High throughput screening for SARS-CoV-2 inhibitors targeting 5 helix bundle.

SARS-CoV-2 and other related viruses enter host cells via receptor recognition and membrane fusion. A crucial part of this is mediated by 5HB which is capable of binding to the viral spike heptad repeats (HR2) making 5HB a potential druggable target of virus entry. Thus, we constructed a 5-Helix Bundle (5HB) pentamer assay for the purpose of identifying potential inhibitors SARS-CoV-2 virus entry. Following implementation and optimization into a 1536 well format, we validated this assay via a pilot HTS and proved we were able to find small molecule inhibitors that appear to compete with the 5HB binding to HR2. This allowed us to push forward and complete the full HTS campaign testing 635,262 compounds. Upon completion of the 5HB pentamer HTS, we also tested and validated a monomer version of the 5HB assay against a pilot screen and then used it to help confirm on-target activity. This allowed for the selection of 130 compounds which were tested in dose titration format against the 5HB pentamer assay. The same compounds were tested in secondary cell-based assays for SARS2 and Machupo virus entry via a dual luciferase transient transfection system. We also incorporated a live/dead cytotoxicity counterscreen. At the conclusion of these screens, 41 compounds were found to be selective inhibitors of the 5HB pentamer assay. From these assays, 31 compounds and analogs were selected which were tested in both the pentamer and monomer assays. 5 compounds emerged which showed good potency in both assays which were then tested in the SARS pseudo virus assay to round out this exercise.