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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

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

Pamela Hamill, Derek Hudson, Richard Y Kao, Polly Chow, Meera Raj, Hongyan Xu, Martin J Richer, François Jean

Biol Chem. 2006 Aug;387(8):1063-74.

PMID: 16895476

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP482379373	Fluorescent red 610		482379-37-3	Price

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