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

Alon Ben David,Lilach Levin,Eyal Epstein,Ran Zichel,Ada Barnea,Eyal Dor,Eran Diamant,Niva Natan,Shira Chapman,Amram Torgeman,Lilach Cherry Mimran

doi:10.3390/molecules26113213

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.

Highlights

The identification of compounds that inhibit the binding of receptor binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) was carried out by high-throughput screening of the LOPAC® 1280 and DiscoveryProbeTM libraries using an assay designed to measure the RBD-ACE2 interaction
This assay consisted of the SARS-CoV-2 receptor ACE2 adsorbed to 96-well plates and soluble hFcRBD (Figure 1)
Following a washing step, the RBD-ACE2 interaction was quantified by the addition of a detecting alkaline phosphatase-conjugated goat anti-human-Fc antibody

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Coronavirus disease 2019 (COVID-19) is a worldwide pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 has infected over 150 million people, and over 3 million deaths have been reported [1]. The emergence of COVID-19 has prompted a global effort to develop vaccines and drugs for prevention and treatment. These efforts have led to the development of vaccines, several of which are being administered worldwide under emergency use authorization [2–4]. Only a few therapeutics (Remdesivir and monoclonal antibodies) are available under emergency-use or conditional marketing authorization by the American and European regulatory authorities [5–7]

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Results

Conclusion