Wm. Kati et al., Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides, ARCH BIOCH, 362(2), 1999, pp. 363-375
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.