Fig 1: EGCG inhibits the infection of 4 mutant S-pseudotyped lentivirus in human ACE2 overexpressing cells. HEK293 cells were transiently transfected with hACE2-mCherry (red). After 24 h, the hACE2 overexpressing cells were infected by RBD wild-type and mutant S-pseudotyped lentivirus (D614G, N501Y, N439K & Y453F) (green) in the presence of 0–100 µM EGCG. The infected cells were then replaced with fresh medium and continually incubated for 48 h. All images were captured by confocal microscopy using a Leica SP8 (×40 oil immersion objective lens). Data of pseudovirus entry inhibition at different concentrations are standardized to the mean of fluorescence intensity (quantified by Image J) at 0 µM concentration and presented as mean ± S.D. of three replicates; *P < 0.05, **P < 0.01, student-t-test analysis.
Fig 2: Structural basis for the proposed “conformation competition” mechanism for the ACE2-blocking activity of DL28. (A) Comparison of the RBD conformations at the RBM between two unbound forms (blue, magenta), the ACE2-bound (Lan et al., 2020) form (yellow), and the DL28-bound form (cyan). The ACE2-interacting residues are shown as green Ca spheres. (B) Alignment of the DL28-RBD structure (green surface and cyan ribbon) with the ACE2-RBD structure (Lan et al., 2020; wheat surface and yellow ribbon). DL28 pushes the boxed loop in (A) toward ACE2, causing clashes between two aromatic residues and the RBD-interacting a-helices in ACE2. (C) Both ACE2 and DL28 use a rigid structure to interact with the boxed loop in (A), making a compromise unlikely to reach. The black box highlights the clash between ACE2 (wheat) and the DL28-bound form of RBD (cyan). The clashing RBD residues in the ACE2-bound form are shown as yellow sticks. The magenta box highlights the interaction between DL28 and the “backrest” region mediated by main-chain interactions. (D) Ca b-factor distribution shown in putty representation using a rainbow ribbon with a radius that increases from the lowest (61.6 Å2; dark blue) to the highest (170.0 Å2; red) B-factor. The average B-factor of the “backrest” region (residue 470–491) is 76.59 Å2 which is lower than that of the whole chain (97.5 Å2), suggesting relative inflexibility.
Fig 3: Dalbavancin inhibits binding of SARS-CoV-2 spike protein to ACE2 in vitro.a ACE2 (0.5 µg) and SARS-CoV-2 spike protein (0.5 µg) were mixed and treated with various candidate peptide drugs (10 µM). Co-precipitated proteins were identified by western blot analysis using anti-ACE2 antibody. Analyzed proteins are indicated on the right. For positive control (bottom), ACE2-His (0.5 µg) and SARS-CoV-2 spike protein (0.5 µg) were mixed and treated with various concentration of ACE2-hFc, and then co-precipitated proteins were identified by western blot analysis using anti-His-tag antibody. b ACE2 (2 µg/ml) was crosslinked to microplates by N-oxysuccinimide esters for ELISA. SARS-CoV-2 spike protein (10 ng/mL) and tested candidate drugs (1 µM) were incubated with ACE2, and extra un-crosslinked ACE2 protein (100 ng/mL) was used as a positive control. SARS-CoV-2 spike protein antibodies were used for chromogenic reaction. c Binding curves of immobilized human ACE2 with SARS-CoV-2 spike protein (left, positive control) and dalbavancin (right). Concentration-response SPR experiment showing binding of dalbavancin to ACE2 with an equilibrium dissociation constant (KD) of ~147 nM.
Fig 4: Integrin and ACE2 are independent receptors for SARS-CoV-2 pseudovirus infection on hACE2 transiently transfected CHO-K1 cells.A, representative images of SARS-CoV-2 pseudovirus particle (PP) infection on hACE2 transiently transfected CHO-K1 cells upon integrin activation by MnCl2 and the inhibition of integrin by inhibitor Cilengitide. The first row, SARS-CoV-2 PP massively infects hACE2 transiently transfected CHO-K1 cells in the absence of MnCl2. The second row, SARS-CoV-2 PP barely infects hACE2 transiently transfected CHO-K1 cells upon integrin activation in the presence of MnCl2. The third and fourth rows, SARS-CoV-2 PP infection on hACE2 transiently transfected CHO-K1 cells upon integrin activation in the presence of MnCl2 is gradually recovered by Cilengitide. B, quantification of the relative infection of three randomly selected regions from each of three independent experiments. Data are averages ± SEM for nine images (three randomly selected images from each three independent experiments). ***p < 0.001. Note: the infect rate here is lower than Figure 4, which is because hACE2 here is transiently transfected while integrin is endogenously expressed. ACE2, angiotensin-converting enzyme 2; hACE2, human ACE2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Fig 5: Molecular basis for neutralization.a, b Alignment of the SR4-RBD (a) or MR17-RBD (b) to the ACE2-RBD structure (PDB ID 6M0J)3 reveals that SR4/MR17 (blue) binds RBD (red) at the motif (dark red) where ACE2 (white) also binds. c, d SR4 (c) and MR17 (d) compete with ACE2 for RBD binding. A sensor coated with streptavidin was saturated with 2 µg mL-1 of biotinylated RBD. The sensor was then soaked in 200 nM of the indicated sybody before further soaked in sybody-containing buffer with (black) or without (red) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2–RBD interaction was monitored in the absence of sybodies (magenta). e, f Alignment of the SR4-RBD (e) and MR17-RBD (f) to the “up” conformation of the RBD from the cryo-EM structure of the trimer S (PDB ID 6VYB)2. The three subunits are colored yellow (A’), white (B’), and light blue (C’). Synthetic nanobodies are colored deep blue. RBM (red) marks the ACE2-binding motif. g, h Binding kinetics of SR4 (g) and MR17 (h) to S. BLI assay was performed with sybodies immobilized and S as analyte at the indicated concentrations (nM). Source data for c, d, g, h are provided as a Source data file. ACE2 angiotensin-converting enzyme 2, BLI biolayer interferometry, RBD receptor-binding domain, RBM receptor-binding motif.
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