Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

Abstract

Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis and screening of a small library of NHH carbamates by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterease-1 (PPT1), mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

“…Recent studies by our lab and others have found using chemical proteomics that small molecules can accumulate in the lysosome to furnish much greater engagement of proteins than would be predicted based on in vitro experiments (Cognetta et al, 2015, Zuhl et al, 2016). We therefore tested whether lysosomal accumulation may underlie the in situ activity of inhibitor 1 /probe 4 against cathepsins by treating CTSC-transfected cells with the lysosomal neutralizing agents ammonium chloride (NH 4 Cl, 10 mM, 15 min) or bafilomycin A1, a vacuolar H + -ATPase inhibitor (10 nM, 15 min), followed by in situ exposure to probe 4 .…”

Chemical Methods for Probing Virus–Host Proteomic Interactions

“…Highthroughput approaches, including fluorescence polarization-based ABPP (FluoPol-ABPP; Figure 4b) have been designed to overcome this limitation. 62 Recently, competitive ABPP has led to the discovery of new classes of inhibitors including carbamate, [63][64][65] aza-β-lactams, 66 β-lactones, 67 and reversible piperazine amide 68 (serine hydrolase inhibitors), α-ketoheterocyclic compounds 69 (diacylglycerol lipase inhibitors) and many others. 68 This technique was applied in a study by Yang et al 48 in which they assessed the potency and identified off-targets of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 orlistat inhibitor of FASN.…”

Expanding the Toolkit for the Serine Hydrolases

“…Thus, the study by Cognetta et al (2015) provides new pharmacological tools that target serine hydrolases PPT1 and ABHD4 with good selectivity in vivo. These enzymes had heretofore been difficult to study because of their lack of reactivity toward broad-based fluorophosphonate activity-based probes.…”

“…In this issue of Chemistry & Biology, Cognetta et al (2015) describe new pharmacological tools, including N-hydroxyhydantoin-containing carbamate inhibitors and an activity-based probe, for palmitoyl protein thioesterase 1 and alpha, beta-hydrolase domain-4 that expand the toolkit for the serine hydrolases.…”

“…(A) The base-activated serine nucleophile of the serine hydrolase is covalently modified by the electrophilic carbamate chemotype (leaving group is shown in red; the carbamylating group is shown in black). (B) Cognetta et al (2015) describe new inhibitors in which the N-hydroxyhydantoin-containing leaving group (shown in red) was synthetically modified to afford new inhibitors that selectively inactivate palmitoyl protein thioesterase 1 (PPT1) and ABHD4. This scaffold was used to generate an alkyne-tagged activitybased chemoproteomic probe (termed ABC45) that reacts directly with PPT1 and ABHD4 in complex proteomes.…”