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In a recent study posted to the bioRxiv* preprint server, researchers investigated the potential of the calpain-2 (CAPN2) molecule as a therapeutic target against (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

Coronavirus disease 2019 (COVID-19) has caused considerable morbidity and mortality across the globe, warranting the development of therapeutic agents against SARS-CoV-2. The authors of the present study previously found that calpain inhibitor molecules effectively inhibited SARS-CoV-2 by targeting the M^pro (main protease) enzyme of the virus, critical for processing SARS-CoV-2 proteins.  

About the study

In the present study, researchers extended their previous analysis by investigating caplain-2 as a probable target for developing anti-SARS-CoV-2 agents.

The authors had recently screened for antiviral compounds using Vero E6 cells and recombinant SARS-CoV-2mNeonGreen virus, voltaren gel precio en mexico wherein several compounds demonstrated anti-SARS-CoV-2 efficacy, and the top 18 hits were validated in the present study. For the validation experiments, recombinant vesicular stomatitis virus (VSV) e-green fluorescent protein (eGFP) reporter viruses encoding either native VSV-G or the spike (S) protein of SARS-CoV-2 were used.

To investigate whether the MG132 compound that targeted host CPN2 could inhibit SARS-CoV-2, calpain inhibitor molecules such as ALLN, calpeptin, E-64d, and calpain inhibitor III were used since the molecules inhibit calpain with varying specificities and target the calpain family differently.

To assess CAPN2 involvement in COVID-19, a genetic knockout (KO) of the gene was performed using lentivirus-facilitated Cas9/CRISPR (clustered regularly interspaced short palindrome repeats) among MA104 cells, that express angiotensin-converting enzyme 2 (ACE2), critical for viral entry.

Further, western blot analysis was performed to validate CAPN2 KO efficiency. CAPN2 KO and wild-type (WT) cells were inoculated with SARS-CoV-2 S protein expressing chimeric VSV (VSV-SARS-CoV-2).

Standard plaque assays of VSV-SARS-CoV-2 infections were performed, and the time point at which CAPN2 exerted pro-viral effects was determined. KO and WT cells were inoculated with VSV-SARS-CoV-2, and the level of viral messenger ribonucleic acid (mRNA) was determined at 1.0- to 6.9 hours post-infection (hpi) by quantitative reverse-transcription-polymerase chain reaction (RT-qPCR).

Further, a recombinant SARS-CoV-2 strain with S mutations, D614G, E484K, and N501Y, was tested. Single-clone CAPN2 KO MA104 cells were generated and confirmed by Sanger sequencing. Furthermore, classical VSV-SARS-CoV-2 cold binding assays were performed to investigate whether VSV-SARS-CoV-2 adsorption was impacted negatively by the lack of caplain-2, and the potential S protein cleave by CAPN2 was evaluated.  

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Co-transfection experiments were performed using ACE2-expressing human embryonic kidney (HEK)293 cells and WA1 strain S protein, with CAPN2, furin, or TMPRSS2 (transmembrane serine protease 2), key proteases reported for cleaving the S protein for efficient host invasion. Subsequently, the team harvested the cellular lysates and quantified the intensities of whole-S protein and the cleaved product, subunit 2 (S2), followed by an assessment of ACE2 levels in CAPN2 KO and WT cells.

Results

Calpain inhibitors (calpain inhibitor III, calpeptin, E-64d, and ALLN) effectively inhibited VSV-SARS-CoV-2 with 50% inhibition effective concentration (EC₅₀) values <1.5 µM, but not Mpro, and contrastingly, calpain inhibitors showed no antiviral activity towards the native proteins of WT VSV. VSV-SARS-CoV-2 infections were significantly lowered among calpain-2 KO cells, and CAPN2 was critical for an initial step in the proliferation of SARS-CoV-2. CAPN2 promoted SARS-CoV-2 binding with cells of the host.

The findings indicated that calpain inhibitor molecules targeted a Mpro-independent pathway to effectively inhibit SARS-CoV-2 and calpain-2 as a new host factor that could be potentially targeted at the host cell invasion step to widen the therapeutic landscape of COVID-19. Most antiviral compounds tested demonstrated dose-dependent infection by VSV and VSV-SARS-CoV-2 inhibition among MA104 cells.

IMBX (3-isobutyl-1-methylxanthine), Nigericin, and brefeldin A showed EC₅₀ values below 2.0 μM against infections of VSV and VSV-SARS-CoV-2. Nitazoxanide showed SARS-CoV-2 inhibition, and MG132 was 100-fold selective in activity against VSV and VSV-SARS-CoV-2 with EC₅₀ values of 44 and 0.6 µM, respectively. No inhibitor except calpeptin showed cytotoxicity. VSV-SARS-CoV-2 mRNA was lowered by 4.0-fold in cells lacking CAPN2. VSV-SARS-CoV-2 plaques were 1.0 mm and 2.0mm in size in KO and WT cells, respectively. However, there were no significant differences in VSV plaque sizes in KO and WT cells.

GFP signals from VSV-SARS-CoV-2 were lowered among CAPN2 knockout cells at six hpi, indicating that CAPN2 aided the replication of SARS-CoV-2. Significantly lesser VSV-SARS-CoV-2 mRNA and S protein levels were noted in KO cells compared to WT cells. Of interest, non-significantly lower levels of mRNA were found in CAPN2 knockout cells, indicating that CAPN2 effects on SARS-CoV-2 probably depended on the S protein nature.

In addition, the levels of viral mRNA in KO and WT cells were similar in the presence of antibody incubation. Co-localization analysis of WGA (wheat germ agglutinin) and ACE2 demonstrated significantly lesser surface ACE2 levels among CAPN2 193 KO cells.

Conclusion

Overall, the study findings showed that calpain-2 positively regulated ACE2 presence at cellular surfaces, thus improving S-mediated binding and infectivity of SARS-CoV-2. Thus, CAPN2 could be considered a new pro-viral factor aiding SARS-CoV-2 host cell entry.

*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.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.