The World Health Organization (WHO), on March 12, 2020, declared a pandemic state of the global epidemic caused by a new coronavirus, SARS-CoV-2. Identifying the Main protease (Mpro) of SARS-CoV-2 as a drug target led to a speed-up drug design and discovery process. However, an effective algorithm is needed to indicate the activity against (Mpro) during virtual screening. In our studies, we proposed a virtual screening protocol combining molecular modeling and machine learning techniques that will allow us to discover novel inhibitors of SARS-CoV-2 Mpro. Based on results from virtual screening, potential Mpro inhibitors were selected for synthesis and further in vitro evaluation. The effectiveness of small-molecule compounds, derivatives of mono-, di- and tri-heterocyclic azole systems with various substituents of an amine, amide, or ester nature were evaluated in the test for antiviral activity. Compounds were first tested on Vero E6 cells to select non-toxic doses on SARS-CoV-2 infected cells. Next, we studied the anti-SARS-CoV-2 activity of selected compounds on infected Vero E6 cells. The most active compound showed 52.6% inhibition of SARS-CoV-2 replication at the dose of 20 µM. In summary, the application of the proposed virtual screening protocol allowed the selection of new compounds to be used as a starting point for developing SARS-Cov-2 Mpro inhibitors.

The project is co-financed by the Polish National Agency for Academic Exchange (PPN/BIT/2021/1/00056/U/00001), Italian Ministry of Foreign Affairs and International Cooperation Executive Programme of Scientific and Technological Cooperation between the Italian Republic and the Republic of Poland (PO22MO07), JUMC grants N42/DBS/000217, N42/DBS/000303.