Potent small molecule inhibitors against SARS-CoV, MERS-CoV and SARS-CoV-2

In a recent study published on bioRxiv*preprint server, researchers in the United States have identified several potent pan-coronavirus (CoV) inhibitors.

CoVs are single-stranded, positive-sense, enveloped RNA viruses of the family Coronaviridae. The viral life cycle begins with the attachment of a trimeric spike to cells, which is cleaved by furin-like proteases into S1 and S2 subunits. The virus uses the receptor binding domain (RBD) of S1 to bind to the host cell receptor, angiotensin converting enzyme 2 (ACE2).

The spike is exposed on the viral surface and is the primary target for vaccine development and neutralizing antibodies (nAbs). However, the RBD is less conserved among CoVs. Therefore, nAbs against SARS-CoV react poorly with SARS-CoV-2. In addition, the RBD harbors many mutations, some of which decrease the effectiveness of antibodies and vaccines. Thus, RBD might not be ideal for developing pan-CoV inhibitors.

To study: Discovery of highly potent small molecule pan-coronavirus fusion inhibitors. Image Credit: Design_Cells / Shutterstock

The study and the conclusions

In the current study, researchers reported small molecules with inhibitory activity against SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome (MERS)-CoV. First, they screened a set of carboxylic acid (COOH)-containing molecules, now called NBCoV, and identified 20 compounds that were not pa-atry ininterference compounds (PAINS).

The antiviral activity of these NBCoVs was evaluated in two cell lines, 293T-ACE2 and A549-AT cells, infected with SARS-CoV-2 WA-1 pseudoviruses pretreated with increasing concentrations of NBCoV. In parallel, the 50% cytotoxicity concentration (CC₅₀) was also evaluated in both cell lines. One of the compounds, NBCoV63, exhibited a half-maximal inhibitory concentration IC₅₀ of 80 nM and 55 nM in 293T-ACE2 and A549-AT cells, respectively, and a CC₅₀ of 50 μM in both cell lines.

NBCoV35 and NBCoV37 exhibited low nanomolar activities in both cell lines. NBCoV37 was more cytotoxic for 293T-ACE2 cells, while NBCoV35 had comparable cytotoxicity in both cell types. The antiviral activity of NBCoV36 was weaker than that of the other three molecules. The remaining molecules had little or no anti-CoV activity.

In addition, the team tested the effectiveness of the best performing NBCoVs (35, 36, 37 and 83) against SARS-CoV-2 D614G pseudoviruses and Gamma, Delta and Omicron variants BA.1 and BA.4 /5 of concern (VOC). NBCoV63 was most potent against SARS-CoV-2 variants tested, with IC₅₀ values ​​of 34-96 nM and 26-105 nM in 293T-ACE2 and A549-AT cells, respectively.

NBCoV35 inhibited Omicron BA.4/5 best in both cell lines, but was weaker against Gamma. NBCoV37 also showed antiviral activity against all variants tested, while NBCoV36 had weaker activity. Additionally, the team tested these compounds in Calu-3 cells, permissive to SARS-CoV, MERS-CoV and SARS-CoV-2. Calu-3 cells were infected with SARS-CoV-2 strain WA-1 or Omicron BA.4/5 variant.

As before, NBCoV63 retained its excellent anti-CoV activity against both, whereas NBCoV 35–37 had a higher CI₅₀ values ​​in Calu-3 cells than in other cell lines. NBCoVs have also been evaluated for activity against SARS-CoV and MERS-CoV pseudoviruses. Again, NBCoV63 was the strongest inhibitor against SARS-CoV and MERS-CoV. NBCoVs (35–37) also had anti-SARS-CoV activity.

In contrast, NBCoV35 and NBCoV36 were less potent against MERS-CoV, but NBCoV37 was very potent. The team selected NBCoV63 based on the results and tested its activity against the live isolate of SARS-CoV-2 Hong Kong (HK), Delta and Omicron in the conventional plaque reduction assay. The IC₅₀ NBCoV63 values ​​were lower for the HK isolate and the Delta variant but higher against Omicron than those for the control compound, remdesivir.

The team noted that NBCoV63 also blocked SARS-CoV-2-mediated cell-cell fusion, indicating its ability to disrupt SARS-CoV-2 spike-ACE2 binding and cell-cell fusion. Slip-based docking of the prefusion tip with active NBCoV63 or inactive NBCoV66 revealed salt bridges/hydrogen bonds between the COOH moiety of NBCoV63 and tip residues K947 and K776.

These interactions between NBCoV66 and the spike were missing due to the absence of COOH moiety in the inhibitor. These results were validated using single mutation pseudoviruses (K947D, K776D, K947L or K776L). This revealed no antiviral activity of NBCoV36 and NBCoV37 against all mutants at the highest concentration tested. On the other hand, IC₅₀ NBCoV63 and NBCoV35 values ​​were more than 15 times elevated against mutants compared to wild type pseudovirus.

Finally, the team looked at the absorption, distribution, metabolism, and excretion (ADME) profile of NBCoV63, assessed by a company, Cyprotex US, LLC. The data suggested that NBCoV63 was less soluble and a low clearance compound, with a half-life greater than 180 minutes. It was 99.5% plasma protein bound and highly tolerant to cytochrome P 450 (CYP) mediated metabolism.

conclusion

In summary, the team identified NBCoV63 as a pan-CoV inhibitor with consistently high potency against SARS-CoV, MERS-CoV, and SARS-CoV-2 and its VOCs. Notably, it was just as effective as remdesivir against the SARS-CoV-2 HK isolate and the Delta variant, but less effective against the Omicron variant. It also inhibited SARS-CoV-2-mediated cell-cell fusion in a dose-dependent manner. ADME data revealed drug-like properties, but its solubility needs to be improved.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.