MINIMAL-SIZE, CONSTRAINED CORTICOTROPIN-RELEASING FACTOR AGONISTS WITH I-(I-LYS AND LYS-GLN BRIDGES(3) GLU)

In three earlier publications (Miranda et al. J. Med. Chem. 1994, 37, 1450-1459; 1997, 40, 3651-3658; Gulyas et al. Proc. Natl, Acad. Sci. U .S.A. 1995, 92, 10575-10579) we have hypothesized that covalent constr aints such as side-chain-to-side-chain lactam rings would stabilize an or-helical conformation shown to be important for the recognition and binding of the CRF C-terminus 30 residues, to CRF receptors. These st udies led to the discovery of useful CRF antagonists such as alpha-hel ical CRF (alpha-hel-CRF) and Astressin both in vitro and in vivo. To t est the hypothesis that such lactam rings may also be modulating activ ation of the receptor when introduced at the N-terminus of CRF, we stu died the influence of the successive introduction from residues 4 to 1 4 of a cyclo(i,i+3)[Lys(i)-Glu((i+3))] and a cyclo(i,i+3)[Glu(i)-Lys(( i+3))] bridge on the in vitro potency of the agonist [Ac-Pro(4),DPhe(1 2),Nle(21,38)]hCRF((4-41)) and related compounds. We have also introdu ced the favored cyclo(Glu(30)-Lys(33)) substitution found to be remark able in several families of antagonists (such as Astressin) and in a n umber of CRF agonists and investigated the role of residues 4-8 on rec eptor activation using successive deletions. Earlier studies had shown that in both oCRF and ct-helical CRF, deletion of residues 1-6, 1-7, and 1-8 led to gradual loss of intrinsic activity (IA) (from 50% IA to <10% IA) resulting in cr-hel-CRF being a patent competitive antagonis t. We show that acetylation of the N-terminus of these fragments gener ally increases potency by a factor of 2-3 with no influence on IA. Whi le cyclo(30-33)[Ac-Leu(8),DPhe(12),Nle(21),Glu(30), Lys(33),Nle(38)]hC RF((8-41)) (30) is the shortest reported analogue of CRF to be equipot ent to CRF (70% IA), the corresponding linear analogue (31) is 120 tim es less potent (59% IA). Addition of one amino acid at the N-terminus {cyclo(30-33)[Ac-Ser(7),DPhe(12),Nle(21),Glu(30) Lys(33),Nle(38)]hCRF( (7-41)) (28)} results in a 5-fold increase in agonist potency and full intrinsic activity (113%). The most favored modifications were also i ntroduced in other members of the CRF family including sauvagine (Sau) , urotensin (Utn), urocortin (Ucn), and alpha-hel-CRF. Parallel and co nsistent results were obtained suggesting that the lactam cyclization at residues 29-32 and 30-33 (for the members of the CRF family with 40 and 41 amino acid residues, respectively) will induce (in the shorten ed agonists) a structural constraint (alpha-helix) that stabilizes a b ioactive conformation similar to that shown in the Astressin family of CRF antagonists and that residue 8 (leucine or isoleucine) bears the sole responsibility for activation of the receptor since deletion of t hat residue leads to potent antagonists (Gulyas et al. Proc. Natl. Aca d. Sci. U.S.A. 1995, 92, 10575-10579).