A comparative NMR conformational analysis of three distinct tetrapeptide inhibitors of the Hepatitis C NS3 protease that differ at the 4-aryloxy-substituted P2 proline position was undertaken. Specifically, transferred nuclear Overhauser effect experiments in combination with restrained systematic conformational searches were used to characterize the orientation of the P2 aryl substituents of these inhibitors when bound to the NS3 protease. Differences between free and bound conformations were also investigated.
View Article and Find Full Text PDFThis work describes the use of NMR as a medicinal chemistry tool for better understanding the binding characteristics of inhibitors of the HCV NS3 protease. The protease-bound structure of a tetrapeptide-like inhibitor that has an acid C-terminus, a norvaline at P1 and a naphthylmethoxy proline at P2 is described. Conformational comparisons are made with a similar compound having a 1-amino-cyclopropylcarboxylic acid at P1 and with a hexapeptide inhibitor.
View Article and Find Full Text PDFSubstrate hydrolysis by human cytomegalovirus (HCMV) protease is essential to viral capsid assembly. The interaction of HCMV protease and the N-terminal cleavage products of the hydrolysis of R- and M-site oligopeptide substrate mimics (R and M, respectively, which span the P9-P1 positions) was studied by NMR methods. Protease-induced differential line broadening indicated that ligand binding is mediated by the P4-P1 amino acid residues of the peptides.
View Article and Find Full Text PDFThe autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible.
View Article and Find Full Text PDFHuman cytomegalovirus (HCMV) protease is a slow-processing enzyme in vitro and its characterization would be facilitated if more efficiently cleaved substrates were available. Here we describe the development of improved fluorogenic peptide substrates for this protease and demonstrate that its indolent nature can be overcome by appropriate modifications within existing substrates. Prior structure-activity studies have indicated that replacement of the Val-Val-Asn sequence corresponding to the P4-P2 residues of the maturation site of the enzyme by the optimized Tbg-Tbg-Asn(NMe2) sequence conferred significant binding to inhibitors (Tbg, t-butylglycine).
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