Abstract
The number of dengue virus infections is increasing and the dengue NS2B–NS3 protease is considered a promising target for the development of antiviral therapies. Therefore, reliable and fast screening systems are needed for the discovery of new lead structures. In this chapter, we describe two dengue virus protease assays based on an internally quenched, high-affinity Förster resonance energy transfer (FRET) substrate (K m = 105 μM). A fluorimetric assay using a microtiter fluorescence plate reader can be used for high-throughput screening of a large number of compounds. Alternatively, an HPLC-based assay with fluorescence detection can be applied to confirm the compound hits and to avoid false-positive results that may arise due to the inner filter effect of some compounds.
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References
Lescar J, Luo D, Xu T et al (2008) Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from dengue virus as a target. Antiviral Res 80:94–101
Yusof R, Clum S, Wetzel M et al (2000) Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J Biol Chem 275:9963–9969
Leung D, Schroder K, White H et al (2001) Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J Biol Chem 276:45762–45771
Li J, Lim SP, Beer D et al (2005) Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries. J Biol Chem 280:28766–28774
Gouvea IE, Izidoro MA, Judice WAS et al (2007) Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates. Arch Biochem Biophys 457:187–196
Niyomrattanakit P, Winoyanuwattikun P, Chanprapaph S et al (2004) Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J Virol 78:13708–13716
Shiryaev SA, Ratnikov BI, Aleshin AE et al (2007) Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis. J Virol 81:4501–4509
Yin Z, Patel SJ, Wang WL et al (2006) Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett 16:40–43
Niyomrattanakit P, Yahorava S, Mutule I et al (2006) Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides. Biochem J 397:203–211
Steuer C, Heinonen KH, Kattner L et al (2009) Optimization of assay conditions for dengue virus protease: effect of various polyols and nonionic detergents. J Biomol Screen 14:1102–1108
Deng J, Li N, Liu H et al (2012) Discovery of novel small molecule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping. J Med Chem 55: 6278–6293
Prusis P, Lapins M, Yahorava S et al (2008) Proteochemometrics analysis of substrate interactions with dengue virus NS3 proteases. Bioorg Med Chem 16:9369–9377
Meldal M, Breddam K (1991) Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem 195: 141–147
Förster T (1948) Zwischenmolekulare Energ-iewanderung und Fluoreszenz. Ann Phys 437: 55–75
Yaron A, Carmel A, Katchalski-Katzir E (1979) Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Anal Biochem 95:228–235
Steuer C, Gege C, Fischl W et al (2011) Synthesis and biological evaluation of alpha-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture. Bioorg Med Chem 19:4067–4074
Nitsche C, Steuer C, Klein CD (2011) Arylcyanoacrylamides as inhibitors of the dengue and West Nile virus proteases. Bioorg Med Chem 19:7318–7337
Nitsche C, Behnam MAM, Steuer C et al (2012) Retro peptide-hybrids as selective inhibitors of the dengue virus NS2B-NS3 protease. Antiviral Res 94:72–79
Mendgen T, Steuer C, Klein CD (2012) Privileged scaffolds or promiscuous binders: a comparative study on rhodanines and related heterocycles in medicinal chemistry. J Med Chem 55:743–753
Liu YY, Kati W, Chen CM et al (1999) Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal Biochem 267:331–335
Steuer C (2011) Medizinische Chemie der Dengue Protease und verwandter flaviviraler Proteasen. PhD thesis, University of Heidelberg
Khumthong R, Angsuthanasombat C, Panyim S et al (2002) In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates. J Biochem Mol Biol 35:206–212
Merrifield RB (1965) Solid-phase peptide synthesis. Endeavour 24:3–7
Chang C-D, Meienhofer J (1978) Solid-phase peptide synthesis using mild base cleavage of N alpha-fluorenylmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int J Pept Protein Res 11:246–249
Champreda V, Khumthong R, Subsin B et al (2000) The two-component protease NS2B-NS3 of dengue virus type 2: cloning, expression in Escherichia coli and purification of the NS2B, NS3(pro) and NS2B-NS3 proteins. J Biochem Mol Biol 33:294–299
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Nitsche, C., Klein, C.D. (2013). Fluorimetric and HPLC-Based Dengue Virus Protease Assays Using a FRET Substrate. In: Gong, E. (eds) Antiviral Methods and Protocols. Methods in Molecular Biology, vol 1030. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-484-5_18
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DOI: https://doi.org/10.1007/978-1-62703-484-5_18
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