Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

Kuzikov, M.; Costanzi, E.; Reinshagen, J.; Esposito, F.; Vangeel, L.; Wolf, M.; Ellinger, B.; Claussen, C.; Geisslinger, G.; Corona, A.; Iaconis, D.; Talarico, C.; Manelfi, C.; Cannalire, R.; Rossetti, G.; Gossen, J.; Albani, S.; Musiani, F.; Herzog, K.; Ye, Y.; Giabbai, B.; Demitri, N.; Jochmans, D.; De Jonghe, S.; Rymenants, J.; Summa, V.; Tramontano, E.; Beccari, A. R.; Leyssen, P.; Storici, P.; Neyts, J.; Gribbon, P.; Zaliani, A.

doi:10.1101/2020.12.16.422677

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bioRxiv · 10.1101/2020.12.16.422677

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

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Abstract

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 M and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 [A], showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/422677v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17ca2aeorg.highwire.dtl.DTLVardef@19c5159org.highwire.dtl.DTLVardef@1a0adf6org.highwire.dtl.DTLVardef@1fd05cd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure.C_FLOATNO Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray. C_FIG

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Kuzikov, M., Costanzi, E., Reinshagen, J., Esposito, F., Vangeel, L., Wolf, M., Ellinger, B., Claussen, C., Geisslinger, G., Corona, A., Iaconis, D., Talarico, C., Manelfi, C., Cannalire, R., Rossetti, G., Gossen, J., Albani, S., Musiani, F., Herzog, K., Ye, Y., Giabbai, B., Demitri, N., Jochmans, D., De Jonghe, S., Rymenants, J., Summa, V., Tramontano, E., Beccari, A. R., Leyssen, P., Storici, P., Neyts, J., Gribbon, P., Zaliani, A.. 2020-12-16. Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen. https://doi.org/10.1101/2020.12.16.422677

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