Project Summary/Abstract: Development of ALG-097431 as a Broad-Spectrum SARS-CoV-2 3CLpro Inhibitor Inhibition of viral proteases with small molecule drugs is a proven successful therapeutic approach and is currently used for the treatment of HCV and HIV infection. The SARS-CoV-2 main, 3-chymotrypsin-like cysteine protease (Mpro, 3CLpro) represents another promising viral protease target for antiviral therapy with no human homolog. The discovery and development of small molecule, covalent binding inhibitors of the 3CLpro is the focus of this program. Together with our collaborators at the Centre for Drug Design and Discovery (CD3) and the Rega Institute at KU Leuven, Belgium, we have developed multiple biochemical and cell-based assays to profile compounds and have access to the hamster model of SARS-CoV-2 infection. Through structure-based optimization, combined with selectivity and SARS-CoV-2 3CLpro biochemical enzymatic assays, we have identified a series of highly selective and potent 3CLpro inhibitors. The inhibition of replication of SARS-CoV-2 by these compounds was confirmed in A549 cells expressing the human ACE-2 receptor with compounds demonstrating low nanomolar EC50 values. No cytotoxicity was observed in A549 cells at concentrations up to 100 μM. The activity of these compounds extended to other human coronaviruses such as the alpha-coronavirus 229E and the beta-coronavirus OC43, demonstrating broad-spectrum anti-coronaviral activity. A representative compound, ALG-097111, was taken into an in vivo efficacy model in which hamsters were challenged with SARS-CoV-2. At two days post-infection, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed in ALG-097111-treated animals. Further compound optimization has resulted in the development of ALG-097431. This compound demonstrates an IC50 of 4 nM vs. the 3CLpro enzyme and an EC50 of 39 nM (selectivity index of >1200) in a A549-ACE2/B.1.1.7 cell-based assay. Further, ALG-097431 exhibits potent activity in a primary human airway epithelial cell, air-liquid interface 3D-culture system (EC90 = 12 nM). The compound demonstrates a clean in vitro ADME-tox profile and shows good oral bioavailability in rats and dogs. Human efficacious dose- projections indicate the potential for twice-daily dosing with a 250 – 600 mg dose without the need for co- administration with ritonavir. The goals of this project are to advance ALG-097431 into human clinical trials while simultaneously optimizing further compounds for enhanced antiviral activity and PK properties. Initially this will involve establishing PK/PD correlations and conducting further selectivity analysis and more extensive PK analysis. Following exploratory safety pharmacology studies, GMP drug synthesis, formulation development and validation of bioanalytical methods will be initiated. Prior to the initiation of clinical ...