A CONTINUOUS FLUORESCENCE-BASED ASSAY OF HUMAN CYTOMEGALOVIRUS PROTEASE USING A PEPTIDE SUBSTRATE

The 28-kDa protease from human cytomegalovirus (hCMV) has been success fully cloned, expressed, and purified to homogeneity. An internally qu enched fluorescent substrate oyl-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-Arg-L eu-Ala-5- [(2'-aminoethyl)-amino]-naphthalene-1-sulfonic acid; DABCYL- CMV-EDANS) based on the maturational cleavage site (M-site) junction w as synthesized in an effort to develop a fluorescence-based assay. Thi s substrate is cleaved specifically between the Ala-Ser peptide bond t hereby liberating the C-terminal peptide-EDANS fragment from the proxi mity quenching effect of the DABCYL group. This results in greater tha n a 10-fold increase in fluorescence that is observed at the EDANS emi ssion wavelength of 495 nm. Human CMV protease efficiently cleaved thi s synthetic substrate permitting continuous assay at peptide concentra tions lower than 10 mu M. At substrate concentrations greater than 10 mu M, linearity was lost due to the ''inner filter effect.'' This repr esents the first fluorescence-based assay for any of the herpes virus proteases. Additionally, a peptidyl inhibitor, Val-Val-Asn-Ala-Psi[CH2 NH]-Ser-Ser-Arg-Leu-Ala-OH, was prepared. This inhibitor was also base d on the same in-site cleavage junction with a nonhydrolyzable reduced peptide bond incorporated at the cleavage site. Using the fluorescenc e-based assay, this reduced peptide bond analog was observed to be an inhibitor of hCMV protease with an inhibition constant of >500 mu M. ( C) 1995 Academic Press, Inc.