title: Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region.
abstract: Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.
authors.affiliation: National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia.
authors.forename: Irena
authors.initials: I
authors.lastname: Fonda
authors.forename: Mojca
authors.initials: M
authors.lastname: Pernus
authors.forename: Vladka
authors.initials: V
authors.lastname: Gaberc-Porekar
authors.forename: Maja
authors.initials: M
authors.lastname: Kenig
authors.forename: Anton
authors.initials: A
authors.lastname: Stalc
authors.forename: Anthony
authors.initials: A
authors.lastname: Meager
authors.forename: Viktor
authors.initials: V
authors.lastname: Menart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019763:Heparin Lyase / Q000494:pharmacology; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D007526:Isoelectric Point; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D019763:Heparin Lyase; D006497:Heparan Sulfate; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; C471050:LK-805