Discoverey of four SARS-CoV-2 Mpro inhibitors, boceprevir, calpain inhibitors II and XII and GC-376

Discoverey of four SARS-CoV-2 Mpro inhibitors, boceprevir, calpain inhibitors II and XII and GC-376

By Dr. Sanchari Sinha Dutta, Ph.D.Nov 3 2020

A team of scientists from the United States has recently revealed that small molecule inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease exhibit potent antiviral activity. These inhibitors show better efficacy when used in combination with antiviral medicine Remdesivir. The study is currently available on the bioRxiv* preprint server.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) pandemic, is a positive-sense RNA virus with a genome size of about 30 kb. The virus spread rapidly from person to person, primarily via respiratory droplets. Since its emergence in December 2019 in China, the deadly SARS-CoV-2 has infected more than 40 million people and claimed more than 1 million lives globally. Currently, no antiviral medication or vaccine is available to specifically and effectively treat SARS-CoV-2 infection.  

The SARS-CoV-2 genome encodes several structural and non-structural proteins, and many of these proteins are currently under investigation as potential antiviral targets. One such example is 3-chymotrypsin-like protease or main protease, which is a non-structural SARS-CoV-2 protein required for viral replication. Small molecule inhibitors of viral main protease, including boceprevir, calpain inhibitors II and XII, and GC-376, have been shown to inhibit SARS-CoV-2 replication in vitro assays. The crystal structure analysis studies have shown that these compounds stably and strongly interact with viral main protease.

Interestingly, calpain inhibitors II and XII exhibit more potent antiviral effects than GC-376, despite having weaker inhibitory effects against SARS-CoV-2 main protease. In in vitro setups, calpain inhibitors II and XII have been found to inhibit human cathepsin L.

Given these observations, the current study scientists hypothesized that stronger antiviral activities of calpain inhibitors II and XII may be attributed to their ability to inhibit host cathepsin L, which is known to facilitate the entry of SARS-CoV-2 into cells by cleaving and activating the viral spike protein.

Current study design

The scientists conducted pseudovirus neutralization assay and drug time-of-addition assay to investigate whether calpain inhibitors II and XII are capable of inhibiting both viral main protease and host cathepsin L.

Moreover, they determined the antiviral efficacy of boceprevir, calpain inhibitors II and XII, and GC-376 against a group of human coronaviruses, including highly infectious SARS-CoV-1 and Middle-East respiratory syndrome coronavirus (MERS-CoV) and seasonal HCoV-OC43, HCoV-NL63, and HCoV-229E. They also tested the efficacy of these compounds in combination with antiviral medicine Remdesivir.  

Important observations

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Through in vitro assays, the scientists observed that calpain inhibitors II and XII significantly inhibit the entry of pseudo-SARS-CoV-2 into Vero E6 cells by inhibiting cathepsin L. However, boceprevir and GC-376 failed to show such effects.

For the next set of experiments, they chose HCoV-OC43 as a candidate virus as it shares a similar mechanism as SARS-CoV-2 to enter host cells. By adding calpain inhibitor XII at different time points of viral replication, they observed that the compound can inhibit viral replication both at early and intermediate stages. In contrast, GC-376 inhibited viral replication only at the intermediate stage. These findings further confirm that calpain inhibitor XII inhibits both viral main protease (cleaves viral polyproteins during replication) and host cathepsin L (involved in the early stage of viral fusion), whereas GC-376 inhibits only viral main protease.

Using differential scanning fluorimetry assay wherein the melting temperature is measured to determine ligand-protein interaction, the scientists confirmed that boceprevir, calpain inhibitors II and XII, and GC-376 are capable of directly interacting with and inhibiting the enzymatic activity of main protease of SARS-CoV-1, MERS-CoV, and HCoV-OC43, and SARS-CoV-2. This indicates that all tested compounds have broad-spectrum antiviral effects.

In addition, all four tested compounds showed significant antiviral effects against different human coronaviruses in a dose-dependent manner. These compounds effectively prevented viral cytopathic effects (infection-induced structural alteration in host cells) and inhibited viral RNA synthesis. However, of all tested compounds, boceprevir showed relatively lower enzyme inhibitory effect and antiviral efficacy. Notably, the scientists observed an additive antiviral effect when these compounds were tested in combination with antiviral medicine Remdesivir.

Taken together, the current study identifies four small-molecule inhibitors of SARS-CoV-2 main protease (boceprevir, calpain inhibitors II and XII, and GC-376) as potential therapeutic candidates to contain viral transmission.

*Important Notice

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


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