Inhibition of human cytomegalovirus protease by monocyclic beta-lactam derivatives: Kinetic characterization using a fluorescent probe

Recent reports have demonstrated the potential of monocyclic beta-lactam de rivatives as inhibitors of human cytomegalovirus (HCMV) protease. Investiga tion of the mechanism of inhibition by NMR and mass spectrometry has reveal ed the presence of an acylenzyme intermediate suggesting that beta-lactams are hydrolyzed by the enzyme and cause inhibition by competing with substra te. The potential of a fluorogenic beta-lactam derivative for convenient ki netic characterization of this mechanism has been evaluated using 4S-(4meth ylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl ) amide (1). Upon acylation of the enzyme, the fluorescent umbelliferone mo iety is released, allowing for continuous monitoring of the hydrolytic proc ess. Examination of a series of progress curves by numerical analysis has p rovided valuable information on acylation and deacylation rates which relat e to the IC50 values observed for beta-lactams. More importantly the potent ial of compound 1 as an active site titrating agent for HCMV protease has b een exploited, and a simple protocol for rapid determination of active enzy me is described. The data are consistent with the HCMV protease dimer being composed of two functional active sites. This titrating agent represents a n important tool that should significantly facilitate the characterization of this novel enzyme.