ABSTRACT: Coronaviruses and togaviruses are major causes of morbidity and mortality worldwide, or are poised to initiate future deadly epidemics. These viruses express their own cysteine proteases, Mpro and nsP2pro, to process viral proteins early in their life cycles. These proteases have structural features distinct from human proteases, and they can be inhibited with uncharged substrate analogs, making them targetable by orally bioavailable drugs. The recent initiation of clinical trials for an oral SARSCoV2 Mpro inhibitor validates this class of targets. A diverse set of protease inhibitors are now needed to counter future resistance and provide treatments for other coronaviruses. Further, no protease inhibitors have entered clinical trials for togaviruses, including the human pathogens Chikungunya and Venezuelean Equine Encephaitis Viruses. The Lin, Bogyo, and Einav Labs have developed innovative approaches to create and test viral protease inhibitors, including (i) novel chemical structures that are cell permeable and specifically inhibit viral proteases, (ii) a rapid luciferase-based assay of protease inhibition in living cells, and (iii) unique human adult lung organoids (ALOs) to study the effects of drugs on virus replication and tissue injury. This team has already generated potent, selective, and orally bioavailable inhibitors of SARSCoV2 Mpro, named ML104m and ML1006m. These compounds exhibit EC50 < 1 μM for SARSCoV2 replication, high plasma and hepatocyte stability, and 10% oral bioavailability in mice, achieving plasma concentrations after oral dosing in the therapeutic range. The main goal of this project is to advance our lead SARSCoV2 Mpro inhibitors into the IND-enabling stage while generalizing our approach to other priority viruses. Aim 1 will optimize the therapeutic index (TI) and pharmacokinetic (PK) profile of SARSCoV2 Mpro inhibitors, and define their therapeutic potential. This includes (A) optimizing current leads for SARSCoV2 Mpro inhibition and generating enzyme-degrading derivatives with Project 4; (B) testing these leads for antiviral efficacy individually and in combination with compounds from Projects 3, 4, and 6; (C) testing efficacy in ALOs and efficacy, pharmacokinetics, and toxicity in mice; and (D) selecting IND candidates. Aim 2 will generalize our methods to other coronaviruses and key togaviruses, and study our inhibitors’ mechanisms of action. This includes (A) optimizing current leads for MERSCoV, (B) creating new inhibitors of CHIKV and VEEV nsP2pro, and (C) studying when protease inhibitors act within the viral life cycle and how resistance emerges. All Aims will leverage the activities of the Translational Acceleration, Pandemic Assistance, and Structural Biology cores of the SyneRx Center. If successful, this project will rapidly develop novel inhibitors for SARSCoV2 and other coronaviruses that are ready to enter clinical trials and that have the potential to be best-in-class. It will also estab...