title: Constrained peptide analogues of transforming growth factor-alpha residues cysteine 21-32 are mitogenically active. Use of proline mimetics to enhance biological potency.
abstract: Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 microM; in the presence of low doses of TGF-alpha, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-alpha alone. These effects are consistent with the peptides acting via the same mechanism as TGF-alpha. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the beta-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.
authors.affiliation: Cancer Research Campaign Medical Oncology Unit, Southampton General Hospital, United Kingdom.
authors.forename: S G
authors.initials: SG
authors.lastname: Chamberlin
authors.forename: K J
authors.initials: KJ
authors.lastname: Sargood
authors.forename: A
authors.initials: A
authors.lastname: Richter
authors.forename: J M
authors.initials: JM
authors.lastname: Mellor
authors.forename: D W
authors.initials: DW
authors.lastname: Anderson
authors.forename: N G
authors.initials: NG
authors.lastname: Richards
authors.forename: D L
authors.initials: DL
authors.lastname: Turner
authors.forename: R P
authors.initials: RP
authors.lastname: Sharma
authors.forename: P
authors.initials: P
authors.lastname: Alexander
authors.forename: D E
authors.initials: DE
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D013237:Stereoisomerism; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C095673:2-azabicyclo(2.2.1)heptane-3-carboxylic acid; D001643:Bridged Bicyclo Compounds; D010455:Peptides; D016211:Transforming Growth Factor alpha; D011392:Proline