Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: Influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by "promiscuous" inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-

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“…Generally, the protease shows lower activity at acidic pH, 16 but the effect of the nonionic detergents is similar to those at pH 9.0.…”

Section: Resultsmentioning

confidence: 89%

Optimization of Assay Conditions for Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Steuer

Heinonen

Kattner³

et al. 2009

SLAS Discovery

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“…In analogy to a number of specificity studies on the NS3 proteases from DEN and WNV [15], [29], [42], [43], we assayed a small set of commercially available fluorogenic peptide substrates, based on P3-P1 sequences of cleavage sites (capsid protein, NS2B/NS3 itself, NS3/NS4A, and NS4B/NS5 cleavage site) of NS2B/NS3 proteases of WNV, YFV, and JEV. In addition, we assayed an internally quenched peptide, Abz-(R)4SAGnY-amide, representing the P4-P3' sequence of the capsid protein cleavage site of the DEN-2 virus [22].…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

et al. 2012

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BackgroundJapanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease.Methodology/Principal FindingsThe full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K m and k cat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k cat /K m was found for Pyr-RTKR-amc (k cat/K m: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (k cat/K m: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro.Conclusions/SignificanceA simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.

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“…A suitable enzymatic substrate was identified by functional profiling using tetra peptide and octapeptide libraries comprising ~13,000 substrates (Li, J., et al, 2005). Detailed specificity studies have led to the design of robust screening assays in high-throughput formats, employing both colorimetric and fluorescent readouts (Nall et al, 2004; Li, J., et al, 2005; Chappell et al, 2005; 2006; 2007; Gouvea et al, 2007; Knox et al, 2006; Lohr et al, 2007; Radichev et al, 2008; Shiryaev et al, 2006; Shiryaev et al, 2007a, b, c; Yin et al, 2006a,b)…”

Section: Targeting Critical Functions Of Individual Flaviviral Prmentioning

confidence: 99%

Molecular targets for flavivirus drug discovery

2009

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Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.

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Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: Influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

Cited by 34 publications

References 27 publications

Optimization of Assay Conditions for Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Optimization of Assay Conditions for Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Molecular targets for flavivirus drug discovery

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