title: Kinetic characterization of newly discovered inhibitors of various constructs of human T-cell leukemia virus-1 (HTLV-1) protease and their effect on HTLV-1-infected cells.
abstract: BACKGROUND
  Human T-cell leukemia virus-1 (HTLV-1) was the first identified human retrovirus and was shown to be associated with diseases such as adult T-cell leukemia lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy. Retroviral proteases (PRs) are essential for viral replication by processing viral Gag and Gag-(Pro)-Pol polyproteins during maturation. Full-length HTLV-1 PR is 125 residues long; whether the C-terminal region is required for catalytic activity is still controversial. In this study, we characterized the effect of C-terminal amino acids of HTLV-1 PR for PR activity and examined the binding of compounds identified by in silico screening. One compound showed inhibition against the virus in infected cells.
  
  
  METHODS
  Truncated (116-, 121- and 122-residue) forms of HTLV-1 PR were prepared and proteins from expression of the genes were purified. In silico screening was performed by docking small molecules into the active site of HTLV-1 PR. The kinetic constants k(cat), K(m), k(cat)/K(m) and inhibition constants K(i) for inhibitors identified by the computational screening were determined. Western blot and ELISA analyses were used to determine the effect of the most potent PR inhibitors on HTLV-1 protein processing in infected cells.
  
  
  RESULTS
  The constructs showed similar catalytic efficiency constants (k(cat)/K(m)); thus HTLV-1 PR C-terminal amino acids are not essential for full activity. Computational screening revealed new PR inhibitors and some were shown to be inhibitory in enzyme assays. In HTLV-1-infected cells, one of the small molecules inhibited HTLV-1 gag cleavage and decreased the amount of HTLV-1 p19 produced in the cells.
  
  
  CONCLUSIONS
  We have identified an HTLV-1 PR inhibitor that is biologically functional. Inhibitor screening will continue to develop possible drugs for therapy of HTLV-1 infection.
authors.affiliation: University of Florida College of Medicine Biochemistry and Molecular Biology, Gainesville, FL, USA.
authors.forename: Ahu
authors.initials: A
authors.lastname: Demir
authors.forename: Raphael M
authors.initials: RM
authors.lastname: Oguariri
authors.forename: Andrew
authors.initials: A
authors.lastname: Magis
authors.forename: David A
authors.initials: DA
authors.lastname: Ostrov
authors.forename: Tomozumi
authors.initials: T
authors.lastname: Imamichi
authors.forename: Ben M
authors.initials: BM
authors.lastname: Dunn
mesh_terms: D000595:Amino Acid Sequence; D044966:Anti-Retroviral Agents / Q000737:chemistry / Q000494:pharmacology*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D015683:Gene Products, gag / Q000378:metabolism; D015684:Gene Products, pol / Q000737:chemistry; D015368:Human T-lymphotropic virus 1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D059748:Proteolysis / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: D044966:Anti-Retroviral Agents; D015683:Gene Products, gag; D015684:Gene Products, pol; D011480:Protease Inhibitors; D054852:Small Molecule Libraries; D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease