Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor

The neuropeptide galanin (1–29) binds with high affinity to hypothalamic receptors (K_D～ 0.9 nM) and regulates feeding behavior. The N-terminal fragments (1–16), (1–16)NH₂ are high affinity (K_D～ 6nM) full agonists in vivo and in vitro.l -Ala substitutions show that amino acid residues Gly¹, Trp², Asn⁵, Tyr⁹, and Gly¹² are important for the high affinity binding of galanin (1–16). Shortening the fragment (1–16) to galanin (1–7) causes a gradual drop of affinity: galanin (1–15), (1–14), and (1–13) have submicromolar K_D values and galanin (1–12) has K_D～ 3 μm . Cyclic analogs of galanin (1–12) of different ring size were synthesized by condensing Gly¹ and Gly¹² without or with spacer groups. These analogs, independent of ring size, had a lower affinity than the linear galanin (1–12). Derivatization of the N-terminus of galanin (1–29), (1–16), and (1–12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N-terminal Gly.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).     

Novel galanin receptor ligands

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(l-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(l-13) was C-terminally elongated with Lys¹⁴; different pharmacologically active small molecules were then attached to the ε-amino group of Lys¹⁴. Several cysteine-substituted linear and ring closed analogs of galanin(l-9) and galanin(l-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.     

Structural comparison of alanine-substituted analogues of the calcitonin gene-related peptide 8-37 Importance of the C-terminal segment for antagonistic activity

Replacement of specific residues of the antagonistic fragment human calcitonin gene-related peptide 8–37 (hCGRP 8–37) by alanine residues produces good antagonists to CGRP1 receptors when the replacement is made at positions 17 and 20 but a poor antagonist when the replacement is made at position 21. The solution structures of hCGRP 8-37 and of the three alanine analogues have been determined by two-dimensional ¹H NMR spectroscopy and molecular modeling. Following the complete assignment of the NMR spectra, a comparison of the chemical shifts and of the temperature dependence of the amide chemical shifts showed that these parameters differed for [Ala¹⁷]-hCGRP 8–37 and [Ala²⁰]-hCGRP 8–37 relative to hCGRP 8–37 in the N-terminal and central segments but not in the C-terminal segment (residues 31–37). In the case of [Ala²¹]-hCGRP 8–37, differences were observed all along the chain. Molecular modeling calculations were performed by distance geometry, simulated annealing and energy minimization using NOE distance constraints. Molecular models showed a structural homology between [Ala¹⁷]-hCGRP 8–37, [Ala²⁰]-hCGRP 8–37 and hCGRP 8–37 in the C-terminal segment Asn³¹-Phe³⁷ as well as hydrogen bonding between Val²⁸ and Asn³¹. These structural similarities are not observed with [Ala²¹]-hCGRP 8–37. Therefore, the structure of the C-terminal segment of hCGRP 8–37 appears to be critical for antagonistic activity at CGRP1 receptors.     

Galanin: structure-dependent effect on pancreatic amylase secretion and jejunal strip contraction

Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1–29) = porcine galanin-(1–19) = galanin-(1–15) = rat galanin-(3–29) > rat galanin-(2–29) = porcine galanin-(2–29) > galanin-(1–10). Fragments of rat galanin-(9–29) and N^α-acetyl-galanin-(9–29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15–29) and rat galanin-(21–29) were unable to produce significant inhibition. Rat and porcine galanin-(1–29), galanin-(1–15) and rat N^α-acetyl-galanin-(9–29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1–29) and porcine galanin-(1–29) have similar potencies. Galanin-(1–15) and galanin-(1–10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly¹ from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3–29) and (9–29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9–29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone

In memoriam Howard S. Tager Within the scope of structure-function studies on the proteohormone insulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26-B30 were synthesized by trypsin-catalyzed coupling reactions of des-(B23-B30)-insulin with synthetic peptides. The peptides were obtained by Fmoc solid-phase peptide synthesis. Insulins with multiple amino acid → glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27-B30 on the self-association of the hormone. The analogues [Gly^{B27,B28,B29,B30}]insulin and [Gly^B27,B28,B30]insulin exhibit relative receptor affinities of 80% and self-associate. The successive extension of [Ala^B26]des-(B27-B30)-insulin-B26-amide (relative receptor binding 273%) with amino xids corresponding to the native sequence B27-B30 showed the influence of the length of the B-chain on receptor affinity: the extension by B27-threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28-side chain on main-chain conformation, self-association and receptor binding was examined with [X^B28]des-(B29-B30)-insulin-B28-amides (X = Phe, Gly, D-Pro). While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [Phe^B28]des-(B29-B30)-insulin-B28-amide (relative binding 50%) shows diminished self-association. The backbone-modified insulin derivative [Sar^B26]des-(B27-B30)-insulin-B26-amide (sarcosine =N-methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding. © Munksgaard 1995.     

The critical C-terminus of the small subunit of herpes simplex virus ribonucleotide reductase is mobile and conformationally similar to C-terminal peptides

The C-terminus of the small subunit of class I ribonucleotide reductases is essential for subunit association and enzymatic activity. ¹H NMR analysis of the small subunit (2 × 38 kDa as a homodimer) of herpes simplex virus ribonucleotide reductase shows that this critical binding site is mobile and exposed in relation to the rest of the protein. Assignments of six C-terminal amino acids are made by comparing the TOCSY and NOESY spectra of the small subunit with the spectra of an identical protein truncated by seven amino acids at the C-terminus and the spectra of an analogous 15 amino acid peptide. The mobility of the C-terminus may be important for subunit recognition and could be general for other ribonucleotide reductases. The spectral comparisons also suggest that the six C-terminal amino acids of the small subunit and peptide are confor-mationally similar. This observation may be important for the design of inhibitors of ribonucleotide reductase subunit association. © Munksgaard 1994.     

Synthesis and biological activity of dynorphin-(1 - 13) and analogs substituted in positions 8 and 10

Dynorphin-(1–13) (Dyn-(1–13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [³H]-ethylketocyclazocine (EKC, k ligand), [³H]-[D-Ala², MePhe⁴-Gly-ol⁵]-enkephalin (DAGO, μ ligand) and [³H]-[D-Ser², Thr⁶]-Leu-enkephalin (DSLET, δ ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the k receptor ([³H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1–13)). On the other hand, the ability of [Ala⁸]-Dyn-(1–13) to displace the binding of [³H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the μ receptor was not greatly affected ([³H]-DAGO displacement; Ki of 0.44nM as compared with 0.50nM for Dyn-(1–13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the k site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro¹⁰]-Dyn-(1–13) was equipotent to [Ala⁸]-Dyn-(1–13) in the GPI but its affinity for the μ binding site was decreased by a factor of 2.7 as compared with Dyn-(1–13). The affinity of [D-Pro¹⁰]-Dyn-(1–13) for the other binding sites (k and δ) was not greatly affected. Replacement of positions 8 or 10 by Trp or D-Trp decreased the activity on the GPI by more than 50% as well as the affinity for most receptor types. These data indicate that the selectivity of Dyn(1–13) for the k opioid receptor can be increased either by increasing its affinity for the K binding site ([Ala⁸]-Dyn-(1–13)) or lowering its potency for other binding sites, [Ala⁸]-Dyn-(1–13) being less potent on δ sites and [D-Pro¹⁰]-Dyn-(1–13) less potent on μ sites.     

Opioid peptides Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) were replaced by N^α-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of μ, Δ, and κ opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the κ binding site. The introduction of N^α-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed μ/δ specificity.     

Comparative biological activities of potent analogues of α-melanotropin

Several α-melanotropin (α-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO₂), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle⁴] -α-MSH_4–11 - NH₂ fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle⁴, Ala⁷]-α-MSH_4–11-NH₂ was more potent than Ac-[Nle⁴, D-Ala⁷]-α-MSH_4–11-NH₂ in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of α-MSH, to biological potency of the hormone.     

Structure activity of C-terminal modified analogs of Ac-CCK-7

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of chole-cystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe³³-NH₂] (2) in which the phenyl ring of the C-terminal Phe-NH₂ has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyi or bicyclic aryl moieties for the phenyl ring of phenylalanine³³ including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10–70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine³³ aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC₅₀s 10^-11-10^-14 M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine³³ and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.     

Structural requirements for dermorphin opioid receptor binding

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) was the most potent competitor in all experiments. The d -configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of d -Ala² with l -Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH₂ and dermorphin-(1-3)-NH₂, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.     

Peptide N-alkylamides by solid phase synthesis*

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH₂ CH₃, NH₂ CH₂ CH₃, NH₂ CH₂ CF₃) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [D Glu⁶, Pro⁹-NHCH₂ CH₃]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [D Glu⁶]-GnRH. Other analogs including [D Trp⁶, Pro⁹-NHCH₂ CH₃]-GnRH, [D Ala⁶, Pro⁹-NHCH₂ CF₃]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

New high affinity peptide antagonists to the spinal galanin receptor.

1. The role of endogenous galanin in somatosensory processing has been studied with galanin receptor antagonists. The new galanin receptor ligands C7, M32, M38 and M40 bind with high affinity (Kd in nanomolar range) to spinal cord galanin receptors and possess oxidative stability as compared to earlier generations of peptide ligands. These peptides have been examined in the spinal flexor reflex model where exogenous galanin exhibited biphasic excitatory and inhibitory effects. 2. Intrathecal administration of C7 [galanin(1-13)-spantide] and M32 [galanin (1-13)-neuropeptide Y(25-36) amide] blocked facilitation of the nociceptive flexor reflex induced by 30 pmol intrathecal galanin in decerebrate, spinalized rats in a dose-dependent manner, thus behaving as antagonists of the galanin receptor. In contrast, M38[galanin(1-13)-(Ala-Leu)3-Ala amide] and M40 [galanin(1-13)-Pro-Pro-(Ala-Leu)2-Ala amide], exhibited only weak antagonism at high doses in this model. Moreover, lower doses of M40 potentiated galanin-induced reflex facilitation. C7 was neurotoxic at high doses in the rat spinal cord. 3. M32 and C7 were potent antagonists of galanin receptors in rat spinal cord, in correlation with their in vitro binding characteristics. In contrast, M38 and M40, despite their high in vitro affinity, exhibited only very weak antagonism. Moreover, M40 may also behave as a partial agonist. 4. Previous studies have shown that the galanin receptor may be heterogeneous. The discrepancy between in vitro binding and in vivo antagonistic potency of M38 and M40 may also suggest the presence of different galanin receptor subtypes within the rat spinal cord. However, other explanations for the discrepancy, such as differences in metabolic stability, diffusion rates and penetration to the site of action are also possible.     

Synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by a β-alanine

Syntheses of analogues of the C-terminal heptapeptide of cholecystokinin are described. These analogues were obtained by replacing glycine 29 by a β-alanine. The C-terminal phenylalanine amide was in some cases substituted by 2-phenylethyl alcohol and/or residues of the C-terminal tetrapeptide by their d -enantiomers. These compounds were tested for their action on stimulation of amylase release from rat pancreatic acini and for their ability to inhibit binding of labeled CCK to rat pancreatic acini and guinea pig brain membranes. Some of these derivatives behaved as CCK receptor antagonists.     

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D 1H NMR and molecular dynamics

Peptides derived from the inhibitor of cAMP-dependent protein kinase, PKI, have been studied by 2D ¹H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala²⁰-Ser²¹] PKI(5-24), and a phosphorylated form of the latter [Ala²⁰-Ser²¹P] PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI (5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly¹⁴-Gly¹⁷ turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp⁹ and Arg¹⁵. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2°C) shows the occurrence of pH-dependent contacts with Phe¹⁰. We conclude that the binding of short inhibitors, such as PKI (5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala²⁰-Ser²¹] PKI (5-24) and the product [Ala²⁰-Ser²¹P] PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. © Munksgaard 1997.     

The role of galanin in modulating stress-related neural pathways

Galanin is a 29 or 30 amino acid peptide with a close anatomical and neurophysiological relationship with neural systems mediating stress. In rodent behavioral studies, the consequences of galanin activity on stress-related behaviors such as anxiety-like and depression-like behavior varies according to site of administration and behavioral assay used. However, galaninergic systems remain a promising novel target for new antianxiety and antidepressant drugs. Current work attempting to delineate which galanin receptor subtypes mediate which effects of galanin will undoubtedly inform the development of these drugs. Further, the well-established deleterious effects of galanin on cognitive function provide support for galanin as a novel link between stress, anxiety and memory. This link suggests that galaninergic drugs could provide a novel therapeutic option for psychopathologies, such as post-traumatic stress syndrome, in which abnormal memory and emotion are intertwined.     

Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists

Galanin, a 29 amino acid peptide, has been reported to possess antinociceptive properties at the spinal site and to potentiate opioid-induced antinociception. Our aim was to investigate whether also endogenous galanin interacts with an exogenously administered opioid, morphine, in the rat spinal cord. This question was investigated by use of the recently developed galanin receptor antagonists galantide [M-15, galanin(1–13)-substance P-(5 -11) amide] and M-35 [galanin-(1–13)-bradykinin-(2–9) amide].Nociception was assessed in the rat tail-flick test using radiant heat and the rat Randall-Selitto model of inflammatory pain using vocalization as the nociceptive criterion. Intrathecal (i.t.) injections were performed in rats under ether anaesthesia. Morphine was administered either i.t. or intraperitoneally (i.p.), and the antagonists were injected i.t. [¹²⁵I]Galanin binding experiments were performed on crude synaptosomal membranes of the rat spinal cord.In the rat tail-flick test, i.t. injection of 3 g morphine evoked antinociception of about 75% of the maximal possible effect (% MPE). Co-injection of either 2 g galantide or 2 g M-35 with morphine almost completely abolished the antinociceptive effect of morphine. I.p. injection of 2.15 mg/kg morphine elicited about 80% MPE when given 10 min prior to i.t. saline injection. Injection of the antagonists instead of saline antagonised the antinociceptive effect of morphine partially thus showing the spinal proportion of the overall antinociceptive effect. In the rat Randall-Selitto test, 3 g morphine, injected i.t., produced antinociception of almost 100% MPE. Coinjection of the antagonists reduced the maximum effect partially by about 25–35%. I.p. injection of 7.5 mg/kg morphine 10 min prior to Lt. injection of saline elicited an antinociceptive effect of 90–100% MPE; injection of the antagonists instead of saline reduced the peak effect to a similar degree as after i.t. injection of 3 g morphine. To exclude a direct interference by morphine with the galanin receptor, in vitro binding of [¹²⁵I]galanin to a spinal synaptosomal fraction was assessed. Morphine, 10 M, did not interfere with the specific [¹²⁵I]galanin binding. These results provide further evidence that galanin is involved in spinal nociceptive processing. It seems to be involved in the mediation of the effects of morphine at this site, either as a co-transmitter, or subsequent to µ-receptor activation on nerve terminals or on interneurones. Correspondence to: W. Reimann at the above address     

Structural requirements for galanin inhibition of pentagastrin-stimulated gastric acid secretion in conscious rats.

The effects of rat galanin, together with a number of its N- and C-terminal fragments, on pentagastrin-stimulated gastric acid secretion were studied in conscious rats equipped with chronic gastric fistulas. Similarly to its porcine counterpart studied previously, at a dose of 3 nmol/kg per h rat galanin was a potent inhibitor of gastric acid secretion. The N-terminal fragments, rat galanin-(1-10) and -(1-15), retained about 60% of the inhibitory potency of the whole galanin sequence whilst the C-terminal fragments, rat galanin-(2-29), -(3-29) and -(9-29), were unable to produce significant inhibition over comparable dose ranges. Surprisingly, however, simply acetylating the alpha-amino group in position 9 of rat galanin-(9-29) restored almost full gastric acid inhibitory activity in a homologous rat model. We speculate that this could be due to a favorable conformational effect on the C-terminal region produced by alpha-acetylation. These results also suggest that structural features within either the N-terminal or C-terminal regions of rat galanin are able to elicit this particular biological response. One possible explanation for this could be the involvement of more than one rat galanin receptor having different ligand recognition requirements.