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Constrained Corticotropin-Releasing Factor (CRF) Agonists and Antagonists with i −( i +3) G lu-Xaa- d Xbb-Ly s Bridges

1998; American Chemical Society; Volume: 41; Issue: 25 Linguagem: Inglês

10.1021/jm980350k

1520-4804

Steven C. Koerber, József Gulyás, Sabine L. Lahrichi, Anne Corrigan, A. Grey Craig, Catherine Rivier, Wylie Vale, Jean Rivier,

Hormonal Regulation and Hypertension

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an α-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20−23)[dPhe12,Glu20,Lys23,Nle21,38]hCRF(12-41) and of astressin {cyclo(30−33)[dPhe12,Nle21,38,Glu30,Lys33]hCRF(12-41)}, two potent CRF antagonists, and of cyclo(30−33)[Ac-Leu8,dPhe12,Nle21,Glu30,Lys33,Nle38]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 Glu20−Lys23 and Glu30−Lys33 lactam rings were favoring an α-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, α-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 [dHis32] (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-Ala13-NH2 scaffold in order to quantify the structural influence of specific l- and dAla6 and l- and dAla7 substitutions in [Glu5,Lys8]Ac-Ala13-NH2 in a standard α-helical configuration. Models of the general form [Glu5,lAla6 or dAla6,lAla7 or dAla7,Lys8]Ac-Ala13-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 α-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 Gln26 in close enough proximity to Lys36 to suggest introduction of a bridge between them. We synthesized dicyclo(26−36,30−33)[dPhe12,Nle21,Cys26,Glu30,Lys33,Cys36,Nle38]Ac-hCRF(9-41) which showed significant α-helical content using circular dichroism (CD) and had low, but measurable potency {0.3% that of 6 or ca. 25% that of [dPhe12,Nle21,38]hCRF(12-41)}. Since the 26−36 disulfide bridge is incompatible with a continuous α-helix, the postulate of a turn starting at residue 31 will need to be further documented.