Hydroxypyridinethione Inhibitors of Human Insulin-Degrading Enzyme.

Insulin-degrading enzyme (IDE) is a human mononuclear Zn2+ -dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid ß-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn2+ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ∼350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn2+ -binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (Ki values of ∼50 µM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE.

Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin.

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.

Cathepsin D regulates cerebral Aß42/40 ratios via differential degradation of Aß42 and Aß40.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid ß-protein (Aß) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aß proteostasis in vivo and to more completely characterize the degradation of Aß42 and Aß40 by CatD. METHODS: We quantified Aß degradation rates and levels of endogenous Aß42 and Aß40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aß42 vs. Aß40, and the degradation of aggregated Aß42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aß42 and Aß40, as well as corresponding p3 fragments. RESULTS: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aß42 and Aß40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aß42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aß42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aß42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aß42 vis-à-vis Aß40. Specifically, Aß42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aß42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aß42 vs. Aß40 also extend to p3 fragments ending at positions 42 vs. 40. CONCLUSIONS: Our findings identify CatD as the principal intracellular Aß-degrading protease identified to date, one that regulates Aß42/40 ratios via differential degradation of Aß42 vs. Aß40. The finding that Aß42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aß42 and downstream pathological sequelae in AD.