Constrained corticotropin-releasing factor (CRF) agonists and antagonists with i-(i+3) Glu-Xaa-DXbb-Lys bridges

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the human corticotrapin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20-23)[DPhe(12),Glu(20),Lys(23),Nle(21,38)]hCRF((12-41)) and of astressin {cyclo(30-33)[DPhe(12),Nle(21,38),Glu(30),Lys(33)]hCRF((12-41))}, two potent CRF antagonists, and of cyclo(30-33)-[Ac-Leu(8),DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((8-41)), the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. 1998, 41, 2614-2620 and references therein). To test the hypothesis that the Glu(20)-Lys(23) and Glu(30)-Lys(33) lactam rings were favoring an alpha-helical conformation rather than a turn, we introduced a D-amino acid at positions 22, 31, and 32 in the respective rings, Whereas the introduction of a D-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a D-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, alpha-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [DHis(32)] (6) or acetylation of the N-terminus (7) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala(13)-NH2 scaffold in order to quantify the structural influence of specific L- and DAla(6) and L- and DAla(7) substitutions in [Glu(5),Lys(8)]Ac-Ala(13)-NH2 in a standard alpha-helical configuration. Models of the general form [Glu(5),LAla(6) or DAla(6),LAla(7) or DAla(7),Lys(8)]Ac-Ala(13)-NH2 were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0-4, 1-5, and 8-12 O-H hydrogen bond donor-acceptor pairs to maintain alpha-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix-turn-helix motif around a turn centered at residue 31. Such a turn brings Gln(26) in close enough proximity to Lys(36) to suggest introduction of a bridge between them. We synthesized dicyclo(26 -36,30 -33)[DPhe(12),Nle(21),Cys(26),Glu(30),Lys(33),Cys(36),Nle(38)]Ac-hCRF((9-41)) which showed significant alpha-helical content using circular dichroism (CD) and had low, but measurable potency {0.3% that of 6 or ca. 25% that of [DPhe(12),Nle(21,38)]hCRF((12-41))}. Since the 26-36 disulfide bridge is incompatible nith a continuous alpha-helix, the postulate of a turn starting at residue 31 will need to be further documented.