title: 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.
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.
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, 3559-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Pamela
authors.initials: P
authors.lastname: Hamill
authors.forename: Derek
authors.initials: D
authors.lastname: Hudson
authors.forename: Richard Y
authors.initials: RY
authors.lastname: Kao
authors.forename: Polly
authors.initials: P
authors.lastname: Chow
authors.forename: Meera
authors.initials: M
authors.lastname: Raj
authors.forename: Hongyan
authors.initials: H
authors.lastname: Xu
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Richer
authors.forename: François
authors.initials: F
authors.lastname: Jean
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical; D007700:Kinetics; D015394:Molecular Structure; D011161:Porifera / Q000737:chemistry* / Q000145:classification*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000145:classification / Q000494:pharmacology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014468:Umbelliferones / Q000737:chemistry / Q000494:pharmacology*; D014709:Vero Cells; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors; D014468:Umbelliferones; D014764:Viral Proteins; C515382:esculetin-4-carboxylic acid ethyl ester; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases