Prediction and experimental validation of drug candidates that bind specifically to the SARS-CoV-2 receptor-binding domain to prevent entry via Angiotensin-converting enzyme 2 (ACE2)

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 (SARS-CoV-2) virus has created enormous public health and economic crises. The SARS-CoV-2 spike protein binds to its host receptor, human angiotensin-converting enzyme 2 (hACE2), through its receptor binding domain (RBD) and is proteolytically activated by human protease TMPRSS2 to process the SARS-CoV-2 spike protein to facilitate host cell entry. To identify new candidate drugs to inhibit binding of the RBD to hACE2 (the first step of virus entry), we used in silico screening (Phase VS from Schrӧdinger) to predict the RBD binding site and energetics for molecules in the ZINC FDA approved Drug Bank database (1,657 drugs), which includes clinical trial drugs approved by FDA. Using a pharmacophore (1 acceptor, 1 donor, 3 negatively charged, 1 positively charged) derived from the 2.5-Å X-ray structure SARS-CoV-2 RBD complexed with hACE2 (PDB ID: 6LZG), we found 29 unique hits. To refine the binding energies and bonding sites, we applied our DarwinDock complete sampling method, predicting that 4 of these 29 should bind strongly to the RBD site. Next, we tested 3 of these experimentally using FLOWER, a label-free optical whispering gallery mode sensing technique that can measure ligand-receptor binding affinities down to attomolar levels. We measure that methotrexate (MTX) and Diethylenetriamine pentaacetate (DTPA) bind with an inhibitory constant (Ki) of 0.2 pM in Tricine buffer, which is 1.832 million fold increase in binding strength compared to hACE2 (Kd: 366.4 nM). We then tested these ligands against common mutations: alpha, delta, and omicron. Our measured binding studies demonstrate that MTX provides inhibition against all four variants of SARS-coV-2. Indeed, the clinical and experimental data have shown that humans taking WT MTX are protected against infection by SARS-CoV-2.