Design of Fluorogenic Peptide Substrates for Human Cytomegalovirus Protease Based on Structure–Activity Relationship Studies☆

Abstract Human 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 P 4 –P 2 residues of the maturation site of the enzyme by the optimized Tbg-Tbg-Asn(NMe 2 ) sequence conferred significant binding to inhibitors (Tbg, t -butylglycine). Incorporation of this improved sequence in a variety of substrates invariably led to improved kinetic parameters compared to homologues containing the natural sequence only. For example, the substrate o -aminobenzoyl-Tbg-Tbg-Asn (NMe 2 )-Ala↓Ser-Ser-Arg-Leu-Tyr(3-NO 2 )Arg-OH (2) displayed a k cat / K m value of 15,940 M −1 s −1 , i.e., more than 60-fold greater than that of the equivalent, nonoptimized substrate 1 under identical conditions. This improved sequence also permitted the development of a sensitive 7-amino-4-methylcoumarin fluorogenic substrate 3 which represents the shortest HCMV protease substrate to date. The kinetic and photometric advantages of these various substrates are discussed along with specific applications.