CONSTRAINED PEPTIDE ANALOGS OF TRANSFORMING GROWTH-FACTOR-ALPHA RESIDUES CYSTEINE-21-32 ARE MITOGENICALLY ACTIVE - USE OF PROLINE MIMETICS TO ENHANCE BIOLOGICAL POTENCY

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro(30) into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys(21)-Cys(32)). 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 EC(50) values in the range 130-330 mu M; 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.