FIG 2.

TMPRSS2-independent membrane fusion induced by the S protein of SARS-CoV-2 is blocked by various metalloproteinase inhibitors. (a) High-throughput screening of the Validated Compound Library (1,630 clinically approved compounds and 1,885 pharmacologically active compounds) obtained from the Drug Discovery Initiative (The University of Tokyo) by the DSP assay using the SARS-CoV-2 S protein. The x axis shows the relative cell fusion value using cells expressing both TMPRSS2 and ACE2 in the presence of each compound (1 μM in DMSO) (n = 1). The y axis shows the relative cell fusion value using cells expressing ACE2 alone in the presence of each compound (1 μM in DMSO) (n = 1). The relative cell fusion value was calculated by normalizing the RL activity for each compound to that of the control assay (DMSO alone; set to 100%). Each dot represents an individual compound. Dots in the red dashed box represent compounds that preferentially inhibit TMPRSS2-independent membrane fusion (<30% inhibition of the relative cell fusion value using the target cells expressing both TMPRSS2 and ACE2 and >40% inhibition of the relative cell fusion value using the target cells expressing ACE2 alone). The compound names for the candidates are indicated. (b) Effects of the metalloproteinase inhibitors on cell fusion in the cocultures of cells expressing SARS-CoV-2 S protein with those expressing ACE2 alone or in combination with TMPRSS2. Relative cell fusion values were calculated by normalizing the RL activity for each coculture to that of the coculture with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO, which was set to 100%. Values are means ± SD (n = 3/group). (c) Expression of ACE2 in target cells (top). Tubulin was used as a loading control (bottom). ACE2-WT, wild-type ACE2; ACE2-NN, enzymatically inactive ACE2 with H374N and H378N mutations. (d) Effects of the metalloproteinase inhibitor on cell fusion in the cocultures of cells expressing SARS-CoV-2 S protein with those expressing wild-type ACE2 (ACE2-WT) or enzymatically inactive ACE2 (ACE2-NN). Relative cell fusion values were calculated by normalizing the RL activity for each coculture to that of the coculture with cells expressing wild-type ACE2 in the presence of DMSO, which was set to 100%. Values are means ± SD (n = 3/group). marima, 1 μM marimastat. **, P < 0.01.