ABSTRACT: Our overall objective is to advance to the clinic oral small molecule inhibitors of NSP4-mediated membrane-associated replication of SARS-CoV-2 and other RNA viruses of pandemic concern. Positive-strand RNA viruses replicate their genomes in association with intracellular membranes or novel membrane structures induced by specific viral non-structural (NS) proteins. SARS-CoV-2 also induces intracellular membrane structures to support its replication and its NSP4 protein has recently been implicated in this process. Inspection of NSP4 revealed an N-terminal amphipathic helix (AH). Addition of the latter to lipid vesicles in vitro specifically induced their aggregation, suggesting this segment may mediate part of NSP4’s membrane altering activity. Excitingly, STF-3577, an optimized analog of an inhibitor we previously identified against a similar function mediated by hepatitis C virus’ NS4B, prevents NSP4 AH-mediated lipid vesicle aggregation in a dose-dependent fashion with an IC50 of 480nM. Cryo electron microscopy and tomography of SARS-CoV-2 infected cells treated with STF-3577 revealed an impairment in the characteristic viral induced intracellular membrane rearrangements and associated nascent virions, along with a corresponding accumulation of possible precursor small individual membrane vesicles. Importantly, addition of STF-3577 to SARS-CoV-2 infected cells inhibited genome replication with an EC50 of 803nM with no effect on cell viability at the highest concentration tested (20 uM). No natural mutations have been observed in the NSP4 AH targeted by STF-3577. STF-3577 has high oral bioavailability, is well tolerated in 7-day repeat dosing, just two doses decreased virus lung titers >3 log in SARS- CoV-2-infected mice, and it has strong in vitro synergy with SARS-CoV-2 protease inhibitors. We hypothesize that: 1) STF-3577 represents an attractive lead molecule for entering IND-enabling studies; 2) a focused medicinal chemistry strategy can identify next generation/back up more potent analogs of STF-3577; 3) the inhibition of lipid vesicle aggregation assay represents an ideal biochemical assay to help guide the medicinal chemistry optimization of potency effort; 4) similar assays with candidate NSP4 peptides from other viruses can be used to guide the development of inhibitors targeting additional RNA viruses of pandemic concern; 5) STF- 3577 and its optimized analogs represent ideal combination partners for other direct-acting anti-SARS-CoV-2 agents (e.g., protease inhibitors); and 6) there may be a high barrier to the development of resistance to STF- 3577. We will test these hypotheses by: 1) optimizing STF-3577’s anti-SARS-CoV-2 potency and pharmacokinetics; 2) determining the in vivo activity of the optimized NSP4 inhibitors against SARS-CoV-2 in mice and hamsters; 3) expanding the virology data package; 4) nominating a NSP4 inhibitor IND candidate; and 5) exploring targeting NSP4 function in other RNA viruses of pandemic pote...