title: Allosteric inhibition of the NS2B-NS3 protease from dengue virus.
abstract: Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.
authors.affiliation: Department of Chemistry, University of Massachusetts , 104 LGRT, 710 N. Pleasant St., Amherst, Massachussetts 01003, United States.
authors.forename: Muslum
authors.initials: M
authors.lastname: Yildiz
authors.forename: Sumana
authors.initials: S
authors.lastname: Ghosh
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bell
authors.forename: Woody
authors.initials: W
authors.lastname: Sherman
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000998:Antiviral Agents / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003716:Dengue Virus / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D062105:Molecular Docking Simulation; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011480:Protease Inhibitors / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D003545:Cysteine; D015335:Molecular Probes; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases