Molecular Determinants of Substrate Specificity in Human Insulin-Degrading Enzyme.

Insulin-degrading enzyme (IDE) is a 110 kDa chambered zinc metalloendopeptidase that degrades insulin, amyloid ß, and other intermediate-sized aggregation prone peptides that adopt ß-structures. Structural studies of IDE in complex with multiple physiological substrates have suggested a role for hydrophobic and aromatic residues of the IDE active site in substrate binding and catalysis. Here, we examine functional requirements for conserved hydrophobic and aromatic IDE active site residues that are positioned within 4.5 Å of IDE-bound insulin B chain and amyloid ß peptides in the reported crystal structures for the respective enzyme-substrate complexes. Charge, size, hydrophobicity, aromaticity, and other functional group requirements for substrate binding IDE active site residues were examined through mutational analysis of the recombinant human enzyme and enzyme kinetic studies conducted using native and fluorogenic derivatives of human insulin and amyloid ß peptides. A functional requirement for IDE active site residues F115, A140, F141, Y150, W199, F202, F820, and Y831 was established, and specific contributions of residue charge, size, and hydrophobicity to substrate binding, specificity, and proteolysis were demonstrated. IDE mutant alleles that exhibited enhanced or diminished proteolytic activity toward insulin or amyloid ß peptides and derivative substrates were identified.

Efficient synthesis of α-fluoromethylhistidine di-hydrochloride and demonstration of its efficacy as a glutathione S-transferase inhibitor.

Histidine decarboxylase (HDC) is an enzyme that converts histidine to histamine. Inhibition of HDC has several medical applications, and HDC inhibitors are of considerable interest for the study of histidine metabolism. (S)-α-Fluoromethylhistidine di-hydrochloride (α-FMH) is a potent HDC inhibitor that is commercially available at high cost in small amounts only. Here we report a novel, inexpensive, and efficient method for synthesis of α-FMH using methyl 2-aziridinyl-3-(N-triphenylmethyl-4-imidazolyl) propionate and HF/pyridine, with experimental yield of 57%. To identify novel targets for α-FMH, we developed a three step in silico work-flow for identifying physically plausible protein targets. The work-flow resulted in 21 protein target hits, including several enzymes involved in glutathione metabolism, and notably, two isozymes of the glutathione S-transferase (GST) superfamily, which plays a central role in drug metabolism. In view of this predictive data, the efficacy of α-FMH as a GST inhibitor was investigated in vitro. α-FMH was demonstrated to be an effective inhibitor of GST at micromolar concentration, suggesting that off-target effects of α-FMH may limit physiological drug metabolism and elimination by GST-dependent mechanisms. The present study therefore provides new avenues for obtaining α-FMH and for studying its biochemical effects, with potential implications for drug development.