A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening.
Kirill GorshkovDesarey Morales VasquezKevin ChiemChengjin YeBruce Nguyen TranJuan Carlos de la TorreThomas MoranCatherine Z ChenLuis Martinez-SobridoWei ZhengPublished in: bioRxiv : the preprint server for biology (2021)
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 (IC 50 ) 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.
Keyphrases
- sars cov
- respiratory syndrome coronavirus
- energy transfer
- high throughput
- coronavirus disease
- clinical trial
- quantum dots
- drug discovery
- single molecule
- healthcare
- living cells
- nitric oxide
- gene expression
- randomized controlled trial
- single cell
- oxidative stress
- protein protein
- copy number
- small molecule
- study protocol
- cell proliferation
- emergency department
- adverse drug
- signaling pathway
- cell cycle arrest
- loop mediated isothermal amplification
- genome wide
- dna methylation
- amino acid
- endoplasmic reticulum stress
- phase ii
- electronic health record
- pi k akt