AI Promoted Virtual Screening, Structure-Based Hit Optimization, and Synthesis of Novel COVID-19 S-RBD Domain Inhibitors

Coronavirus disease 2019 (COVID-19) is caused by a new, highly pathogenic severe-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) that infects human cells through its transmembrane spike (S) glycoprotein. The receptor-binding domain (RBD) of the S protein interacts with the angiotensin-converting enzyme II (ACE2) receptor of the host cells. Therefore, pharmacological targeting of this interaction might prevent infection or spread of the virus. Here, we performed a virtual screening to identify small molecules that block S-ACE2 interaction. Large compound libraries were filtered for drug-like properties, promiscuity and protein-protein interaction-targeting ability based on their ADME-Tox descriptors and also to exclude pan-assay interfering compounds. A properly designed AI-based virtual screening pipeline was applied to the remaining compounds, comprising approximately 10% of the starting data sets, searching for molecules that could bind to the RBD of the S protein. All molecules were sorted according to their screening score, grouped based on their structure and postfiltered for possible interaction patterns with the ACE2 receptor, yielding 31 hits. These hit molecules were further tested for their inhibitory effect on Spike RBD/ACE2 (19-615) interaction. Six compounds inhibited the S-ACE2 interaction in a dose-dependent manner while two of them also prevented infection of human cells from a pseudotyped virus whose entry is mediated by the S protein of SARS-CoV-2. Of the two compounds, the benzimidazole derivative <b>CKP-22</b> protected Vero E6 cells from infection with SARS-CoV-2, as well. Subsequent, hit-to-lead optimization of <b>CKP-22</b> was effected through the synthesis of 29 new derivatives of which compound <b>CKP-25</b> suppressed the Spike RBD/ACE2 (19-615) interaction, reduced the cytopathic effect of SARS-CoV-2 in Vero E6 cells (IC₅₀ = 3.5 μM) and reduced the viral load in cell culture supernatants. Early in vitro ADME-Tox studies showed that <b>CKP-25</b> does not possess biodegradation or liver metabolism issues, while isozyme-specific CYP450 experiments revealed that <b>CKP-25</b> was a weak inhibitor of the CYP450 system. Moreover, <b>CKP-25</b> does not elicit mutagenic effect on <i>Escherichia coli</i> WP2 uvrA strain. Thus, <b>CKP-25</b> is considered a lead compound against COVID-19 infection.