Fig. 4. Evidence that γ-lactam 16a inhibits Mpro by selective reversible covalent reaction with Cys145. (A) γ-Lactam 16a does not covalently react with the covalent Mpro:nirmatrelvir alkyne derivative 2442 complex obtained via irreversible covalent reaction of Mpro Cys145 with 24,42 indicating that γ-lactams selectively react with the nucleophilic thiolate of Cys145 under the tested conditions; (B) addition of an excess of 2442 to a mixture containing the covalent Mpro:16a complex results in stoichiometric formation of the corresponding covalent Mpro:2442 complex, substantial levels of the Mpro:16a complex were not detected using SPE-MS implying that the reaction of γ-lactams with Mpro is reversible and/or that the acyl–enzyme complex is not stable towards hydrolysis. Assays were performed using SPE-MS, as described in the Experimental section, employing SARS-CoV-2 Mpro (3.0 μM), and, if appropriate, γ-lactam 16a (15 μM) and/or nirmatrelvir alkyne 2442 (50 μM) in buffer (20 mM HEPES, pH 7.5). Representative spectra of technical duplicates are shown.
