Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors

Abstract

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

Peptidic inhibitors of insulin-degrading enzyme with potential for dermatological applications discovered via phage display

“…Among the modified versions of these parent peptides, an additional five exhibited K i values <10 μM; thus nine of the twenty-five peptides tested (36%) showed good activity. These hit rates are markedly higher than those obtained through high-throughput compound screening [ 41 , 45 , 48 ] or other approaches [ 29 ]. Notably, the potency of P12-1 (K i = 800 nM) compares favorably to that of the highly optimized and extensively characterized macrocyclic IDE inhibitor, 6bK , which shows a IC 50 value of ~100 nM against insulin [ 29 ].…”

Targeting Insulin-Degrading Enzyme to Treat Type 2 Diabetes Mellitus

“…Toward this goal, ML345 and its derivatives, a thiol-reacting IDE inhibitor that covalently links to IDE C819 with up to 50 nM affinity has also been developed recently [78, 79]. Its development stems from the notion that IDE is sensitive to thiol-mediated inactivation by oxidation and nitrosylation [62, 80, 81].…”

Development of ebsulfur analogues as potent antibacterials against methicillin-resistant Staphylococcus aureus

“…13 Clinical applications of ebselen are hypothesized to be related to its ability to covalently bind to cysteine residues on targeted proteins or its antioxidant activity via mimicking glutathione peroxidase. 14,15 In addition, ebselen was found to inhibit the growth of various Gram-positive and Gram-negative bacterial strains. 16 In 2014, the crystal structure of antigen 85C, a putative drug target in Mycobacterium tuberculosis ( Mtb ), was solved and revealed that ebselen covalently inhibit the antigen 85 complex and hence, explained its activity against Mtb .…”