Refine
Year of publication
- 2023 (2) (remove)
Document Type
- Article (1)
- Doctoral Thesis (1)
Language
- English (2)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Keywords
- SARS-CoV-2 (1)
- Virologie (1)
- Virology (1)
Institute
- Medizin (2)
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
The impact of the Covid-19 pandemic called for rapid responses in face of unprecedented challenges. In this context, earning more about the causative agent SARS-CoV-2 becomes imperative. Therefore, clinical virus isolates were studied with focus on infectivity, replication kinetic, and caspase activity.
Firstly, clinical specimens collected from patients were tested for infectivity in cell culture. Combined with polymerase chain reaction results, a formula predicting infectivity in cell culture based on abundance of viral RNA was developed. Additionally, analysis of different specimen types, sources, and material, elucidate the question of infectivity. Here, infectivity was demonstrated in specimens derived from different parts of the respiratory tract, including specimens collected from deceased persons. A protocol for virus isolation on human airway epithelium in air-liquid interface culture was established.
Secondly, replication kinetics of 20 clinical isolates were compared, including a subset of seven sequenced isolates. All isolates replicated in the colon epithelial cell culture model. Within the subset, differences between isolates carrying the D614G amino acid exchange and with original spike protein were observed.
Lastly, elevated caspase activity was demonstrated in two cell culture models including human airway epithelium in air-liquid interface culture.
Subsequently, caspase inhibition by small-molecule compound Emricasan and its effects on the cytopathic effect observed in cell culture were studied. Here, increased cell survival in a colon epithelial cell line was shown with unimpaired virus replication. Elevated caspase activity was identified as early marker of infection and validated by testing across 20 clinical virus isolates.
This study offers information on infectivity that can help shape the understanding of transmission risk. As such, parts of the data collected here were used for validation of rapid antigen tests. The insights gained by studying caspase activity contributed in part to the development of a drug screening method by Bojkova et al.,41 thus aiding routine laboratory workflow. It was demonstrated that Emricasan exhibits no antiviral effect, while the finding of increased cell survival in cell culture could give rise to further research on prevention of tissue damage.