title: Creation of interferon-alpha8 mutants with amino acid substitutions against interferon-alpha receptor-2 binding sites using phage display system and evaluation of their biologic properties.
abstract: In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.
authors.affiliation: Biomedical Institute, Research Center, Hayashibara Biochemical Laboratories, Inc., Fujisaki, Okayama, Japan.
authors.forename: Kouzo
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Madoka
authors.initials: M
authors.lastname: Taniai
authors.forename: Kakuji
authors.initials: K
authors.lastname: Torigoe
authors.forename: Shigeto
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Norie
authors.initials: N
authors.lastname: Arai
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Suemoto
authors.forename: Kenshi
authors.initials: K
authors.lastname: Yoshida
authors.forename: Takanori
authors.initials: T
authors.lastname: Okura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mori
authors.forename: Noboru
authors.initials: N
authors.lastname: Fujioka
authors.forename: Tadao
authors.initials: T
authors.lastname: Tanimoto
authors.forename: Manabu
authors.initials: M
authors.lastname: Miyata
authors.forename: Harumi
authors.initials: H
authors.lastname: Ariyasu
authors.forename: Chie
authors.initials: C
authors.lastname: Ushio
authors.forename: Mitsukiyo
authors.initials: M
authors.lastname: Fujii
authors.forename: Toshio
authors.initials: T
authors.lastname: Ariyasu
authors.forename: Masao
authors.initials: M
authors.lastname: Ikeda
authors.forename: Tsunetaka
authors.initials: T
authors.lastname: Ohta
authors.forename: Masashi
authors.initials: M
authors.lastname: Kurimoto
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Fukuda
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D053633:Receptor, Interferon alpha-beta / Q000378:metabolism*; D011994:Recombinant Proteins; D012845:Sindbis Virus / Q000187:drug effects; D020349:Surface Plasmon Resonance; D020298:U937 Cells; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D006131:Growth Inhibitors; C507964:IFNAR1 protein, human; C508033:IFNAR2 protein, human; D007370:Interferon Type I; D016898:Interferon-alpha; D019151:Peptide Library; D011994:Recombinant Proteins; C056556:methionyl human consensus alpha interferon; D053633:Receptor, Interferon alpha-beta