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

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-metallopepitidase 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 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 (IC50app = 63 nM), nontoxic at concentrations up to 100 mu M, 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.