Alkyne Derivatives of SARS-CoV‑2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule
inhibitor that, in combination with ritonavir, is used to treat infections
by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir
interrupts the viral life cycle by inhibiting the SARS-CoV-2 main
protease (Mpro), which is essential for processing viral
polyproteins into functional nonstructural proteins. We report studies
which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned
similarly to the electrophilic nitrile of nirmatrelvir can efficiently
inhibit isolated Mpro and SARS-CoV-2 replication in cells.
Mass spectrometric and crystallographic evidence shows that the alkyne
derivatives inhibit Mpro by apparent irreversible covalent
reactions with the active site cysteine (Cys145), while the analogous
nitriles react reversibly. The results highlight the potential for
irreversible covalent inhibition of Mpro and other nucleophilic
cysteine proteases by alkynes, which, in contrast to nitriles, can
be functionalized at their terminal position to optimize inhibition
and selectivity, as well as pharmacodynamic and pharmacokinetic properties.