Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

Wioletta Rut; Katarzyna Groborz; Linlin Zhang; Xinyuanyuan Sun; Mikolaj Zmudzinski; Rolf Hilgenfeld; Marcin Drag

including the RNA-dependent RNA polymerase (RdRp, Nsp12) and helicase (Nsp13). The inhibition of M pro would prevent the virus from replication and therefore constitutes one of the potential anti-coronaviral strategies. [6] [7] Due to the close phylogenetic relationship between SARS-CoV-2 and SARS-CoV, [2, 8] their main proteases share many structural and functional features. From the perspective of the design and synthesis of new M pro inhibitors, a key feature of both of the enzymes is their ability to cleave the peptide bond following Gln. The SARS-CoV M pro cleaves polyproteins within the Leu-Gln↓(Ser, Ala, Gly) sequence (↓ indicates the cleavage site), which appears to be a conserved pattern of this protease. [6a, 7, 9] The ability of peptide bond hydrolysis after Gln residues is also observed for main proteases of other coronaviruses [10] but is unknown for human enzymes. This observation, along with further studies on the M pro , can potentially lead to new broad-spectrum anti-coronaviral inhibitors with minimum side effects. [11] In the present study, we applied the HyCoSuL (Hybrid Combinatorial Substrate Library) approach to determine the full substrate specificity profile of SARS-CoV M pro and SARS-CoV-2 M pro proteases. The use of natural and a large number of unnatural amino acids with diverse chemical structures allowed an in-depth characterization of the residue preference of the binding pockets within the active sites of the proteases. The results from library screening enabled us to design and synthesize ACC-labeled substrates with improved catalytic efficiency in comparison to a substrate containing only natural amino acids.