RESUMEN
Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.
Asunto(s)
Virus del Dengue/enzimología , Serina Endopeptidasas/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Antivirales/química , Cromatografía de Afinidad , Escherichia coli , Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Inhibidores de Proteasas/química , Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
West Nile Virus (WNV) has spread rapidly during the last decade across five continents causing disease and fatalities in humans and mammals. It highlights the serious threat to both our health and the economy posed by viruses crossing species, in this case from migratory birds via mosquitoes to mammals. There is no vaccine or antiviral drug for treating WNV infection. One attractive target for antiviral development is a viral trypsin-like serine protease, encoded by the N-terminal 184 amino acids of NS3, which is only active when tethered to its cofactor, NS2B. This protease, NS2B/NS3pro, cleaves the viral polyprotein to release structural and non-structural viral proteins that are essential in viral replication and assembly of new virus particles. Disruption of this protease activity is lethal for virus replication. The NS3 protein also has other enzymes within its sequence (helicase, nucleoside triphosphatase, RNA triphosphatase), all of which are tightly regulated through localisation within membranous compartments in the infected cell. This review describes the various roles of NS3, focussing on NS2B-NS3 protease and its function and regulation in WNV replication and infection. Current advances towards development of antiviral inhibitors of NS2B/NS3pro are examined along with obstacles to their development as an antiviral therapy.
Asunto(s)
Antivirales/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virus del Nilo Occidental/enzimología , Antivirales/química , Antivirales/uso terapéutico , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , ARN Helicasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Fiebre del Nilo Occidental/tratamiento farmacológicoRESUMEN
BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.
Asunto(s)
Mycoplasma/genética , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa/normas , ADN Bacteriano/análisis , ADN Bacteriano/genética , Humanos , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Proyectos de Investigación , Replicación de Secuencia Autosostenida , Sensibilidad y EspecificidadRESUMEN
Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.
Asunto(s)
Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/metabolismo , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Cinética , Proteínas Recombinantes/farmacología , Especificidad por Sustrato , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.