DESIGN OF FLUOROGENIC PEPTIDE-SUBSTRATES FOR HUMAN CYTOMEGALOVIRUS PROTEASE BASED ON STRUCTURE-ACTIVITY RELATIONSHIP STUDIES

Human cytomegalovirus (HCMV) protease is a slow-processing enzyme in v itro and its characterization would be facilitated if more efficiently cleaved substrates were available. Here we describe the development o f improved fluorogenic peptide substrates for this protease and demons trate that its indolent nature can be overcome by appropriate modifica tions within existing substrates. Prior structure-activity studies hav e 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 o ptimized Tbg-Tbg-Asn(NMe2) sequence conferred significant binding to i nhibitors (Tbg, t-butylglycine), Incorporation of this improved sequen ce in a variety of substrates invariably led to improved kinetic param eters compared to homologues containing the natural sequence only, For example, the substrate o-aminobenzoyl-Tbg-Tbg-Asn (NMe2)-Ala down arr ow Ser-Ser-Arg-Leu-Tyr(3-NO2)Arg-OH (2) displayed a k(cat)/K-m value o f 15,940 M-1 s(-1), i.e., more than 60-fold greater than that of the e quivalent, nonoptimized substrate 1 under identical conditions, This i m proved sequence also permitted the development of a sensitive 7-amin o-4-methylcoumarin fluorogenic substrate 3 which represents the shorte st HCMV protease substrate to date, The kinetic and photometric advant ages of these various substrates are discussed along with specific app lications. (C) 1998 Academic Press.