R5 - Identifying and Targeting Essential Host Pathways for Next Generation Antivirus Therapeutics Development

Introduction: :

Introduction: Although many very effective vaccines, such as recent mRNA vaccines for COVID-19 and quadrivalent flu vaccine, have contributed significantly to controlling viral infections and pathogenesis. However, the not fully protected individuals, especially some populations, such as children and older populations, and the potential immune escape of viral mutants have highlighted the utmost need to understand the virus life cycle and develop novel anti-virus therapeutics with broad antiviral activity and low drug resistance. Recently, discovering and targeting host factors has become a very attractive approach for antiviral therapeutics development. We proposed and have been working a novel strategy for anti-virus infections by blocking essential host-viral interactions and manipulating host immune responses by suppressing overreactive immune responses, such as cytokine storms. for future anti-virus therapeutics..

Materials and

Methods: : Material and

Methods: We developed a qFRET-based high-throughput screening assay consisting CyPet-SUMO1, YPet-RanGap1c, SUMOylation E1 activating enzyme Aos1/Uba2, E2 conjugating enzyme Ubc9, and conducted this HTS assay campaign for more than 220,000 compounds. The hits were selected according to the 3xSD below the average signal. The hits were validated and confirmed through SUMOylation assays in vitro and in cells. We then performed plaque assays of influenza viruses to determine the activities of the conformed SUMOylation inhibitor. For manipulating immune responses, we collaborated with NIAID/NIH to determine the effects of Sphigosine-1-phosphate receptor-1(S1P1) agonist, siponimod from Novartis, in the mouse models of SARS-CoV-2 and influenza virus infections for both survival and clinical behavior monitoring.

Results, Conclusions, and Discussions::

Results: We discovered a specific and potent SUMOyaltion inhibitor through our qFRET-based HTS and the SUMOylation inhibitor not only completely blocked IAV/IBV life cycles but also did not induce any drug resistance after a long-term selection. In contrast, both the current market drug oseltamivir and a viral endocytosis inhibitor Apilimod induce significant drug resistance mutations. More importantly, one of the in vitro identified-SUMOyaltion sites of the IAV/IBV M1 protein, critical for viral RNA genome packaging and virus particle assembly and required for the viral life cycles. In addition, we have demonstrated the novel immunosuppressant siponimod developed from our previous discovery can not only significantly reduce death of mice infected with SARS-CoV-2 viruses but also influenza viruses but also mitigated clinical symptoms, suggesting a successful approach for manipulating viral infections.

Conclusion: Novel strategies of identifying and blocking essential host pathways and prevention of over-reactive human immune responses for future anti-virus infections have been formed and supported by the experimental data in both SUMOylation and S1P1 lipid signalings.
Fig 1. A novel strategy for anti-virus infections by targeting host factors or immune responses. A. qFRET-based HTS for SUMOylation cascade inhibitor screening. B. The summary of HTS flow. C. Completely inhibition of IAV and IBV by the SUMOylation inhibitor. D. Significant rescue of mice infected with lethal dose of IAV by immunosuppressant siponimod. E. Siponimod reduced clinical symptom of mice infected with SARS-CoV-2 virus. F. Significant rescue of mice infected with lethal dose of SARS-CoV-2 virus by immunosuppressant siponimod.

Discussion: Although vaccines, such as a quadrivalent vaccine for influenza A&B viruses (IAV or IBV, respectively) and novel mRNA vaccines for SARS-CoV-2, have contributed significantly to the control of viral infections/severe conditions, vaccines are often less effective for younger and older populations. The current drugs for viral inhibition are often effective at the early stage of infections and not effective to viral mutants. Novel strategies are highly needed. Our approaches not only inhibit virus without potential drug resistance development but also prevent damages from host overreactive immune responses, representing novel methods of anti-virus therapeutics development in the future.

Acknowledgements and/or References (Optional)::