Fig. 3. β-Lactams inhibit Mpro. IC50s for (A) 1 (penicillin V sulfone C3 benzyl ester) and (B) 2 (C6-methoxy penicillin G sulfoxide C3 p-nitrobenzyl ester) determined using SPE-MS; data are a mean of technical duplicates with (a) 30 min and (b) 60 min preincubation. IC50s are means of two independent repeats each composed of technical duplicates (n = 2 ± SD). Proposed reaction of 1 (C) and 2 (D) with Mpro. (E) A single molecule 1 covalently modifies Mpro. (F) 2 does not efficiently modify Mpro through covalent reaction. Conditions: 1 μM Mpro, 20 μM β-lactam, 20 mM HEPES, pH 7.5, 50 mM NaCl. (G) 1 does not covalently modify Mpro preincubated with N3, suggesting 1 reacts with Cys-145. (H) 2 does not efficiently react with Mpro preincubated N3. Conditions: 1 μM Mpro preincubated with 3 μM N3, 20 μM 1 or 2, 20 mM HEPES, pH 7.5, 50 mM NaCl. Black spectra: wild-type Mpro (33 796 Da).
