Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides

The gene coding for the 3C protease from human rhinovirus strain 1B was eff iciently expressed in an Escherichia coli strain which also overexpressed t he rare argU tRNA. The protease was isolated from inclusion bodies, refolde d, and exhibited a k(cat)/K-m = 3280 M-1 s(-1) using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the br omomethylketonehydrazides. Compounds containing the bromomethylketonehydraz ide backbone and a glutamine-like side chain at the P1 position were potent , time-dependent inhibitors of rhinovirus 3C protease with k(inact)/K-inact values as high as 23,400 M-1 s(-1). The inhibitory activity of compounds c ontaining modified P1 side chains suggests that the interactions between th e P1 carboxamide group and the 3C protease contributes at least 30-fold to the k(inact)/K-inact rate constants for bromomethylketonehydrazide inhibiti on of 3C protease. Electrospray ionization mass spectrometry measurements o f the molecular weights of native and inhibited 3C protease have establishe d an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromom ethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhib ited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 res idue. The bromomethylketonehydrazides were fairly weak inhibitors of chymot rypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture. (C) 1999 Academic Press.