In vitro screening of potential inhibitors targeting SARS-CoV-2 Main protease

In December 2019, a new virus named SARS-CoV-2 started spreading rapidly around the world, causing several waves of COVID-19, one of the deadliest pandemics after the Spanish flu of 1919. New vaccine strategies and new antiviral drugs were quickly developed. Up to now, due to SARS-CoV-2 persistence through new variants or in animal reservoirs, it remains essential to discover new effective antiviral drugs. Among the possible strategies, we proposed the design of small molecules to block a key protein of SARS-Cov-2 virus: the Main protease, denoted as Mpro or 3CLpro. This target has been considered a top choice as it has no human homolog and is well conserved among coronaviruses. Furthermore, Mpro is required to release viral proteins for particle assembly and is thus essential to the virus replication cycle. Expertise in targeting other viral proteases fuelled this approach, and the approval of the drug Paxlovid® validated the targeting Mpro as a strategy to limit SARS-CoV-2 replication. This thesis work has its root on a project dedicated to a virtual screening campaign of non-covalent inhibitors. Biochemical screening, highlighted the most promising hits which are investigated here through stability studies. Surprisingly, among the three compounds examined (M-A23, M-B28, M-C23), the most unstable compound was shown to be M-A23, which is also the compound with the highest inhibitory potential. Considering possible fragmentation pathways, a new project started with the aim of examining M-A23 derivatives, namely M-A23a, M-A23b and M-A23R, which corresponds to M-A23 in the reduced form, replicating the assays put in place in our original biochemical screening. This thesis experimental work started with the validation of the original protocols employing new fresh batches and sources of reagents employed for the biochemical assays, respectively two new proteins and a new labelled substrate. Extensive evaluation and troubleshooting work have been carried out on these new materials to ensure the consistency and repeatability within the historical standards. After this comparability exercise, the most appropriate protein and substrate conditions to carry out the FRET-based inhibition assay were identified and finally validated using two positive control compounds reported in the literature as inhibitors of Mpro activity. In perspective, the validation of the inhibition assay represents the starting point to confirm the inhibitory potential of M-A23 and to evaluate the activity of the new derivatives, eventually leading to the optimization of the hit structure.