Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19

Abstract

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

The Vascular Endothelium and Coagulation: Homeostasis, Disease, and Treatment, with a Focus on the Von Willebrand Factor and Factors VIII and V

“…Current treatment of certain congenital coagulopathies is based on the use of extended half-life exogenous recombinant coagulation factors [ 127 ] capable of neutralizing the action of matrix-degrading proteases. They are also based on the activation and inactivation of the proteases involved in the maturation and activation of, for example, transforming growth factor, myostatin, complement C3 and C5, or IL-1b and IL-18 [ 128 ], and on the inactivation of the retroviral proteases of HIV [ 129 ] or SARS-CoV-2, both at the level of the intracellular life cycle of the virus (protease 3CL [ 130 , 131 ]) and by facilitating the entry of the virus into the cell (TMPRSS2 [ 132 ]).…”

The Functional Landscape of SARS-CoV-2 3CL Protease

“…HEK293T cells and Vero E6 cells were obtained from ATCC (Catalog #CRL-3216 and #CRL-1586, respectively). Huh7-ACE2 cells (Huh7 cells overexpression human ACE2) were generated previously by transduction of Huh7 cells with lentivirus encoding human ACE2, packaged using pLEX307-ACE2-blast (Addgene plasmid #158449), pMD2.G (Addgene plasmid #12259, gift of Didier Trono), and psPAX2 (Addgene plasmid #12260, gift of Didier Trono), and selecting for stable expression with 5□µg/mL blasticidin (Liu et al, 2022a). Morphology was visually confirmed prior to use and all cell lines tested mycoplasma negative.…”

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

“…Vero E6 cells were obtained from ATCC (Catalog #CRL-1586), HEK293T cells were obtained from ATCC (Catalog #CRL-3216), and Vero E6-TMPRSS2-T2A-ACE2 cells were obtained from BEI Resources (Catalog #NR-54970). Huh7-ACE2 cells were generated previously 33,39 . Cell morphology was visually confirmed prior to use and all cell lines tested mycoplasma negative.…”