Discovery of Highly Potent Fusion Inhibitors with Potential Pan-Coronavirus Activity That Effectively Inhibit Major COVID-19 Variants of Concern (VOCs) in Pseudovirus-Based Assays

Abstract

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell–cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

Discovery of highly potent small molecule pan-coronavirus fusion inhibitors

“…We conclusively established the structure-activity relationship (SAR). We demonstrated that despite the presence of the same ene-rhodanine moiety in the 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 . However, the NBCoV molecules contain an ene-rhodanine scaffold, a well-known frequent hitter, which suggests that they might be pan assay interference compounds (PAINS) 47-49 .…”

“…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 cell-to-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.…”

“…Pseudoviruses capable of single-cycle infection were prepared by transfecting 8×10 6 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 ΔEnv -NanoLuc DNA and the Envs SARS-CoV-2 and its VOCs, SARS-CoV and the MERS-CoV to obtain the respective pseudoviruses.…”