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 LYPAL1 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.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.kinetic stabilizer | high-throughput screen | dimerization | structural biology | proteotoxicity S ecretion of an Ig light chain (LC) by a clonally expanded plasma cell population can lead to the disease LC amyloidosis (AL)-both a cancer and a proteinopathy (1, 2). "Free" LCs secreted without an associated antibody heavy chain (HC) initially adopt a well-defined homodimeric structure, wherein the monomers may be covalently linked by an interchain disulfide bond ( Fig. 1A) (3). LC monomers comprise an N-terminal variable (V) domain attached to a C-terminal constant (C) domain (SI Appendix, Fig. S1). Each patient's clonal plasma cells secrete a single, unique LC sequence. Most LCs are rapidly removed by the kidney.However, since amyloidogenic full-length (FL) LCs are generally less stable than nonamyloidogenic FL LCs, they can misfold, or misfold and misassemble, into nonnative species including cross-β-sheet amyloid fibrils, which are a hallmark of AL (4-8). Sequence also seems to play a role, as not all destabilized FL LCs aggregate in patients (4-8). How aggregation occurs in patients is not known, but several processes have been described in vitro, including destabilization-dependent endoproteolysis that releases amyloidogenic LC fragments (4,9,10). LC fragments including V domains are observed in patient deposits alongside FL LCs (11-13).Since we do not understand the structure-proteotoxicity relationships driving AL, a conservative strategy is to block FL LC misfolding at its origin by stabilizing the FL LC native state. Such a strategy has been effective at ameliorating the transthyretin amyloidoses (14-19). A small molecule that stabilizes FL LC dimers should prevent any misfolding and/or endoproteolysis that lead to LC aggregation and organ toxicity. We refer to such molecules as kinetic stabilizers, since they reduce the rate at which LCs transiently visit nonnative, aggregation-prone, and protease-sensitive conformations (20). The interfaces between the domains of the LC dimer are an important...

National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC 50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

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