Identification of receptor-binding pharmacophores of growth-hormone-releasing factor in rat adenopituitary.

Previous structure-activity studies on growth-hormone-releasing factor (GRF) have mainly been carried out in pituitary cell culture assays. In such systems, the molecular features necessary to increase GRF receptor affinity cannot be fully distinguished from those that improve proteolytic resistance. To assess the affinity of GRF analogues, we have recently characterized [125I-Tyr10]hGRF(1-44)NH2 binding to rat adenopituitary, developing a reliable binding assay in which GRF-carboxamide-related peptides are stable. In the present study, we have determined the binding affinity of two series of analogues in which the entire sequence of hGRF(1-29)NH2 was scanned with D-amino acid and alanine substitutions. To further document their potency, we have evaluated the ability of representative candidates of each series to stimulate cAMP production. In the first series, a D-amino acid substitution at Ala4, Ile5, Phe6, Thr7, Val13, Gln16, Leu17, Ala19, Arg20 and Ile26 decreased drastically the binding affinity of hGRF(1-29)NH2 while it induced a smaller decrease at Tyr1, Asp3, Ser9, Tyr10, Arg11, Lys12, Leu14, Ala15, Ser18, Lys21, Leu22, Leu23, Gln24, Met27 and Ser28. Interestingly, a D-substitution in position 8 generated an analogue exhibiting a significantly greater binding affinity than hGRF(1-29)NH2, while it had no influence on hGRF(1-29)NH2 affinity at Ala2, Asp25 and Arg29. Adenylate cyclase activities of [D-Tyr1], [D-Tyr10] and [D-Arg20]hGRF(1-29)NH2 correlate with their binding affinity. In the second series, the largest decrease of binding affinity was observed with an alanine substitution at Tyr1, Asp3, Ile5, Phe6, Tyr10, Arg11, Lys12, Leu14, Leu17, Arg20 and Lys21.(ABSTRACT TRUNCATED AT 250 WORDS)