Assuntos
Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/química , Estrutura Secundária de Proteína , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Humanos , Metaloproteases/química , Metaloproteases/metabolismo , Peptídeo Hidrolases/química , Inibidores de Proteases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.
Assuntos
RNA Helicases/química , RNA Helicases/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cinética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Eletricidade Estática , Especificidade por SubstratoRESUMO
West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.
