Project Summary – Project 1 The COVID-19 pandemic is exerting an unprecedented health and socioeconomic impact. Despite the rapid generation of vaccines, vaccination hesitancy, emergence of viral variants of concern (VOC), and breakthrough cases have fueled continued community transmission, creating utmost urgency for the development of next- generation antiviral drugs. Typically narrow therapeutic windows define a clear directive to treat early and rapidly build high tissue exposure to maximize the benefit of pharmacological intervention in acute RNA virus infections. Groundbreaking expansion of diagnostic capacity combined with efficient contact tracing have established infrastructure to identify SARS-CoV-2 transmission before the onset of specific clinical signs. Oral bioavailability and high tolerability are in our view key drug properties to efficiently serve the needs of an outpatient group. Direct-acting antivirals appear to be best suited to combine high potency with an appropriate safety profile. We have previously identified the orally efficacious nucleoside analog inhibitor EIDD-2801/molnupiravir and established proof-of-concept for pharmacological suppression of SARS-CoV-2 spread to untreated contacts using the ferret transmission model. It is the overarching goal of this AC/DC project to harness our demonstrated expertise in the development of applicable therapeutics and de-risk a mechanistically and structurally distinct companion drug to molnupiravir and at least one independent alternative to the stage of formal development. In pilot studies, we have identified a novel uridine analog that shows outstanding oral pharmacokinetic (PK) properties in different species, broad-spectrum antiviral activity in cultured cells and primary human airway epithelium organoids, acts through induction of delayed polymerase chain termination, and is orally efficacious against SARS-CoV-2, VOC, and several other viral pathogens of pandemic potential. To broaden our anti-CoV portfolio, we will in a multi-pronged approach simultaneously advance an early-stage adenosine analog anti-CoV hit and, having pioneered recombinant SARS-CoV-2 reporter virus technology and miniaturized a high- throughput screening (HTS) protocol, launch an anti-SARS-CoV-2 campaign using our established high- biocontainment HTS facilities. To prepare for formal development, the nucleoside analog classes will be subjected to full mechanism of action characterization, resistance profiling, and assessment of off-target effects (aim1). Non-nucleoside anti-CoV hit candidates from HTS will be validated through direct and orthogonal counterscreens, viral target identification, and indication spectrum and mechanistic profiling (aim 2). Confirmed nucleoside analog and non-nucleoside CoV inhibitors will be synthetically optimized in iterative rounds, driven by antiviral potency, PK properties, tolerability, and insight into the molecular docking pose (aim 3). Emerging leads will be de-risk...