Since 2020, many compounds have been investigated for their potential use in the treatment of SARS-CoV-2
infection. Among these agents, a huge number of natural products and FDA-approved drugs have been evaluated as potential therapeutics for SARS-CoV-2 using virtual screening and docking studies. However, the identification of the molecular targets involved in viral replication led to the development of rationally designed anti-SARS-CoV-2 agents. Among these targets, the main protease (Mpro) is one of the key enzymes needed in the replication of the virus. The data gleaned from the crystal structures of SARS-CoV-2 Mpro complexes with small molecule covalent inhibitors has been used in the design and discovery of many highly potent and broadspectrum Mpro inhibitors. The current review focuses mainly on the covalent type of SARS-CoV-2 Mpro inhibitors. The design, chemistry, and classification of these inhibitors were also in focus. The biological activity of
these inhibitors, including their inhibitory activities against Mpro, their antiviral activities, and the SAR studies,
were discussed. The review also describes the potential mechanism of the interaction between these inhibitors
and the catalytic Cys145 residue in Mpro. Moreover, the binding modes and key binding interactions of these
covalent inhibitors were also illustrated. The covalent inhibitors discussed in this review were of diverse
chemical nature and origin. Their antiviral activity was mediated mainly by the inhibition of SARS-CoV-2 Mpro,
with IC50 values in the micromolar to the nanomolar range. Many of these inhibitors exhibited broad-spectrum
inhibitory activity against the Mpro enzymes of other coronaviruses (SARS-CoV-1 and MERS-CoV). The dual
inhibition of the Mpro and PLpro enzymes of SARS-CoV-2 could also provide higher therapeutic benefits than
Mpro inhibition. Despite the approval of nirmatrelvir by the FDA, many mutations in the Mpro enzyme of SARSCoV-
2 have been reported. Although some of these mutations did not affect the potency of nirmatrelvir, there is
an urgent need to develop a second generation of Mpro inhibitors. We hope that the data summarized in this
review could help researchers in the design of a new potent generation of SARS-CoV-2 Mpro inhibitors