Fig. 4.
Hit compounds 36 and 37 inhibit SARS-CoV-2 Mpro in a cellular context. 293T cells were transfected to express either NanoLuc-nsp4-Mpro fusion protein (NLuc-Mpro) or its catalytically inactive mutant form (C145A). Whole cell lysates obtained from cells collected at 16 h post-transfection were analyzed by Western Blot with an antibody recognizing SARS-CoV-2 Mpro. The expression of NLuc-Mpro in DMSO-treated cells resulted in the cleavage of Mpro from the NanoLuc tag and in the release of an authentic Mpro protein (Mpro, with a molecular mass of ∼33 kDa), which was not detected when the inactive mutant C145A (with a molecular mass of ∼52 kDa) was expressed. The dose-dependent inhibitory effect of hit compounds 36 and 37 on Mpro activity was detected through the accumulation of the 52-kDa, uncleaved NLuc-Mpro fusion protein. Nontransfected 293T cells (NT) and transfected cells treated with 100 μM boceprevir (BOC) were included as controls. β-actin was used as a loading control. Molecular masses in kDa are indicated on the left.