Discovery of new small molecules as anti-SARS-CoV-2 agents inhibiting ACE2-spike binding.

The severe and acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a plus-strand RNA virus closely related to SARS and MERS, rapidly spreading worldwide. This virus is responsible for an ongoing global pandemic of coronavirus disease 2019 (COVID-19) causing many casualties in the human population. For these reasons, it is imperative to find drugs able to solve this global health issue as fast as possible. Interaction between the spike (S) protein of SARS-CoV-2 and the cell surface receptor angiotensin-converting enzyme 2 (ACE2) is responsible of the infectivity of the host, allowing the entry of the virus into the host cells. Indeed, ACE2 acts as a ligand-receptor pair that initiates the viral attachment and cellular entry of the virus. In particular, the receptor-binding domain (RBD) of the S protein binds the membrane-distal portion of the ACE2 protein. Notably, current vaccines induce antibody responses to S protein, and most neutralising antibodies bind to the RBD. Therefore, targeting the binding between the S protein and the ACE2 receptor is a promising approach for virus entry. Inhibitors of the protein-protein interaction between the S protein and human ACE2 are of considerable interest as potential antiviral agents because the interaction between S and ACE2 initiate membrane fusion and virus entry, taking place at an accessible extracellular site. While development of protein-protein interaction inhibitors with small molecules is more challenging than antibodies, small-molecule inhibitors could offer alternatives that are less strain- and mutation-sensitive, suitable for oral or inhaled administration, and more controllable/less immunogenic. Indeed, this strategy has already been successfully applied to inhibit the viral entry of other viruses, as in the case of two FDA-approved drugs maraviroc and enfuvirtide. This work was aimed at finding potent antiviral drugs against SARS-CoV-2 by targeting well identified proteins essential to the viral cycle, using HTS techniques. A flagged version of the recombinant RBD of the SARS-CoV-2 and ACE2 receptor with two different tags constructed by collaborators IP Paris was used for the HTS to identify molecules that interfere with the interaction between the RBD and ACE2. 21 candidates were selected from HTS based on a robust HTRF assay and, among them, two hits were identified, showing % of inhibition of 51.4 and 25.6 and IC50 of 1 and 2.42 µM vs ACE2 and of 0.44 and 3.58 µM against RBD. However, the two hits proved to be cytotoxic against SARS-CoV-2 infected cells. Therefore, in order to reduce the cytotoxic profile of these compounds and to improve their druggability, some analogues were designed. The data coming from the biological assays will be shown and discussed.