POTENT, STRUCTURALLY CONSTRAINED AGONISTS AND COMPETITIVE ANTAGONISTSOF CORTICOTROPIN-RELEASING FACTOR

Predictive methods, physicochemical measurements, and structure activi ty relationship studies suggest that corticotropin-releasing factor (C RF; corticoliberin), its family members, and competitive antagonists ( resulting from N-terminal deletions) usually assume an cu-helical conf ormation when interacting with the CRF receptor(s). To test this hypot hesis further, we have scanned the whole sequence of the CRF antagonis t [D-Phe(12),Nle(21,38)]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, no rleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys s caffold. We have found astressin -Phe(12),Nle(21,38),Glu(30),Lys(33)]r /hCRF(12-41)} to be approximately 30 times more potent than [D-Phe(12) ,Nle(21,38)] r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary c ell culture assay. Astressin has low affinity for the CRF binding prot ein and high affinity (K-i = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-I-125-Tyr(12)] astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more po tent than any previously tested antagonist in reducing hypophyseal cor ticotropin (ACTH) secretion in stressed or adrenalectomized rats. The clo(30-33)[Ac-Pro(4),D-Phe(12),Nle(21,38),Glu(30), Lys(33)] r/hCRF-(4- 41) agonist and its linear analog are nearly equipotent, while the ant agonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural con straint in the antagonists that is normally induced by the N terminus of the agonist.