Figure 5.

Nirmatrelvir alkyne derivatives inhibit SARS-CoV-2 M^pro via covalent reaction with Cys145. (a) Reaction of M^pro (bottom) with nirmatrelvir (1, center) and its alkyne derivative 13 (top); SPE-MS analysis indicates near-quantitative reaction of 1 or 13 with M^pro (∼500 Da mass shifts). (b) Addition of a 10-fold excess of alkyne 28 to the covalent complexes of M^pro with 1 or 13, followed by 30 min incubation; SPE-MS analysis of the mixtures reveals formation of a covalent complexes of M^pro with 28 only for the M^pro complex with 1 (∼66 Da mass shift, center), but not with 13 (top), as compared with unreacted M^pro (bottom). (c) SPE-MS analysis of the mixtures of the covalent complexes of M^pro with 1 or 13 with 28 indicates that 28 reacts slowly with the M^pro complex with 1 (∼66 Da mass shift, center), but not with 13 (top), as compared with unreacted M^pro (bottom). M^pro assays were performed using SPE-MS as described in the Experimental Section employing SARS-CoV-2 M^pro (2.0 μM) in buffer (20 mM HEPES, pH 7.5).^43,62