Structure–Activity Studies on the Corticotropin Releasing Factor Antagonist Astressin, leading to a Minimal Sequence necessary for Antagonistic Activity

Abstract

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.

NMR Structure of the First Extracellular Domain of Corticotropin-releasing Factor Receptor 1 (ECD1-CRF-R1) Complexed with a High Affinity Agonist

“…On the one hand, structureactivity studies of the CRF family ligands showed that the first six residues of the N-terminal portion of the peptide are not necessary for receptor signaling as long as ␣-helicity is maintained (3, 4, 16 -18). On the other hand, the C-terminal (ϳ15) residues contribute significantly to receptor binding affinities (16,19). Similar data, both on signaling and binding properties, have been reported for the other members of the B1 GPCR family and their corresponding ligands (20 -24).…”

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

“…These data are reflected in the 20 conformers representing the 3D structure of ECD1-CRF-R2␤-astressin complex for which continuous helical hydrogen bonds are observed from CO i to NH iϩ4 for residues 27-37 of astressin. The C-terminal four residues prefer to be in a 3 10 -helical conformation, which is supported by the presence of ␣H i -HN iϩ2 NOEs and the missing Nle-38(␣H)-Ile-41(␤H) NOE (SI Fig. 5B).…”

“…5B). The preference for a 3 10 helix over an ␣-helix toward the C terminus of helical peptides and proteins is often reported (26). Of special interest are the hydrogen bonds between the carbonyl of Glu-39 and the C-terminal NH 2 group, and between the carbonyl of Ile-41 and the amide of Val-113 of ECD1.…”

“…These belong to the peptide hormone B1 family (family B1 GPCRs), comprising receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, and parathyroid hormone. Two CRF receptors, CRF-R1 and CRF-R2, have been cloned in mammals (6, 7).Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was also observed for the receptors (11,12).…”

“…Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was also observed for the receptors (11,12).…”