PROJECT SUMMARY This COMPETITIVE REVISION application is being submitted to expand the scope of our ongoing NIH grant UH3HL141797 in order to leverage our human organ-on-a-chip (Organ Chip) microfluidic culture devices for the rapid development and assessment of potential therapeutic agents for COVID-19. Our ongoing UH3 grant supports the development of human Lung Chips as in vitro preclinical tools for rapid discovery of new therapeutics for viral pandemics caused by influenza. In recent studies, we showed that highly differentiated human cells in our Lung Chips, as well as human intestinal cells within Intestine Chips we developed, express high levels of ACE2 and TMPRSS2 that mediate SARS-CoV-2 virus (CoV2) infection. We also were able to infect these Organ Chips with CoV2 spike protein-expressing viral pseudoparticles (CoV2pp) that closely mimic the effects of native CoV2 virus when tested against multiple FDA approved drugs in cell-based assays. Human Lung Chips were also shown to be more stringent models for assessing potential COVID19 inhibitory activity as only a subset of these drugs significantly inhibited entry of the CoV2pp when administered under flow on-chip at their maximum concentration (Cmax) in human blood reported in clinical studies. Here, we propose to use human Intestine and Lung Chips in combination with computational discovery and synthetic chemistry approaches to develop broad-spectrum coronavirus therapeutics that would both help infected COVID19 patients now, and allow us to be prepared to prevent infections by related pandemic viruses that emerge in the future. In preliminary studies, multiple novel compounds designed with our computational tools exhibited significant inhibitory activities when tested against both CoV2pp and native CoV2 virus in cell based assays. Thus, our Specific Aims include: 1) to use computational and synthetic chemistry approaches to create new compounds that are predicted to inhibit infection by CoV2 virus and related coronaviruses, 2) to prioritize active molecules by analyzing their structure-activity relationships in cell-based assays infected with native CoV2 and related coronaviruses, 3) to identify lead compounds and effective doses based on inhibition of infection and host inflammatory responses in human Organ Chips using native coronaviruses, and 4) to carry out pharmacokinetic studies in mice coupled with iterative chemical synthesis and testing in cell-based assays to optimize the pharmaceutical properties and safety of the lead compounds, while retaining efficacy. Through this effort, we will identify new compounds that demonstrate broad spectrum inhibiting activities against CoV2 as well as related coronaviruses, and generate pharmacokinetic data necessary to move these drugs into animal validation studies and, eventually, human clinical trials. This work will also further establish the value of human Organ Chips as preclinical tools for accelerating drug development.

PROJECT NARRATIVE The COVID-19 crisis is the greatest public health challenge that exists today worldwide. This project, which leverages Organ-on-a-Chip microfluidic culture technology to mimic human organ-level responses to viral infection and drug therapies, has the potential to identify broad- spectrum coronavirus therapeutics that would both help infected COVID19 patients now and allow us to be prepared to prevent infections by related pandemic viruses that emerge in the future.