural space that is poorly accessed by commercial compound collections. Although at the time of their original synthesis (23) Synthesis of ABLs. See SI Materials and Methods for details. it may not have been possible to predict whether these ABLs would show specific biological activity, their incorporation into ACKNOWLEDGMENTS. We thank Tianyang Ji, David Milliken, Kim Masuda, the NIH library provided a forum for screening against many Benjamin Kipper, and Jaclyn M. Murphy for technical assistance. This work proteins and cellular targets, culminating in their identification was supported by National Institutes of Health grants CA132630 (B.F.C.), MH084512 (H.R.), GM57034 (G.C.F.), and GM086040 (postdoctoral fellowship as PME-1 inhibitors. We then used advanced chemoproteomic to J.T.M.); the National Science Foundation (predoctoral fellowship to assays to confirm the remarkable selectivity displayed by ABLs D.A.B.); the California Breast Cancer Research Program (predoctoral fellow- for PME-1 across (and beyond) the serine hydrolase superfamily. ship to D.A.B.); and The Skaggs Institute for Chemical Biology.

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