Characterization of the human cytomegalovirus protease as an induced-fit serine protease and the implications to the design of mechanism-based inhibitors

The conformational properties of the N-tert-butylacetyl-L-tert-butylglycyl-L-N-delta,N-delta-dimethylasparagyl-L-alanyl methyl ketone (MK) 1 and its terminal N-isopropylacetyl analogue 2 were investigated. Whereas these compounds are weak (mM IC50 range) inhibitors of the human cytomegalovirus (HCMV) protease, their activated carbonyl analogues are 1000-fold more potent (e.g., trifluoromethyl ketone 3, IC50 = 1.1 mu M). A combination of NMR techniques demonstrated that MK 2 exists in solution as a relatively rigid and extended peptide structure and that the bulky side chains, notably the P3 tert-butyl group, greatly contribute to maintaining this solution conformation. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provided an enzyme-bound conformation of MK 2 that was found to be similar to its free solution structure and compares very well to the X-ray crystallographic structure of a related peptidyl inhibitor complexed to the enzyme. The fact that Ligands such as MK 2 exist in solution in the bioactive conformation accounts, in part, for the observed inhibitory activity of activated ketone inhibitors bearing comparable peptidyl sequences. Comparison of the X-ray structures of HCMV protease apoenzyme and that of its complex with a related peptidyl alpha-ketoamide inhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme upon complexation of inhibitors such as 1 and 3. The above observations indicate that HCMV protease is a novel example of a serine protease that operates by an induced-fit mechanism for which complexation of peptidyl Ligands results in structural changes which bring the enzyme to a catalytically active (or optimized) form. Kinetic and fluorescence studies are also consistent with an induced-fit mechanism in which a considerable proportion of the intrinsic ligand-binding energy is used to carry out the conformational reorganization of the protease. Issues related to the rational design of both mechanism and nonmechanism-based inhibitors of HCMV protease,notably in light of the peptidyl ligand-induced optimization of its catalytic functioning, are discussed.