Academic cross-fertilization by public screening yields SPECIAL FEATURE a remarkable class of protein phosphatase 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 enzyme 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 protein phosphatase 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 substrate 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 serine hydrolase 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 proteins (1). probes can form the basis for a fluorescence polarization-activity- A much more limited number of phosphatases 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 enzymes (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.