A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Abstract

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 μM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, β), Brazilian/Japanese variant P.1 (Gamma, γ), and Californian (Epsilon, ε), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro

“…A variety of screening methods have been reported for the screening and discovery of small molecule inhibitors of M pro , including virtual screening [35] , FRET technology [36] , [37] , [38] , [39] , cell model screening [31] , and phenotypic screening [40] , etc . We constructed a nine-peptide-fluorogenic molecular as the substrate of M pro , which recognize the sequence of -AVLQ/SGFRK- (the cleavage site is indicated by /) [41] .…”

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

“…In addition, numerous cell-based screening assays with SARS-CoV-2 have also been recently reported 12,15−18 . Unlike the S and E proteins, however, the structural nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 protein in infected cells, has not been fully exploited as a target for drug development against SARS-CoV-2 19 .…”