A Novel Cell-based System for the Search of SARS-CoV-2 ORF3a Inhibitors against COVID-19

Abstract The COVID-19 pandemic caused by SARS-CoV-2 has presented unprecedented challenges due to its rapid spread, mutability, high death rate, and long-term complications. Vaccines have shown efficacy but wane over time, and new variants emerge. Antiviral drugs offer an effective alternative, but their success has been variable, and the development of drug resistance is a concern. Hence, there is a continued need to identify novel antiviral drugs for the treatment of COVID-19 and its post-COVID complications. Through a comprehensive genome-wide study utilizing a surrogate fission yeast system, we have identified the ORF3a protein as a promising therapeutic target. ORF3a is known to play a significant role in viral pathogenesis, contributing to cellular damage in the lungs and kidneys, induction of the NLRP3 inflammasome and cytokine storm, and the severity of COVID-19. Specifically, our research demonstrates that ORF3a expression in lung and kidney epithelial cells induces innate cellular oxidative stress and proinflammatory immune responses, leading to NF-kB-mediated cytokine production of TNFα and IL-6, ultimately resulting in apoptotic cell death. We refer to these effects collectively as "the cytopathic ORF3a effects." Consistent with clinical observations linking the emergence of ORF3a mutants to COVID-19 severity, we have also observed distinct variations in the cytopathic activities of emerging ORF3a mutants. As oxidative stress and inflammation contribute to cell death, tissue damage, and the severity of COVID-19, and ORF3a triggers cytokine storm and upregulation of TNFα and IL-6, which are strong predictors of COVID-19 severity, targeting ORF3a's cytopathic effects becomes crucial for mitigating the impact of the disease. The objective of our study is to develop an integrated system utilizing fission yeast and human cells to identify human suppressive cellular proteins (hSCPs) and small molecule inhibitors (SMIs) that specifically target ORF3a. Our approach consists of two main aims: (1) conducting a genome-wide search to identify hSCPs capable of suppressing ORF3a, and (2) employing a fission yeast cell-based high-throughput screening (HTS) system to rapidly identify SMIs with therapeutic potential. We hypothesize that direct inhibition of ORF3a using an SMI will effectively counteract its cytopathic effects, mitigate tissue damage, and reduce the severity of COVID-19. Our research team comprises experts in yeast biology, virology, viral infection in BSL-3 containment, HTS drug discovery, and medicinal chemistry. The novelty of our project lies in the absence of known inhibitors targeting ORF3a, underscoring the significance of this study. In our preliminary HTS runs, we have identified a SMI S3080, an FDA-approved antiviral drug Etravirine, as an inhibitor of ORF3a in both fission yeast and mammalian cells, thereby supporting the feasibility of our proposed HTS study. Successful completion of this research will enable the identification of hSCPs and SMIs capable of inhibiting the cytopathic effects of ORF3a, thereby laying the foundation for the development of effective antiviral drugs against COVID-19.