The 3CLPRO inhibitor ALG-097111 potently inhibits SARS-CoV-2 replication in hamsters

Background: There is an urgent need for potent drugs for the treatment or prevention of SARS-CoV-2 infections. Inhibition of viral proteases has been proven a successful therapeutic strategy for infections with HIV and HCV. Most reported inhibitors of the SARS-CoV-2 3-chymotrypsin-like (3CL) cysteine protease also target cathepsin L;the latter is involved in the SARS-CoV-2 entry process. We aim to develop potent and selective 3CL protease inhibitors devoid of cathepsin L inhibition. Methods: Structure based optimization and biochemical profiling, resulted in ALG-097111, a potent and selective SARS-CoV-2 3CL protease inhibitor. ALG-097111 was profiled in vitro in SARS-CoV-2 and CoV-OC43 cellular assays. In vitro microsomal stability and in vivo PK evaluation in rodents, in presence of the CYP-inhibitor ritonavir, was followed by the evaluation of ALG-097111 in a SARS-CoV-2 infection model in hamsters. Results: ALG-097111 exhibits potent SARS-CoV-2 3CLpro activity (IC50 = 0.007 μM) with no associated cathepsin L inhibition (IC50 > 10 μM). This selectivity extended to other human proteases, displaying less than 50 % inhibition at 10 μM, as well as receptor and kinase panels. While ALG-097111 is stable in human and dog microsomes (t1/2= >60 min) and hepatocytes (t1/2= >360 min), ALG-097111 showed lower stability in hamsters specifically (t1/2= 15 min). Addition of ritonavir to the hamster microsome assay increased the in vitro half-life (t1/2= > 60 min). When administered subcutaneously with oral co-dosing of ritonavir, ALG-097111 shows high plasma and lung exposures. Dosing hamsters with ALG-097111, followed by intranasal SARS-CoV-2 infection led to a significant reduction of infectious virus titers by 3.7 log10 (TCID50/mg) and viral RNA by 3.5 log10 (RNA copies/mg) in the lungs as compared to the vehicle group. In the same experiment, the nucleoside analogue Molnupiravir (EIDD-2801), used as a reference inhibitor, reduced infectious virus titers by 4.1 log10 (TCID50/mg) and viral RNA load by 2.0 log10 (RNA copies/mg). Conclusion: We demonstrate that ALG-097111, a potent and selective inhibitor of the SARS-Cov-2 3CL protease that is devoid of any cathepsin L activity, reduces infectious virus titers in the lungs of SARS-CoV-2 infected hamsters to (almost) the detection limit. This validates the 3CL protease as an excellent target for the treatment of SARS-CoV-2 infections.