CONVEYANCE OF PARTIAL AGONISM ANTAGONISM TO BOMBESIN GASTRIN-RELEASING PEPTIDE ANALOGS ON SWISS 3T3 CELLS BY A CARBOXYL-TERMINAL LEUCINE INSERTION

Gastrin-releasing peptide (GRP) is a neuroendocrine hormone that may be involved in the pathophysiology of small cell lung carcinoma. We describe carboxyl-terminal peptide analogues of GRP and bombesin, a 14-residue amphibian homologue, that were modeled after the antagonist [Leu13-psi(CH2NH)-Leu14]bombesin and retained the psi bond. Three novel peptides contained a Leu insertion amino to the psi bond, i.e. ...Leu13Leu14psiX (residues numbered after bombesin) where X = LeuNH2 or norleucine-NH2). The Leu-insertion analogues behaved as pure partial agonists/antagonists when examined for the ability to stimulate [H-3]thymidine incorporation into quiescent Swiss 3T3 cells (agonist activity) and to diminish the agonist response of GRP (antagonist activity). A time course of [H-3]thymidine incorporation into quiescent cells indicated maximal incorporation at 20-h post-peptide addition for bombesin and GRP and a Leu-insertion peptide, but the extent of the incorporation for the Leu-insertion peptide was half that of GRP and bombesin. The agonist dose responses of the Leu-insertion peptides (EC50 values of 1-10 nM) paralleled GRP and bombesin, but the maximal response of the Leu-insertion peptides, even at concentrations as high as 10(-4) M, was half the maximal value of GRP or bombesin. High concentrations of the Leu-insertion peptides antagonized 10 nM GRP (a concentration that produced a near-maximal GRP response) yielding a response that was half the maximal value of GRP and equivalent to the maximal response of the Leu-insertion peptides alone. Analogues of the form ... Leu13psiX behaved as complete antagonists. The K(D) values of the Leu-insertion peptides for competitive binding versus I-125-GRP (2-50 nM) were as potent as parent...Leu14 agonists. Stability studies indicated that peptide potencies for both agonist and antagonist activities diminished upon peptide incubation in medium or on cells. The results suggested that, for the Leu-insertion peptides, degradation into distinct products with different activities was not responsible for their partial agonist/antagonist behavior. Computer-generated molecular modeling studies indicated that the novel structures could adopt energy minimized conformations for either an agonist or an antagonist as proposed earlier (Coy, D. H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W. T., Gardner, J. D., and Jensen, R. T. (1988) J. Biol. Chem. 263, 5056-5060).