Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC^®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Keywords: COVID-19; SARS-CoV-2; angiotensin-converting enzyme 2 (ACE2), high-throughput screening; drug repurposing; receptor binding domain (RBD); small molecule inhibitors (SMIs); spike protein.

Figure 1

Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were coated with ACE2 (100 ng/well) and blocked with TSTA buffer. Each compound (10 µM) from the libraries was incubated with Fc-RBD (1 µg/mL) for 1 h at 25 °C. Following incubation, mixtures were added to the ACE2-coated plates and incubated for 1 h at 37 °C. After washing, the plates were incubated for 1 h at 37 °C with the alkaline phosphatase-conjugated goat anti-human Fc fragment. The plates were then washed, and a colorimetric reaction was developed by the addition of pNPP (measured at 405 nm). Left panel: a maximum signal of an uninterrupted RBD-ACE2 interaction was obtained in the presence of noninhibitory compounds. Right panel: a reduced signal of an inhibited RBD-ACE2 interaction was obtained in the presence of inhibitory compounds.

Figure 2

High-throughput screening of the LOPAC^®1280 and DiscoveryProbe^TM compound libraries for inhibitors of the RBD-ACE2 interaction. Each compound was tested at 10 µM. (A) Distribution of the relative RBD-ACE2 binding values in the presence of the compounds. (B) Distribution of the Z-prime values of each plate. The purple and blue dots correspond to LOPAC^®1280 and DiscoveryProbe^TM, respectively.

Figure 3

Dose–response relationships between the hit compound concentrations and RBD-ACE2 binding. Inhibition profiles of the RBD-ACE2 interaction in the presence of increasing concentrations of heparin sodium (A), ATA (B), and ellagic acid (C), determined by the binding assay. The values are the average of triplicates ± SEM. The IC₅₀ values are listed in Table 1.

Figure 4

Inhibition of viral infection of cells by heparin sodium and ATA. rVSV-SARS-CoV-2-S was preincubated for 1 h with the indicated concentrations of heparin sodium (A) and ATA (B). Mixtures were then added to Vero E6 cells and incubated for 72 h. Cells were then stained using crystal violet, and the relative plaque reduction was determined. The results are expressed as the percent inhibition relative to that of the virus-only control incubated with the corresponding solvent (infection medium without or with 2% DMSO for heparin sodium or ATA, respectively). The values are presented as the mean of 6-well replicates ± SD. The lower images are three representative wells with the indicated compound concentrations.

Figure 5

A docking model of the binding of heparin sodium and ATA to the RBD. (A) RBD-ACE2 interaction (pdb code 6M0J). The RBD forms a concave surface with a protruding loop on one side, to which ACE2 binds (ACE2 is colored in light blue and is shown in a ribbon presentation; RBD is shown in surface (gold colored) and ribbon (red colored) representations). Heparin sodium (one disaccharide unit) and ATA were docked to the RBM using the SwissDock server [20]. The compounds are shown in stick representation, where oxygen, sulfur, and hydrogen are colored in red, yellow, and white, respectively, and carbon atoms are colored in magenta for heparin sodium (B) and green for ATA (C). The figure was prepared using UCSF Chimera [21].