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 (t_1/2) 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.

Keywords: COVID-19; MERS-CoV; SARS-CoV; SARS-CoV-2; fusion inhibitor; middle east respiratory syndrome (MERS); pan-coronavirus; severe acute respiratory syndrome (SARS).

Figure 1

The structures of all of compounds derived from 5-((5-4-cholorophenyl)furan-2-yl)methylene)-3-phenethyl-2-thioxothiazolidin-4-one that were tested against SARS-CoV-2, SARS-CoV, and MERS-CoV.

Figure 2

Validation of the SARS-CoV-2, SARS-CoV, and MERS-CoV pseudoviruses and ACE2 and DPP4 expression in different cell lines. (a) Immunoblot to validate the incorporation of the spike (S) protein in the SARS-CoV and SARS-CoV-2 pseudoviruses (b) Immunoblot to validate the incorporation of the spike (S) protein in the MERS-CoV pseudovirus Infection of cells expressing different levels of angiotensin-converting enzyme 2 (ACE2) with (c) the same amounts of SARS-CoV-2 or (d) SARS-CoV pseudovirus. (e) Infection of cells expressing different levels of dipeptidyl peptidase 4 (DPP4), with the same amounts of MERS-CoV pseudovirus. Columns represent the means ± standard deviations (n = 4). (f) Immunoblot of cell lysates to evaluate ACE2 expression (Blot 1 and Blot 2) and DPP4 expression (Blot 3). β-Actin was used as a loading control.

Figure 3

Evaluation of binding affinity of NBCoV1 and NBCoV2 to SARS-CoV-2 active trimer and SARS-CoV-2 S1 subdomain by SPR. Kinetics fitting curve (sensogram) of SARS-CoV-2 trimer to (a) NBCoV1; (b) NBCoV2. Kinetics fitting curve (sensogram) of SARS-CoV-2 S1 subdomain to (c) NBCoV1 and (d) NBCoV2. The binding affinity K_D and kinetic parameters k_on and k_off of (e) NBCoV1 and NBCoV2.

Figure 4

SARS-CoV-2 mediated cell-to-cell fusion inhibition assay. Jurkat cells expressing the SARS-CoV-2 full Spike wild-type gene from Wuhan-Hu-1 isolate and the luciferase gene were used as donor cells and the 293T/ACE2 as acceptor cells. Jurkat cells were preincubated for 1 h with different concentrations of NBCoV small molecules. Positive represent 293T/ACE2 cells cocultured with Jurkat cells in the absence of NBCoVs. Jurkat-Luc represents 293T/ACE2 cells cocultured with Jurkat cells expressing the luciferase gene only, in the absence of NBCoVs. Columns represent the means ± standard deviations (n = 2–4).