Nirmatrelvir (PF-07321332)

Nirmatrelvir (PF-07321332) is an reversible covalent inhibitor of SARS-CoV-2 main protease (M^pro, also referred to as 3CL protease) with an ki of 3.11 nM. PF-07321332 binds directly to the catalytic cysteine (Cys145) residue of the enzyme.

• Enzyme kinetics assay

  Test compounds and SARS-CoV-2 Mpro enzyme were incubated at a 1:1 molar ratio of 2 μM compound and 2 μM enzyme in assay buffer for 20 minutes. The mixture was then diluted 50-fold into assay buffer followed by a transfer of 5 μl to wells of a black low volume 384-well assay plate. Enzyme activity was monitored on a BMG Pherastar at Ex/Em of 340 nm/460 nm after addition of 5 μl 60 μM peptide substrate. Final reaction conditions were 20 nM enzyme with 20 nM compound and 30 μM peptide substrate.

• Cell lines

  HCoV-229E in Huh-7 cells

• Concentrations

  0.62 µM

• Incubation Time

  2 days

• Method

  hCoV-229E experiments were performed on Huh-7 cells at an MOI of 0.002 and infection determined by in-cell ELISA 2 days later

• Animal Models

  Female C57BL/6 Cg-Tg2Prlmn/J mice 

• Dosages

  20 mg/kg

• Administration

  Oral