Discovery of the Cryptic Sites of SARS-CoV-2 Papain-like Protease and Analysis of Its Druggability.

In late 2019, a new coronavirus (CoV) caused the outbreak of a deadly respiratory disease, resulting in the COVID-19 pandemic. In view of the ongoing pandemic, there is an immediate need to find drugs to treat patients. SARS-CoV-2 papain-like cysteine protease (PLpro) not only plays an important role in the pathogenesis of the virus but is also a target protein for the development of inhibitor drugs. Therefore, to develop targeted inhibitors, it is necessary to analyse and verify PLpro sites and explore whether there are other cryptic binding pockets with better activity. In this study, first, we detected the site of the whole PLpro protein by sitemap of Schrödinger (version 2018), the cavity of LigBuilder V3, and DeepSite, and roughly judged the possible activated binding site area. Then, we used the mixed solvent dynamics simulation (MixMD) of probe molecules to induce conformational changes in the protein to find the possible cryptic active sites. Finally, the TRAPP method was used to predict the druggability of cryptic pockets and analyse the changes in the physicochemical properties of residues around these sites. This work will help promote the research of SARS-CoV-2 PLpro inhibitors.

Glycosylation is key for enhancing drug recognition into spike glycoprotein of SARS-CoV-2.

The emergence of COVID-19 caused by SARS-CoV-2 and its spread since 2019 represents the major public health problem worldwide nowadays, which has generated a high number of infections and deaths. The spike protein (S protein) is the most studied protein of SARS-CoV-2, and key to host-cell entry through ACE2 receptor. This protein presents a large pattern of glycosylations with important roles in immunity and infection mechanisms. Therefore, understanding key aspects of the molecular mechanisms of these structures, during drug recognition in SARS-CoV-2, may contribute to therapeutic alternatives. In this work, we explored the impact of glycosylations on the drug recognition on two domains of the S protein, the receptor-binding domain (RBD) and the N-terminal domain (NTD) through molecular dynamics simulations and computational biophysics analysis. Our results show that glycosylations in the S protein induce structural stability and changes in rigidity/flexibility related to the number of glycosylations in the structure. These structural changes are important for its biological activity as well as the correct interaction of ligands in the RBD and NTD regions. Additionally, we evidenced a roto-translation phenomenon in the interaction of the ligand with RBD in the absence of glycosylation, which disappears due to the influence of glycosylation and the convergence of metastable states in RBM. Similarly, glycosylations in NTD promote an induced fit phenomenon, which is not observed in the absence of glycosylations; this process is decisive for the activity of the ligand at the cryptic site. Altogether, these results provide an explanation of glycosylation relevance in biophysical properties and drug recognition to S protein of SARS-CoV-2, which must be considered in the rational drug development and virtual screening targeting S protein.