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 t erminal N-isopropylacetyl analogue 2 were investigated. Whereas these compo unds 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 techn iques demonstrated that MK 2 exists in solution as a relatively rigid and e xtended peptide structure and that the bulky side chains, notably the P3 te rt-butyl group, greatly contribute to maintaining this solution conformatio n. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provi ded an enzyme-bound conformation of MK 2 that was found to be similar to it s free solution structure and compares very well to the X-ray crystallograp hic structure of a related peptidyl inhibitor complexed to the enzyme. The fact that Ligands such as MK 2 exist in solution in the bioactive conformat ion accounts, in part, for the observed inhibitory activity of activated ke tone inhibitors bearing comparable peptidyl sequences. Comparison of the X- ray structures of HCMV protease apoenzyme and that of its complex with a re lated peptidyl alpha-ketoamide inhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme upon complexat ion of inhibitors such as 1 and 3. The above observations indicate that HCM V protease is a novel example of a serine protease that operates by an indu ced-fit mechanism for which complexation of peptidyl Ligands results in str uctural changes which bring the enzyme to a catalytically active (or optimi zed) form. Kinetic and fluorescence studies are also consistent with an ind uced-fit mechanism in which a considerable proportion of the intrinsic liga nd-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 pept idyl ligand-induced optimization of its catalytic functioning, are discusse d.