Inhibitors of protein-protein interaction between the human receptor ACE2 and the viral protein spike: a new frontier in the fight against SARS-CoV-2.

More than 4 years after its first appearance, the SARS-CoV-2 caused over 7 million of death globally, according to WHO, and, despite numerous efforts, the number of infections or re- infections continues to grow. Several efforts have been focused on vaccine development and the success of this approach in reducing deaths and hospitalization is increasingly evident. However, in addition to vaccines, development of therapy with specific inhibitors of viral replication is of paramount importance. The interaction between the spike (S) protein of SARS-CoV-2 and the human cell surface receptor angiotensin-converting enzyme 2 (ACE2) facilitates the virus's infectivity, enabling its entry into host cells. 1 Therefore, disrupting the binding between the S protein and the ACE2 receptor is promising as approach to impede virus entry. 2 Inhibitors targeting the protein-protein interaction between the S protein and human ACE2 are of significant interest as potential antiviral agents because this interaction initiates membrane fusion and virus entry, occurring at an accessible extracellular site. 3 While developing protein-protein interaction inhibitors with small molecules presents challenges compared to antibodies, small-molecule inhibitors could offer alternatives that are less susceptible to strain variations and mutations, suitable for oral or inhaled administration, and more controllable and less immunogenic. In this study, we identified a novel scaffold as a potent antiviral drug against SARS-CoV-2 by targeting well-established proteins crucial to the viral cycle. A recombinant RBD of SARS-CoV-2 and ACE2 receptor was utilized for HTS to identify molecules interfering with the interaction between the RBD and ACE2. Twenty-one candidates were selected from HTS based on a robust homogeneous time-resolved fluorescence (HTRF) assay. Among them, two hits were identified, demonstrating inhibitions of 51.4% and 25.6% and IC 50 values of 1 and 2.42 µM against ACE2 and 0.44 and 3.58 µM against RBD, respectively. However, both hits exhibited cytotoxicity against SARS-CoV-2 infected cells. Therefore, to mitigate the cytotoxicity and enhance druggability, analogues of these compounds were designed. The data from the biological assays will be presented and discussed.