PT  - JOURNAL ARTICLE
AU  - Wioletta Rut
AU  - Katarzyna Groborz
AU  - Linlin Zhang
AU  - Xinyuanyuan Sun
AU  - Mikolaj Zmudzinski
AU  - Bartlomiej Pawlik
AU  - Wojciech Młynarski
AU  - Rolf Hilgenfeld
AU  - Marcin Drag
TI  - Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging
AID  - 10.1101/2020.03.07.981928
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.03.07.981928
4099  - http://biorxiv.org/content/early/2020/06/08/2020.03.07.981928.short
4100  - http://biorxiv.org/content/early/2020/06/08/2020.03.07.981928.full
AB  - In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.Competing Interest StatementWroclaw University of Science and Technology has filed a patent application covering compounds: Ac-Abu-Tle-Leu-Gln-VS, Biotin-PEG(4)-Abu-Tle-Leu-Gln-VS and Cy5-PEG(4)-Abu-Tle-Leu-Gln-VS as well as related compounds with W.R. and M.D. as inventors.Abu2-aminobutanoic acid;2-Abz2-(amino)benzoic acid;3-Abz3-(amino)benzoic acid;ACC7-amino-4-carbamoylmethylcoumarin;Dab2,4-diaminobutyric acid;Dhtdihydrotryptophan;HyCoSuLHybrid Combinatorial Substrate Library;Ornornithine;RFUrelative fluorescence unit;D-PhgD-phenylglycine;Thzthiazolidine-4-carboxylic acid;Tletert-leucine;