Peripheral CRF-R1/CRF-R2 antagonist, astressin C, induces a long-lasting blockade of acute stress-related visceral pain in male and female rats.

Peptide CRF antagonists injected peripherally alleviate stress-induced visceral hypersensitivity (SIVH) to colorectal distension (CRD) in rodents. Here we further evaluated the dose and time-dependent inhibitory activity of several long-acting peptide CRF receptor antagonists related to astressin on SIVH, focusing on astressin C (AstC), which previously showed high efficacy on stress-related alterations of HPA axis and gut secretomotor functions. Male and female Sprague-Dawley rats pretreated subcutaneously (SC) with AstC were injected intraperitoneally (IP) with CRF 15 min later.

A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin.

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity.

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.

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.

Unexpected sensitivity of sst2 antagonists to N-terminal radiometal modifications.

Chelated somatostatin agonists have been shown to be sensitive to N-terminal radiometal modifications, with Ga-DOTA agonists having significantly higher binding affinity than their Lu-, In-, and Y-DOTA correlates. Recently, somatostatin antagonists have been successfully developed as alternative tracers to agonists. The aim of this study was to evaluate whether chelated somatostatin antagonists are also sensitive to radiometal modifications and how.

Novel dimeric DOTA-coupled peptidic Y1-receptor antagonists for targeting of neuropeptide Y receptor-expressing cancers.

Background: Several peptide hormone receptors were identified that are specifically over-expressed on the cell surface of certain human tumors. For example, high incidence and density of the Y1 subtype of neuropeptide Y (NPY) receptors are found in breast tumors. Recently, we demonstrated that the use of potent radiolabeled somatostatin or bombesin receptor antagonists considerably improved the sensitivity of in vivo imaging when compared to agonists.

N-imidazolebenzyl-histidine substitution in somatostatin and in its octapeptide analogue modulates receptor selectivity and function.

Despite 3 decades of focused chemical, biological, structural, and clinical developments, unusual properties of somatostatin (SRIF, 1) analogues are still being uncovered. Here we report the unexpected functional properties of 1 and the octapeptide cyclo(3-14)H-Cys-Phe-Phe-Trp(8)-Lys-Thr-Phe-Cys-OH (somatostatin numbering; OLT-8, 9) substituted by imBzl-l- or -d-His at position 8. These analogues were tested for their binding affinity to the five human somatostatin receptors (sst(1-5)), as well as for their functional properties (or functionalities) in an sst(3) internalization assay and in an sst(3) luciferase reporter gene assay.

Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog.

Purpose: Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues.

The proposed sst(1) pharmacophore (J. Med. Chem.

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR.

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity.

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide.

Ring size in octreotide amide modulates differently agonist versus antagonist binding affinity and selectivity.

H-DPhe (2)-c[Cys (3)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Thr (15)-NH2 (1) (a somatostatin agonist, SRIF numbering) and H-Cpa (2)-c[DCys (3)-Tyr (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Nal (15)-NH2 (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst 2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst s.

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors.

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In.

Novel sst2-selective somatostatin agonists. Three-dimensional consensus structure by NMR.

The 3D NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II' beta-turn reported in the literature for sst2/3/5-subtype-selective analogues.

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2).

Somatostatin receptor 1 selective analogues: 4. Three-dimensional consensus structure by NMR.

The three-dimensional NMR structures of six analogues of somatostatin (SRIF) are described. These analogues with the amino acid 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 exhibit potent and highly selective binding to human SRIF subtype 1 receptors (sst(1)). The conformations reveal that the backbones of these analogues have a hairpin-like structure similar to the sst(2)-subtype-selective analogues.

Somatostatin receptor 1 selective analogues: 3. Dicyclic peptides.

The binding affinity of short chain somatostatin (SRIF) analogues at the five human SRIF receptors (sst) was determined to identify sterically constrained somatostatin receptor subtype 1 (sst(1)) selective scaffolds. Des-AA(1,2,4,13)-[d-Trp(8)]SRIF (2) retained high binding affinity at all receptors but sst(1), Des-AA(1,2,4,5)-[d-Trp(8)]SRIF (3) at sst(4) and sst(5), and Des-AA(1,2,4,5,13)-[d-Trp(8)]SRIF (4) at sst(2) and sst(4) (AA = amino acid). Des-AA(1,2,4,12,13)-[d-Trp(8)]SRIF (6) was potent and sst(4)-selective (>25-fold); Des-AA(1,2,5,12,13)-[d-Trp(8)]SRIF (7) and Des-AA(1,2,4,5,12,13)-[d-Trp(8)]-SRIF (9, ODT-8) were most potent at sst(4) and moderately potent at all other receptors.

Somatostatin receptor 1 selective analogues: 2. N(alpha)-Methylated scan.

Des-AA(1,2,5)-[d-Trp(8)/d-Nal(8),IAmp(9)]SRIF (AA = amino acid, Nal = 3-(2-naphthyl)-alanine, IAmp = 4-(N-isopropyl)-aminomethylphenylalanine, SRIF = somatostatin), with or without a tyrosine or monoiodotyrosine, were scanned with the introduction of a backbone N-methyl group and tested for binding affinity at the five human somatostatin receptors (sst(1)(-)(5)). N(alpha)-Methylation resulted in loss of sst affinity (2- to >5-fold) when introduced at residues Lys(4) (6), Phe(6) (7), Phe(7) (8), Thr(10) (11), and Phe(11) (12) of the parent compound Des-AA(1,2,5)-[d-Nal(8),IAmp(9)]SRIF (4). N(alpha)-Methylation was tolerated at residues Cys(3) (5), d-Nal(8) (9), Thr(12) (13), and Cys(14) (15) with retention of binding sst affinity and selectivity and resulted in an increase in sst binding affinity at positions IAmp(9) (10) and Ser(13) (14).

Novel sst(4)-selective somatostatin (SRIF) agonists. 4. Three-dimensional consensus structure by NMR.

The three-dimensional NMR structures of eight cyclic octapeptide analogues of somatostatin (SRIF) are described. These analogues, with the basic sequence H-c[Cys(3)-Phe(6)-Xxx(7)-Yyy(8)-Lys(9)-Thr(10)-Zzz(11)-Cys(14)]-OH (the numbering refers to the position in native SRIF), with Xxx(7) being Phe/Ala/Tyr, Yyy(8) being Trp/DTrp/D-threo-beta-Me2Nal/L-threo-beta-Me2Nal, and Zzz(11) being Phe/Ala, exhibit potent and highly selective binding to human SRIF type 4 (sst(4)) receptors. The conformations reveal that the backbones of these analogues do not have the usual type-II' beta-turn reported in the literature for sst(2)-subtype-selective analogues.

Novel sst(4)-selective somatostatin (SRIF) agonists. 3. Analogues amenable to radiolabeling.

After our discovery that H-c[Cys-Phe-Phe-DNal-Lys-Thr-Phe-Cys]-OH (ODN-8) had high affinity and marginal selectivity for human sst(3) (part 2 of this series: Erchegyi et al. J. Med.

Novel sst(4)-selective somatostatin (SRIF) agonists. 2. Analogues with beta-methyl-3-(2-naphthyl)alanine substitutions at position 8.

We present a family of human sst(4)-selective, high-affinity (IC(50) = 2-4 nM) cyclic somatostatin (SRIF) octapeptides. These peptides result from the substitution of dTrp(8) in H-c[Cys(3)-Phe(6)-Phe(7)-DTrp(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)]-OH (SRIF numbering) (ODT-8) by one of the four conformationally biased stereoisomers of beta-methyl-3-(2-naphthyl)alanine (beta-Me2Nal). Whereas H-c[Cys-Phe-Phe-DNal-Lys-Thr-Phe-Cys]-OH (ODN-8, 2) has high affinity and marginal selectivity for human sst(3) (Reubi et al.

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.

Six novel gonadotropin-releasing hormones are encoded as triplets on each of two genes in the protochordate, Ciona intestinalis.

GnRH is the key regulator of the reproductive axis in vertebrates, but little is known about GnRH before the origin of vertebrates. We have identified two genes encoding GnRH in a protochordate, Ciona intestinalis, thought to be related to the ancestral animal that gave rise to vertebrates. Each gene, Ci-gnrh1 and Ci-gnrh2, encodes in tandem three GnRH peptides, each of which is unique compared with known forms.