Fig. 5.

Melatonin inhibits SARS-CoV-2-cell entry by interfering with the spike-ACE2 interaction. A SARS-CoV-2 cell entry assessed by pseudo-virus luciferase reporter assay in HEK293 cells transfected with a human ACE2 expression plasmid or mock transfected. Data are expressed as mean ± SEM of 3 independent experiments, each performed in duplicates. B Concentration–response curves of melatonin on SARS-CoV-2 pseudo-virus cell entry in HEK293 cells expressing human ACE2. Data are expressed as mean ± SEM of 3 independent experiments, each performed in triplicates. C Inhibition of SARS-CoV-2 pseudo-virus cell entry by melatonin (100 µM) in hCMEC/D3 cells transduced with human ACE2. Data are expressed as mean ± SEM of 3 independent experiments, each performed in triplicates. Bottom panel show ACE2 expression in transduced and non-transduced cells by immunofluorescence (red = ACE2; blue = DAPI-stained nucleus; scale bar = 10 µm). D In vitro binding assay of biotinylated S1 spike protein (50 nM) to immobilized human ACE2 in the absence or presence of melatonin at 0.5 10 and 100 μM; a competition by an excess of non-labeled S1 (400 nM) determines the specific signal. E Scheme illustrating the TR–FRET-based binding assay of RBD-d2 to SNAP-tagged ACE2 labeled with Lumi4-Tb at the cell surface. F Change in TR–FRET signal of RBD-d2 (5 nM) binding to Lumi4-Tb-SNAP-ACE2 by increasing concentrations of melatonin. Competition by an excess of non-labeled RBD (300 nM) determines the specific signal. Data are expressed as mean ± SEM of 3 independent experiments, each performed in triplicates. G Docking and molecular dynamic-based prediction of melatonin binding to the crystal structure of ACE2 in complex with the RBD domain of SARS-CoV-2 virus. At t0 (left panel), the molecular docking of melatonin into ACE2/RBD complex (6M17) shows a unique entry channel for melatonin located in close proximity to the interaction surface between ACE2 and the viral spike protein (left panel). At t = 500 ns of simulation using glycosylated ACE2/RBD complex (6VW1), a global view of the complex shows the migration of MLT to the interior side of two ACE2 helices interfacing RBD (central and right panel). Most hydrophobic interactions are exhibited in a zoomed view centered onto the binding site (right panel). H ACE2 enzyme activity in the absence or presence of melatonin at 0.5 and 10 μM; DW600 inhibitor (10 μM) is shown as a positive control. I Scheme illustrating the assay probing conformational change within ACE2 based on intramolecular TR-FRET between Lumi4-Tb-labeled SNAP-ACE2 and d2-labeled anti-FLAG tag antibody. J ACE2 conformational change in HEK293 cells in the presence of non-labeled RBD (5 nM), or melatonin (100 µM). Data are expressed as change of TR-FRET signal (ΔTRFRET ± SEM) of 4 independent experiments, each performed in triplicates. *p < 0.05; **p < 0.01: ***p < 0.005: *****p < 0.0005 by one-way ANOVA, followed by Dunnett’s multiple comparisons test compared to vehicle group