Evidence of a Conformational Change in the Human Cytomegalovirus Protease upon Binding of Peptidyl-Activated Carbonyl Inhibitors

Abstract

A series of N-tert-butylacetyl-l-tert-butylglycyl-l-Ngamma, Ngamma-dimethylasparagyl-l-alanyl-derived inhibitors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl ketone 3, and alpha-ketoamide 4, with respective KI values of 1.1, 0.1, 2100, and 0.2 microM) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fluorescence and CD properties of the enzyme. In the presence of saturating concentrations of compounds 1, 2, and 4, an identical blue shift in the fluorescence maximum of the enzyme upon specific tryptophan excitation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not involve formation of a covalent adduct as evidenced by 13C NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also observed. For both compounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset of inhibition of substrate hydrolysis correlate well with the kobs values of the time-dependent change in the emission spectra. Studies employing a double mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the principal fluorescence reporter. Taken together with information provided by our recent elucidation of the crystallographic structure of the enzyme [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagacé, L. (1996) Nature 383, 272], these observations are consistent with the inhibition of HCMV protease by peptidyl ketones involving a conformational change of the protease. A mechanism involving a kon limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct formation, is proposed for activated inhibitors such as 1 and 2.

Exploiting Ligand and Receptor Adaptability in Rational Drug Design Using Dynamics and Structure-Based Strategies

“…Also setting it apart from classical serine proteases is its existence as a dimer, which is believed to be the sole active species [12,13]. Finally, our spectroscopic studies [14] demonstrated that the binding of substrate-based competitive inhibitors results in a conformational change in the enzyme and that catalysis by HCMV protease can be best described in terms of an "induced-fit" model [15,16].…”

“…The expected boost in potency was observed when this approach was applied, in that compound 1 was found to inhibit HCMV protease with an IC 50 of 1.8 µM (Fig. 2) [14,25], as compared to an IC 50 of > 1000 µM found for the corresponding Mproduct peptide (Fig. 2) that possessed a C-terminal carboxylate.…”

On the application of simple explicit water models to the simulations of biomolecules

“…The conformational changes observed for the dimer-BILC 821 covalent complex were reproduced by the MD simulations of the noncovalent complex between the dimer and the peptidyl-activated carbonyl inhibitor selected from the literature, [94] suggesting that the HCMV protease is a novel example of serine protease that operates by an induced-fit mechanism. The MD simulations also showed that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole, in agreement with mutagenesis studies [98].…”

“…Fig. 6a compares the calculated B-factors for the dimeric form of assemblin complexed noncovalently to a peptidyl-activated carbonyl inhibitor, [94] very similar structurally to BILC 821, with the experimental B-factors for the covalent complex between BILC 821 and the dimer of assemblin [95]. Figs.…”

Selective Nonpeptidic Inhibitors of Herpes Simplex Virus Type 1 and Human Cytomegalovirus Proteases.

“…21,34,35) While a low molecular weight inhibitor with in vitro antiviral activity and stability in human plasma has been found, 29) there are likely to be problems involved with the peptidomimetic inhibitors, which display poor cell penetration and are easily subjected to degradation in cell culture. 21,35) These facts suggest that the low molecular weight inhibitors, compounds 1-4 are more suitable than the peptidomimetic inhibitors as antiherpesvirus agents.…”