In a recent study posted to the bioRxiv* preprint server, researchers introduced a phenotypic screening assay for identifying compounds with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibition potential based on caspase 3/7 activation.
Study: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform. Image Credit: Nhemz/Shutterstock
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Several types of assays have been developed for the detection of SARS-CoV-2 variants, including antibody-based assays for SARS-CoV-2 antigen detection and genetically modified assays; however, the assays have several drawbacks, such as manual handling requirements or a narrow range of detection. Therefore, robust and broad yet simple to perform cell culture assays are required to identify anti-SARS-CoV-2 drugs.
About the study
In the present study, researchers developed a high-throughput SARS-CoV-2 phenotypic screening assay for identifying anti-SARS-CoV-2 compounds based on the activation of caspase 3/7.
The human colon adenocarcinoma (Caco-2) cell line Caco-2-F03 was used for the cell culture experiments. Additionally, Caco-2 cells were obtained from several sources and designated as Caco-2A, -2B, and -2C to discriminate the authors’ original Caco-2 cell line (Caco-2-F03) from the others. The team compared Caco-2F03 to other cell culture models that could be used for identifying potential anti-SARS-CoV-2 drugs.
The susceptibility of Calu-3 (human lung carcinoma cell line), Caco-2-F03, A549-ACE2 (angiotensin-converting enzyme 2) and Vero cells to SARS-CoV-2 strains such as G614, D614, Alpha, Beta, and Delta were compared and confirmed by S immunostaining. Further, caspase activity in the Caco-2-F03 cell line infected with different SARS-CoV-2 isolates was assessed at 24- and 48 hours post-infection (hpi).
The kinase inhibitor library comprising 1796 kinase inhibitors was screened for drug compounds that decreased SARS-CoV-2-induced caspase activation by >90%, including known and potential drugs such as dual specificity protein kinase (CLK-1), phosphoglycerate dehydrogenase (PHGDH), and colony-stimulating factor 1 receptors (CSFR) inhibitors. Remdesivir (10 µM) was used as a positive control. The team also investigated if combined therapy of NCT-503 and 2DG could further enhance anti-SARS-CoV-2 effects compared to single drug therapy.
Caspase 3/7 activity and SARS-CoV-2 S staining by different drug candidates were compared to those of drugs with known anti-SARS-CoV-2 efficacy such as remdesivir, EIDD-1931, ribavirin, nirmatrelvir, and nafamostat using G614-infected Caco-2-F03 cells. The validity of caspase 3/7 assay results was confirmed by comparing anti-SARS-CoV-2 nucleocapsid (N) protein titers and Western Blot analysis.
Air-liquid interface (ALI) cultures of bronchial epithelial (HBE) cells were examined for cellular disruption during SARS-CoV-2 infection by transepithelial electrical resistance (TEER) and SARS-CoV-2 cytotoxicity was assessed based on lactic dehydrogenase (LDH) release. SARS-CoV-2 genomic RNA levels were assessed based on copy numbers detected by polymerase chain reaction (PCR).
Results
Caco-2-F03 demonstrated the best performance since it contained a stable phenotype susceptible to SARS-CoV-2 [indicated by high SARS-CoV-2 S levels and cytopathic effect (CPE) formation] and did not produce false-positive hits due to drug-induced phospholipidosis. Caco-2-F03 cells remained SARS-CoV-2-permissive for 30 passages with high levels of cellular SARS-CoV, SARS-CoV-2 receptor ACE2 and transmembrane serine protease 2 (TMPRSS2).
Caspase-3/7 showed six-fold to eight-fold higher activity compared to caspase-8 and 9, indicating greater robustness therefore, caspase 3/7 detection was chosen for the screening. Caco-2-F03 cell infection with different SARS-CoV-2 isolates effectively activated caspase 3/7, reflected by SARS-CoV-2 S and RNA levels. Caspase 3/7 activity enabled monitoring of SARS-CoV-2 replication in cell lines such as Vero cells and replication of human coronaviruses (hCoVs) such as the Middle East Respiratory syndrome CoV (MERS-CoV), HCoV-229E and SARS-CoV in CaCo-2-F03 cells.
Further, SARS-CoV-2-induced caspase 3/7 activation and CPE were observed in human pluripotent cardiomyocytes (CMS) and hepatocytes. Caspase 3/7 enabled the evaluation of neutralizing antibody (nAb) titers in sera of seven donors obtained 14 days after the second messenger RNA (mRNA)-1273 vaccination. Greater G614 neutralization was observed than Alpha and Gamma neutralization and the remdesivir-resistant strain demonstrated enhanced sensitivity to ribavirin and EIDD-1931. Gly671Ser substitution in the RNA polymerase probably mediated remdesivir resistance.
In the screening, 81 compounds with anti-SARS-CoV-2 activity were identified with the most hits by CaMK (calmodulin-dependent protein kinase), mTOR (mammalian target of rapamycin), ULK (unc-51 like autophagy activating kinase 1), CLK-1, TOPK (T-LAK cell-originated protein kinase), CSF-1R, and PAK (p21-activated kinases). NCT-503 (PHGDH inhibitor) inhibited Delta- and Omicron-induced caspase 3/7 activation and also inhibited SARS-CoV-2 replication in ALI cultures and the activity of NCT-503 increased on combining with the 2-deoxy-D-glucose (2DG, a hexokinase 2 inhibitor).
Overall, the study findings showed that caspase 3/7 activation by different SARS-CoV-2 variants (including remdesivir-resistant strains and other hCoVs) could be used as a simple phenotypic high-throughput screening platform for identifying anti-SARS-CoV-2 drug candidates with a broad range of cell culture models, independent of CPE.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Journal references:
- Preliminary scientific report.
Denisa Bojkova, Philipp Reus, Leona Panosch, et al. (2022). Identification of novel antiviral drug candidates using an optimized 2 SARS-CoV-2 phenotypic screening platform. bioRxiv. doi: https://doi.org/10.1101/2022.07.17.500346 https://www.biorxiv.org/content/10.1101/2022.07.17.500346v1
- Peer reviewed and published scientific report.
Bojkova, Denisa, Philipp Reus, Leona Panosch, Marco Bechtel, Tamara Rothenburger, Joshua D. Kandler, Annika Pfeiffer, et al. 2023. “Identification of Novel Antiviral Drug Candidates Using an Optimized SARS-CoV-2 Phenotypic Screening Platform.” IScience 26 (2). https://doi.org/10.1016/j.isci.2023.105944. https://www.cell.com/iscience/fulltext/S2589-0042(23)00021-4.
Article Revisions
- May 18 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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