Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

NMR structure and peptide hormone binding site of the first extracellular domain of a type B1 G protein-coupled receptor.

The corticotropin-releasing factor (CRF) ligand family has diverse effects on the CNS, including the modulation of the stress response. The ligands' effects are mediated by binding to CRF G protein-coupled receptors. We have determined the 3D NMR structure of the N-terminal extracellular domain (ECD1) of the mouse CRF receptor 2beta, which is the major ligand recognition domain, and identified its ligand binding site by chemical-shift perturbation experiments. The fold is identified as a short consensus repeat (SCR), a common protein interaction module. Mutagenesis reveals the integrity of the hormone-binding site in the full-length receptor. This study proposes that the ECD1 captures the C-terminal segment of the ligand, whose N terminus then penetrates into the transmembrane region of the receptor to initiate signaling. Key residues of SCR in the ECD1 are conserved in the G protein-coupled receptor subfamily, suggesting the SCR fold in all of the ECD1s of this subfamily.

Novel sst(4)-selective somatostatin (SRIF) agonists. 1. Lead identification using a betide scan.

Hypothesizing that structural constraints in somatostatin (SRIF) analogues may result in receptor selectivity, and aiming to characterize the bioactive conformation of somatostatin at each of its five receptors, we carried out an N(beta)-methylated aminoglycine (Agl) scan of the octapeptide H-c[Cys(3)-Phe(6)-Phe(7)-dTrp(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)]-OH (SRIF numbering) (ODT-8) that is potent at all SRIF receptor subtypes (sst's) but sst(1). We found that H-c[Cys-LAgl(N(beta)Me,benzoyl)-Phe-DTrp-Lys-Thr-Phe-Cys]-OH (4), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-Trp-Lys-Thr-Phe-Cys]-OH (6), H-c[Cys-Phe-LAgl(N(beta)Me,benzoyl)-dTrp-Lys-Thr-Phe-Cys]-OH (8), and H-c[DCys-Phe-LAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (10) had high affinity (IC(50) = 14.3, 5.4, 5.2, and 3.4 nM, respectively) and selectivity for sst(4) (>50-fold over the other receptors). The l-configuration at positions 7 and 8 (l(7), l(8)) yields greater sst(4) selectivity than the l(7), d(8) configuration (6 versus 8). Peptides with the d(7), l(8) (7) and d(7), d(8) (9) configurations are significantly less potent at all receptors. H-c[Cys-Phe-Phe-DTrp-LAgl(betaAla)-Thr-Phe-Cys]-OH (16), H-c[Cys-Phe-Phe-DTrp-DAgl(betaAla)-Thr-Phe-Cys]-OH (17), and their N(beta)Me derivatives at position 9 (18, 19) were essentially inactive. Potent but less sst(4)-selective were members of the Agl-scan at positions 10, H-c[Cys-Phe-Phe-dTrp-Lys-lAgl(N(beta)Me,HO-Ac)-Phe-Cys]-OH (20, IC(50) = 6.5 nM), and 11, H-c[Cys-Phe-Phe-DTrp-Lys-Thr-LAgl(N(beta)Me,benzoyl)-Cys]-OH (22, IC(50) = 6.9 nM), while the d-configuration at positions 10 (21) and 11 (23) led to reduced affinity. One of our best analogues, 8, is an agonist when tested for its ability to inhibit forskolin-stimulated cAMP accumulation in sst(4)-transfected CCL39 cells (EC(50) = 1.01 nM). All Agl-containing analogues were first synthesized using unresolved Fmoc-Agl(N(beta)Me,Boc)-OH, and the diastereomers were separated using HPLC. Chiral assignment at the Agl-containing residue was subsequently done using enzymatic degradation and by de novo synthesis in the cases of H-c[Cys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (9) and H-c[DCys-Phe-DAgl(N(beta)Me,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH (11), starting with the papain-resolved Fmoc-DAgl(Boc). These results suggested that the orientation of side chains at position 6, 7, or 11 with respect to the side chains of residues 8 and 9 may be independently responsible for sst(4) selectivity.