Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK (Alpha), South Africa (Beta), and India (Delta)

Abstract

We report the discovery of a series of benzoic acid-based inhibitors that show highly potent pancoronavirus activity. Some compounds also show complete inhibition of CPE (IC100) at 1.25 μM against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants initially identified in the UK and South Africa. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data show druglike characteristics, and in vivo PK in rats demonstrated excellent half-life (t½) of 11.3 h, mean resident time (MRT) of 14.2 h, and orally bioavailable. Despite the presence of ene-rhodamine moiety, we conclusively demonstrated that these inhibitors target the viral spike protein and are not promiscuous or colloidal aggregators. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.

Discovery of highly potent small molecule pan-coronavirus fusion inhibitors

“…Instead, we were confident that despite the presence of a PAINS scaffold in these molecules, their antiviral activity must be due to the distinct pharmacophores of the molecules. We conclusively established the structure-activity relationship compounds used as controls, they showed no antiviral activity at all 46 . We were confident about the SAR information from that study and used that to search compounds from commercial small molecule drug-like databases.…”

“…Peptide-based pan-coronavirus fusion inhibitors were recently shown to possess potent inhibitory activity against HIV-1, HIV-2, and SIV 45 . These encouraging results motivated us to screen a set of COOH-containing molecules, which we referred to as NBCoVs and tested them against SARS-CoV-2, SARS-CoV, and MERS-CoV in spike-pseudotyped antiviral assays 46 .…”

“…These controversies and counterarguments led nine American Chemical Society (ACS) editors to recommend the necessary steps to rule out any artifacts from PAINS-containing compounds in biological activities 57 . Even though we followed the recommendations of the ACS editors 57 and confirmed through concurrent and orthogonal experiments that NBCoV molecules are not promiscuous and aggregators 46 , we decided not to pursue those leads to avoid any future controversy. Instead, we were confident that despite the presence of a PAINS scaffold in these molecules, their antiviral activity must be due to the distinct pharmacophores of the molecules.…”

“…It facilitates the infection of adjacent cells without producing free viruses and, at the same time, contributes to tissue damage and syncytia formation. We previously described a novel cellto-cell fusion inhibition assay using Jurkat cells expressing a luciferase gene and the wild-type S gene from the SARS-CoV-2 Wuhan-Hu-1 isolate as donor cells and 293T/ACE2 cells as acceptor cells 46 . We used 293T/ACE2 cells cultured with or without Jurkat cells as a positive and negative control, respectively.…”

“…HEK293T/17 cells with a proviral backbone plasmid and an envelope expression vector by using FuGENE HD (Promega, Madison, WI) and following the manufacturer's instructions as previously described 46 . We used the HIV-1 Env-deleted proviral backbone plasmid pNL4-3 ∆Env -NanoLuc DNA and the Envs SARS-CoV-2 and its VOCs, SARS-CoV and the MERS-CoV to obtain the respective pseudoviruses.…”