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

In a recent study posted on Bio Rxiv*Preprint Server, US researchers have identified several potent pan-coronavirus (CoV) inhibitors.

CoVs are single-stranded, positive-sense, enveloped RNA viruses belonging to the Coronaviridae family. The viral life cycle begins with the attachment of the trimeric spike to the cell. This spike is cleaved into S1 and S2 subunits by a furin-like protease. 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 a prime target for vaccine development, Neutralizing antibody (nAbs). However, RBD is less conserved across his CoV. As a result, nAbs against SARS-CoV are poorly cross-reactive with SARS-CoV-2.In addition, RBD has many mutations, some of which decrease efficacy of Antibodies and Vaccines. Therefore, RBD may not be ideal for developing pan-CoV inhibitors.

study: Discovery of highly potent small-molecule pan-coronavirus fusion inhibitorsImage credit: Design_Cells / Shutterstock

Research and Findings

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 series of carboxylic acid (COOH)-containing molecules (hereafter referred to as NBCoV) and identified 20 compounds that were not. pAnn-aTo tell ofinterfering compoundss (pain).

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 pseudovirus pretreated with increasing concentrations of NBCoV . In parallel, 50% cytotoxic concentration (CC₅₀) was also evaluated in both cell lines. His one of the compounds, NBCoV63, showed an IC of half the maximal inhibitory concentration.₅₀ 80 nM and 55 nM in 293T-ACE2 and A549-AT cells, respectively, and CC₅₀ 50 μM in both cell lines.

NBCoV35 and NBCoV37 exhibited low nanomolar activity in both cell lines. NBCoV37 was more cytotoxic to 293T-ACE2 cells, whereas NBCoV35 was equally cytotoxic in both cell types. The antiviral activity of NBCoV36 was weaker than her other three molecules. The remaining molecules had little or no anti-CoV activity.

In addition, the team demonstrated efficacy of the highest performing NBCoVs (35, 36, 37, and 83) against SARS-CoV-2 D614G and suspected gamma, delta, and omicron BA.1 and BA.4/5 variants pseudoviruses. sex tested. (VOCs). NBCoV63 is the most potent against SARS-CoV-2 variants tested and 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 weaker against gamma. NBCoV37 also showed antiviral activity against all variants tested, whereas NBCoV36 showed lower activity. Tested on Calu-3 cells that are permissive to CoV-2. Calu-3 cells were infected with SARS-CoV-2 strain WA-1 or Omicron BA.4/5 variant.

As before, NBCoV63 retained good anti-CoV activity against both, but NBCoV 35–37 had higher IC₅₀ values ​​for Calu-3 cells over other cell lines. NBCoV was also evaluated for activity against SARS-CoV and MERS-CoV pseudoviruses. Again, NBCoV63 was the most potent inhibitor against SARS-CoV and MERS-CoV. NBCoV (35 – 37) also had anti-SARS-CoV activity.

In contrast, NBCoV35 and NBCoV36 were less potent against MERS-CoV, while NBCoV37 was highly potent. Based on the results, the team selected NBCoV63 and tested its activity against live SARS-CoV-2 Hong Kong (HK) isolates, Delta, and Omicron in conventional plaque reduction assays. I C₅₀ NBCoV63 values ​​were lower for HK isolates and Delta variants, but higher for Omicron than for the control compound remdesivir.

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

These interactions between NBCoV66 and Spike were absent due to the lack of COOH moieties in the inhibitors. These findings were validated using pseudoviruses with a single mutation (K947D, K776D, K947L, or K776L). This revealed no antiviral activity of NBCoV36 and NBCoV37 against all mutants at the highest concentrations tested. In contrast, IC₅₀ NBCoV63 and NBCoV35 values ​​were elevated more than 15-fold for mutants compared to wild type pseudovirus.

Finally, the team examined the absorption, distribution, metabolism and excretion (ADME) profile of NBCoV63 as assessed by Cyprotex US, LLC. The data suggested that NBCoV63 is a poorly soluble, poorly cleared compound with a half-life of >180 minutes. It bound 99.5% of plasma proteins and was highly resistant 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, SARS-CoV-2 and their VOCs. In particular, it was as effective as remdesivir against SARS-CoV-2 HK isolates and Delta variants, but less effective against Omicron variants. It also dose-dependently inhibited SARS-CoV-2-mediated cell-to-cell fusion. ADME data revealed drug-like properties, but its solubility needs improvement.

*Important Notices

bioRxiv publishes non-peer-reviewed, preliminary scientific reports and should not be considered conclusive, to guide clinical practice/health-related actions, or to be treated as established information .