Study of the inhibitory effect of a pyrazolopyridone derivative against SARS-CoV-2 infection
Objective To investigate the inhibitory effect and mechanism of 3-(4-chlorophenyl)-1,4-diphenyl-1,4,5,7-tetrahydro-6H-pyrazolo[3,4-b]pyridin-6-ketone(named J1)against SARS-CoV-2 infection in vitro.Methods The cytotoxicities of J1 on Vero-E6,ACE2/293T and HRT18 cells were evaluated using MTT assay.Subsequently,the anti-coronavirus activity of J1 was assessed using qRT-PCR and western blotting assay.Pseudovirus-based entry inhibition and time-of-addition assays were conducted to explore the antiviral mechanism of J1.The S protein-mediated cell-cell fusion assay determined whether J1 could block SARS-CoV-2 entry by inhibiting viral fusion.The interaction between the S protein and J1 was further explored through molecular docking and SPR assay.Results The compound had low cytotoxicity against Vero-E6 cells,ACE2/293T cells and HRT18 cells with a concentration of cytotoxicity 50% (CC50)up to 100 μmol·L-1.Both the SARS-CoV-2 wild-type strain and the delta variant were significantly inhibited by J1,and the concentration for 50% of maximal effect(EC50)were 6.07 μmol·L-1 and 2.21 μmol·L-1,respectively.Furthermore,it was found that J1 inhibited HCoV-OC43 infection and reduced the expressions of NP protein and mRNA.Molecular docking results showed that J1 bound stably to the active site of the SARS-CoV-2 S protein through hydrogen bonding and hydrophobic forces interactions.Moreover,J1 inhibited the entry of SARS-CoV-2 PsV into the ACE2/293T cells with half maximal inhibitory concentration(IC50)of 1.57 μmol·L-1 and exhibited a dose-dependent inhibition of SARS-CoV-2 S protein-mediated cell-cell fusion.SPR assay further confirmed that J1 exhibited a relatively strong binding affinity to SARS-CoV-2 S protein.Conclusion The pyrazolopyridone derivative J1 can effectively inhibit the entry of SARS-CoV-2 into cells by targeting S proteins.
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