Click-generated triazole ureas as ultrapotent in vivo–active serine hydrolase inhibitors

Abstract

Serine hydrolases (SHs) are a diverse enzyme class representing > 1% of all human proteins. The biological functions for most SHs remain poorly characterized due to a lack of selective inhibitors to probe their activity in living systems. Here, we show that a substantial number of SHs can be irreversibly inactivated by 1,2,3-triazole ureas, which exhibit negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the SH superfamily, including peptidases (acyl-peptide hydrolase or APEH), lipases (platelet-activating factor acetylhyrolase-2 or PAFAH2), and uncharacterized hydrolases (α, β-hydrolase 11 or ABHD11), with exceptional potency in cells (sub-nM) and mice (< 1 mg/kg). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T-cells. These data designate 1,2,3-triazole ureas as a pharmacologically privileged chemotype for SH inhibition that shows broad activity across the SH class coupled with tunable selectivity for individual enzymes.

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

“…Compound 4 was tested in an FP-Rh/inhibitor competition assay using mouse brain lysate as the source of serine hydrolases and shown to selectively block APT1 and APT2 at 30 nM (Fig. 1B) as reported previously for other similar compounds (32).…”

“…T. gondii genomic DNA was prepared using the Wizard genomic DNA purification kit (Promega). The development and use of fluorophosphonate-rhodamine (FP-Rh) has been described (32,41).…”

“…The separated function of APT1 and APT2 is likely to be due in part to their differential expression between cell types (24,31). To study the importance of depalmitoylation in various cell types, inhibitors were designed to block APT1 and APT2 function with the objective to specifically perturb palmitoylation dynamics and reduce the function of palmitoylated proteins in vitro and in vivo (32,33). The dynamic palmitoylation/depalmitoylation cycle has been demonstrated for a number of substrates in mammalian cells including H-and N-Ras (16,34) and most recently Rac1 (35).…”

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

“…The inhibition by pyrrophenone occurred at concentrations often used (1-2 M) to "selectively" inhibit cPLA 2 ␣ complicating its use to investigate the role of cPLA 2 ␣ in mediating calciuminduced arachidonic acid release. The other small molecule inhibitor that blocked cell death, KT195, is a derivative of 1,2,3-triazole urea that inhibits serine hydrolases by covalent, irreversible carbamoylation of the active site serine nucleophile (37,65). The triazole ureas show promise for the development of selective serine hydrolase inhibitors (65).…”

“…The other small molecule inhibitor that blocked cell death, KT195, is a derivative of 1,2,3-triazole urea that inhibits serine hydrolases by covalent, irreversible carbamoylation of the active site serine nucleophile (37,65). The triazole ureas show promise for the development of selective serine hydrolase inhibitors (65). Other classes of serine hydrolase inhibitors were ineffective such as methylarachidonoyl fluorophosphonate (data not shown), which broadly targets a large number of serine hydrolases (66).…”