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

To determine the SARS-CoV M pro and SARS-CoV-2 M pro substrate preferences, we natural and over 100 unnatural amino acids, Figure 1 ). We incorporated glutamine at the P1 position, because the available crystal structures of SARS-CoV M pro revealed that only glutamine (and, at a very small number of cleavage sites, histidine) residue can occupy the S1 pocket of this enzyme. [9, 12] The imidazole of His163, located at the very bottom of the S1 proteases, which explains less stringent specificity. [11] The S2 pocket can form hydrophobic interactions with P2 residues that are not only limited to leucine. The S3 pocket of SARS M pro . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.07.981928 doi: bioRxiv preprint is not very well defined which is also reflected in our P3 substrate specificity profile. The S4 pocket can be occupied by small residues due to crowded cavity formed by Pro168, L167 at the bottom and T190, A191 at the top wall. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.07.981928 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.07.981928 doi: bioRxiv preprint To validate the results from library screening, we designed and synthesized ACClabeled substrates containing the most preferred amino acids in each position. Then, we measured the rate of substrate hydrolysis relevant to each protease (Figure 3) . The data clearly demonstrate that SARS-CoV M pro and SARS-CoV-2 M pro exhibit the same activity toward tested substrates. The results are consistent with the HyCoSuL screening data. The most preferred substrate, Ac-Abu-Tle-Leu-Gln-ACC, is composed of the best amino acids in each position. Kinetic parameters were determined for the two best substrates (Ac-Abu-Tle-Leu-Gln-ACC, Ac-Thz-Tle-Leu-Gln-ACC) and one containing the best recognized natural amino acids (Ac-Val-Lys-Leu-Gln-ACC) ( Table 1 ) toward SARS-CoV-2 M pro . Due to substrates precipitation due to high concentration needed in the assay, kinetic parameters toward SARS-CoV M pro could not be determined. Analysis of kinetic parameters revealed that these three substrates differ in the k cat value, while K M values are comparable. The copyright holder for this preprint (which was not peer-reviewed) is the . In summary, we established substrate specificity profiles at the P4-P2 positions of the SARS-CoV M pro and SARS-CoV-2 M pro proteases using a combinatorial approach. Our data clearly demonstrate that these two enzymes display very similar substrate preferences.