Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata

Biol Chem. 2006 Aug;387(8):1063-74. doi: 10.1515/BC.2006.131.

Abstract

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (Km and kcat values of 14 microM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antiviral Agents / chemistry*
  • Antiviral Agents / pharmacology
  • Cell Proliferation / drug effects
  • Chlorocebus aethiops
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / isolation & purification
  • Drug Evaluation, Preclinical
  • Kinetics
  • Molecular Structure
  • Porifera / chemistry*
  • Porifera / classification*
  • Protease Inhibitors / chemistry*
  • Protease Inhibitors / classification
  • Protease Inhibitors / pharmacology
  • SARS Virus / drug effects
  • SARS Virus / enzymology*
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / methods*
  • Structure-Activity Relationship
  • Time Factors
  • Umbelliferones / chemistry
  • Umbelliferones / pharmacology*
  • Vero Cells
  • Viral Proteins / antagonists & inhibitors
  • Viral Proteins / chemistry*
  • Viral Proteins / isolation & purification
  • Virus Replication / drug effects
  • Virus Replication / physiology

Substances

  • Antiviral Agents
  • Protease Inhibitors
  • Umbelliferones
  • Viral Proteins
  • esculetin-4-carboxylic acid ethyl ester
  • 3C-like proteinase, Coronavirus
  • Cysteine Endopeptidases