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

Abstract SARS-coronavirus (SARS-CoV) encodes a main protease, 3CL pro , 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 3CL pro 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 3CL pro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 ( K m and k cat values of 14 μM and 0.65 min -1 ). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CL pro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CL pro -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 3CL pro inhibitor (IC 50 =46 μM) and anti-SARS agent (EC 50 =112 μM; median toxic concentration >800 μM) from the tropical marine sponge Axinella corrugata .