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.

SARS coronavirus main proteinase 3.4.22.69

“…[3] P rhodamine 110 + (Ala-Arg-Leu-Gln-NH)-rhodamine S (CAL fluor red 610)-TSAVLQSGFRK(BHQ1) + H 2 O <2> (Reversibility: ?) [8] P (CAL fluor red 610)-TSAVLQ + SGFRK(BHQ1) S 2-aminobenzoyl-SVTLQSG-Tyr(NO 2 )Arg + H 2 O <2> (Reversibility: ?) [28] P ?…”

“…It is suggested 3CLPro inhibitors need small polar groups to decrease the energy barrier for alkylation reaction. The results can be useful for the development of new compounds against SARS [39]; <2> extracts from Rheum palmatum have a high level of inhibitory activity against 3CL protease with IC50 of 0.01376 mg/ml and an inhibition rate of up to 96% [48]) [ [26]; <2> mutant enzyme S139A/Q299A [26]) [26] 0.0069 <2> (o-aminobenzoyl-TSAVLQSGFRK-2,4-dinitrophenyl amide, <2> mutant enzyme S123a/R298A [26]) [26] 0.0153 <2> ((Ala-Arg-Leu-Gln-NH) 2 [9] 0.27 <2> (Ser-Ala-Val-Leu-Gln-Met-Gly-Phe-Arg-Lys, <2> T25G mutant protein [49]) [49] 0.31 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> E166A mutant protein [47]) [47] 0.38 <2> (o-aminobenzoyl-TSAVLQSGFRY(NO2)G) [8] 0.48 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> R298L mutant protein [47]) [47] 0.6 <2> (SITSAVLQ-p-nitrophenyl ester) [7] 0.63 <2> (Thr-Ser-Ala-Val-Leu-Gln-p-nitroanilide, <2> wild-type protein [47]) [47] 0.847 <2> (TFTRLQSLENV, <2> pH 7.3, room temperature [27]) [27] 0.86 <2> (SITSAVLQ-p-nitroanilide) [ [49] 76 <2> (GPFVDRQTAQAAGTDT-NH 2 , <2> pH 7.5, 37 C, mutant enzyme R188I [31]) [31] 455 <2> (KVATVQSKMSD-NH 2 , <2> pH 7.5, 37 C, mutant enzyme R188I [31]) [31] 4753 <2> (TSAVLQSGFRK-NH 2 , <2> pH 7.5, 37 C, mutant enzyme R188I [31]) [31] Additional information <2> (<2> steady-state analysis of the solvent isotope effects on k cat [7]) [7] K m -Value (mM) 0.004 <2> (o-aminobenzoyl-TSAVLQSGFRK-2,4-dinitrophenyl amide, <2> mutant enzyme D(303-306) [26]) [26] 0.00...…”

“…The dimer may be the biological functional form of the protein [27]; <2> in solution the wild type protease exhibits both forms of monomer and dimer and the amount of the monomer is almost equal to that of the dimeric form [37]; <2> wild type and D(300-306) proteases exist with dimer as the major form. The major form becomes monomeric in D(299-306), D(298-306) and D(297-306) [26]) [16,25,26,27,37] 5 Isolation/Preparation/Mutation/Application Purification <1> [19] <2> [6,20,21,24,27,37] <2> (E166A mutant protein: immobilized metal ion affinity chromatography (Ni 2+ )) [47] <2> (Ni-NTA column chromatography) [49] <2> (ammonium sulfate precipitation and anion-exchange column chromatography) [48] <2> (ammonium sulfate precipitation, anion-exchange chromatography) [48] <2> (commercial preparation) [45] <2> (fused to maltose-binding protein, removing the maltose-binding protein by cleavage with factor Xa, purification by Phenyl Sepharose column chromatography) [1] <2> (glutathione S-transferase column chromatography and HiTrap 26/10 QFF column chroamtography) [38] <2> (immobilized metal ion affinity chromatography (Ni 2+ )) [49] <2> (purification of proteolysis-resistant mutant R188I of the SARS 3CL protease) [31] <2> (recombinant His-tagged SARS-CoV 3CL protease) [8] <2> (recombinant enzyme) [34] <2> (strong cation column chromatography connected in series to a strong anion column chromatography) [3] <2> (wild-type enzyme and C-terminally truncated proteases) [26] <3> (glutathione Sepharose column chromatography and Superdex 75 gel filtration) [51] Renaturation <2> (reversible unfolding of SARS-CoV main protease in guanidine-HCl. In the presence of 6 M of guanidine-HCl, the enzyme is completely unfolded in 10 min.…”

“…porcine transmissible gastroenteritis virus Mpro severe acute respiratory syndrome coronavirus 3C-like protease <2> [41,42] severe acute respiratory syndrome coronavirus main protease <2> [21] severe acute respiratory syndrome coronavirus main proteinase <2> [33] CAS registry number 218925-73-6 37353- 2 Source Organism <1> alphacoronavirus [19] <2> SARS coronavirus [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,…”

Pharmacophores and biological activities of severe acute respiratory syndrome viral protease inhibitors

“…These two large compounds (structures not shown) belong to a group of natural polyphenols in tea [36]. Also from a natural product library, an esculetin-4-carboxylic acid ethyl ester (MC8; 12, Figure 1) with an IC 50 value of 46 µM against SARS protease was derived from the marine sponge Axinella corrugate [37]. The screening was performed by using a sensitive red-shifted internally quenched fluorogenic substrate for SARS protease, also based on the FRET between the CAL Fluor Red 610 and Black Hole Quencher-1 as a donoracceptor pair.…”