A continuous assay for foot-and-mouth disease virus 3C protease activity

Jaulent, Agnès M.; Fahy, Aodhnait S.; Knox, Stephen R.; Birtley, James R.; Roqué-Rosell, Núria; Curry, Stephen; Leatherbarrow, Robin J.

doi:10.1016/j.ab.2007.05.026

Cited by 18 publications

(16 citation statements)

References 28 publications

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“…FRET protease assays have been used in characterizations of several viral proteases, such as those of the SARS-Coronavirus (Blanchard et al, 2004; Grum-Tokars et al, 2008), hepatitis C virus (Mao et al, 2008; Santos et al, 2009), yellow fever virus (Kondo et al, 2011), and foot-and-month disease virus (Jaulent et al, 2007). During the preparation of the manuscript, Chang et al also reported the characterization and inhibition of proteases of two norovirus genogroups using FRET assays (Chang et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Substrate specificity of Tulane virus protease

2013

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Tulane virus (TV) is a cultivable calicivirus isolated from rhesus monkeys. In this study, we characterized the substrate specificity of TV protease in trans using recombinant proteases and TV polyprotein fragments containing the predicted proteolytic cleavage sites. Cleavage products have been obtained from 4 of the 5 fragments containing 573Q-S574 between the helicase and 3A-like protein, 712E-A713 between the 3A-like protein and Vpg, 802E-G803 between Vpg and the protease, and 976E-G977 between the protease and RdRp. We also characterized the enzymatic activities of the recombinant proteases of TV and Norwalk virus using synthetic fluorogenic peptide substrates. Under optimal conditions for enzymatic assays, partial cross-reactivities on reciprocal substrates were observed between TV and Norwalk virus proteases. The apparently shared substrate specificities between TV and Norwalk virus proteases suggested that the cultivable TV could be used as a model for in vivo evaluation of lead candidates of protease inhibitors for human norovirus.

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Section: Discussionmentioning

confidence: 99%

Substrate specificity of Tulane virus protease

2013

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“…The mixtures were incubated at 37 °C, and fluorescence readings were obtained on a microplate reader at every 5 min for up to 30 min. The c orrection factors to compensate for the inner filter effects were determined empirically as described previously (Chang et al, 2012b; Cuerrier et al, 2005; Jaulent et al, 2007). The kinetic constant K m was determined by the fitting initial rates to the Michaelis-Menten equation using GraphPad Prism software.…”

Section: Methodsmentioning

confidence: 99%

Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease

et al. 2013

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Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems.

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“…Cyclotide activity against FMDV 3C pro was determined as described previously, using 617 nM, 50 µL FMDV 3C pro , 50 30 µM, 100 µL FRET substrate. 59 Initial rate data were then fitted to determine K i using the GraFit Software package (http://www.erithacus.com/grafit). All assays were reproduced 3 times with fresh solutions of all reagents, and the standard deviation is given where appropriate.…”

Section: Methodsmentioning

confidence: 99%

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

et al. 2008

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The naturally-occurring cyclic cystine-knot microprotein trypsin inhibitors MCoTI-I and MCoTI-II have been synthesised using both thia-zip native chemical ligation and a biomimetic strategy featuring chemoenzymatic cyclisation by an immobilised protease. Engineered analogues have been produced containing a range of substitutions at the P 1 position that redirect specificity 10 towards alternative protease targets whilst retaining excellent to moderate affinity. Furthermore, we report an MCoTI analogue that is a selective low-µM inhibitor of foot-and-mouth-disease virus (FMDV) 3C protease, the first reported inhibitor of this important viral enzyme.

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A continuous assay for foot-and-mouth disease virus 3C protease activity

Cited by 18 publications

References 28 publications

Substrate specificity of Tulane virus protease

Substrate specificity of Tulane virus protease

Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

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