Opiate receptor binding affinities of some D-amino acid substituted beta-casomorphin analogs

beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.     

Structure, biological activity and membrane partitioning of analogs of the isoprenylated a-factor mating peptide of Saccharomyces cerevisiae.

Previous biochemical investigations on the Saccharomyces cerevisiae a-factor indicated that this lipopeptide pheromone [YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II beta-turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a-factor, in which residues at positions 4 and 5 were replaced with: L-Pro4(I); D-Pro4(II); L-Pro4-D-Ala5(III); D-Pro4-L-Ala5(IV); or Nle4(V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a-factor analogs I and III were found to be almost 50-fold more active than the diastereometric homologs II and IV and were equally active to wild-type a-factor. Replacement of Lys4 with the isosteric Nle4 almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II beta-turn compensated for the loss of the favorable contribution of the Lys4 side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H2O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a-factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity.     

Spinal actions of substance P analogues on cardiovascular responses in the rat: a structure-activity analysis

Ten substance P (SP) analogues were tested for their effects on mean arterial pressure and heart rate following intrathecal administration in the pentobarbital anaesthetized rat. The 10 analogues are [D-Pro4,D-alpha Npa7,9,10]SP(4-11) (A-I), (D-alpha Npa7,9,10]SP (A-II), [D-Trp7,9,10]SP (A-III), [D-Pro4,D-Npa7,9,Phe11]SP(4-11) (A-IV), [D-Pro4,D-beta Npa7,D-alpha Npa9,D-Phe11]SP(4-11) (A-V), [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP(4-11) (A-VI), [D-Pro4,D-Trp7,9,10,Phe11]SP(4-11) (A-VII), [D-Pro4,D-Trp7,9,10,Trp11]SP(4-11) (A-VIII), [D-Trp7,9,10,Trp11]SP (A-IX), and [D-Pro4,D-Phe7,9,10,Phe11]SP(4-11) (A-X). At 6.5 nmol, the analogues containing the amino acid D-Npa (A-I, A-II, A-IV, and A-V) or D-Phe (A-X) in positions 7, 9, or 10 of SP or its C-terminal octapeptide are devoid of the long-lasting cardio- and vaso-depressor effects, which are otherwise seen with analogues containing the amino acid D-Trp (A-III, A-VI, A-VII, A-VIII, and A-IX) in the same positions. Some of the analogues containing D-Npa maintain the initial hypotensive effect seen with SP while the analogue containing D-Phe produces only a small hypertensive response. The 10 analogues when tested at a dose that failed to alter basal mean arterial pressure and heart rate did not block the cardiovascular responses elicited by SP and no cross desensitization was observed between SP and these analogues. It appears that these SP analogues exert cardiovascular effects in the rat spinal cord probably without interacting with SP receptors.     

Species differences in the behavioral toxicity produced by intrathecal substance P antagonists: relationship to analgesia

Intrathecal administration of [D-Pro2,D-Trp7,9]-substance P to rats produced an irreversible flaccid paralysis of the hind limbs (paraplegia) which was irreversible with an ED50 of 2.3 micrograms. At 5 micrograms intrathecally, [D-Pro2,D-Phe7,D-Trp9]-substance P, [D-Trp7,9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P octapeptide also produced paraplegia (70-80%). Surprisingly, intrathecal administration of up to 20 micrograms of these analogs to the mouse produced no paraparesis or paraplegia. In the guinea pig or rabbit, 20 micrograms of [D-Pro2,D-Trp7,9]-substance P or [D-Pro4,D-Trp7,9,10]-substance P octapeptide were also devoid of paraparetic effects. Lidocaine hydrochloride, on the other hand, was equieffective across species in producing paraplegia (which was reversible) suggesting that interspecies susceptibility is not a factor in the marked species differences between substance P analogs. In the mouse, intrathecal [D-Pro2,D-Trp7,9]-substance P was active in tail-flick and hot-plate tests at doses showing no overt behavioral effects but in the rat was not analgesic at sub-paraplegic doses. Lidocaine hydrochloride (i.t.) was analgesic in mouse and rat tail-flick tests at doses two times less than paraplegic doses; however, there was an overlap in analgesic and paraplegic doses. Based on these data, we suggest that the rat is unique in being extremely sensitive to the paraplegic effects of intrathecal neurokinin antagonists and may simply be a poor species in which to study the spinal functionality of neurokinins.     

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.     

Long-acting oxytocin antagonists: effects of 2-D-stereoisomer substitution on antagonistic potency and duration of action

Recently we reported the discovery of a series of 2-O-alkyltyrosine- (or 2-p-alkylphenylalanine), 4-threonine-, and 8-ornithine-substituted analogs of [1-penicillamine]oxytocin [( Pen1]OT) which possess prolonged anti-OT activity. In this study, we attempt to improve the potency and the duration of action of this series of OT antagonists by exploring the effects of D-stereoisomer substitution in the 2 position. We compare the in vitro anti-OT potency, expressed in pA2 values, and the duration of in vivo inhibitory action, expressed in recovery t1/2, of [Pen1]OT, [Pen1,Orn8]OT, [Pen1,Thr4]OT, [Pen1,Tyr(OMe)2,Thr4, Orn8]OT, [Pen1, Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,D-Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,Phe2,Thr4]OT, [Pen1,Phe(Me)2,Thr4,Orn8]OT, [Pen1,D-Phe(Me)2,Thr4,Orn8]OT, [Pen1,Phe(Et)2,Thr4,Orn8]OT, and [Pen1,D-Phe(Et)2,Thr4,Orn8]OT. The results show that modifications of the amino acid in position 2 by alkylation of the aromatic ring and use of D-stereoisomerism produce nonparallel effects on the in vitro potency and duration of action of OT antagonists. Time-action curve determinations show that long-acting OT antagonists exhibit delayed peak inhibitory action. Long action is not coupled with high potency in all cases. This dissociation between potency and duration of action gives support to our hypothesis that the potency and duration of action of these peptides may each have different conformational structure requirements.     

Horse urinary kallikrein, II. Effect of subsite interactions on its catalytic activity

The effect of secondary-subsite interactions on the catalytic efficiency of horse urinary kallikrein was studied using as substrates oligopeptides and peptidyl-4-nitroanilides with L-Arg at P1. The known secondary specificity of tissue kallikreins for hydrophobic residues at P2 was also demonstrated for horse urinary kallikrein and a higher preference of this enzyme for L-Phe over L-Leu at P2 was evident. Interaction of subsites S3 with D-Pro and D-Phe enhanced the catalytic efficiency but tripeptidyl-4-nitroanilides with acetyl-D-Pro, L-Pro and acetyl-L-Pro at P3 were no better substrates than acetyl-dipeptidyl-4-nitroanilides. The importance of the leaving group for the catalysis was proved by higher kcat/Km values for the peptides in relation to peptidyl-4-nitroanilides containing a common acyl-chain. The low kcat value for the peptide with L-Pro at P'2 stresses the importance of a hydrogen bond between P'2 amide and the carbonyl group at S'2. One L-arginine residue at the leaving group, specially at the P'2 position, decreases the value of the apparent Km. This effect resulting of side-chain interactions with S'2, is impaired by a second L-Arg at P'1.     

Influence of modified casomorphins on yawning behavior of rats.

Apomorphine-induced yawning was completely suppressed in animals treated with 5 nmol [D-Pro4]casomorphin (CM) (ICV), 10 nmol [D-Phe3]CM (ICV) or 10 nmol [D-Pip4]CM (ICV). The apomorphine-induced yawning was also decreased, by des-Tyr analogs, but only by about 50%. Physostigmine (0.15 mg/kg, IP) induced yawning. The physostigmine-induced yawning was suppressed by 5 nmol [D-Pro4]CM and 10 nmol [D-Phe3]CM. Both [des-Tyr-D-Phe3]CM and [des-Tyr-D-Pip4]CM were without effect, whereas [des-Tyr-D-Pro4]CM increased significantly the physostigmine-induced yawning. The results suggest that dopaminergic transmission can be modulated by beta-casomorphin derivatives, thus resulting in a decrease in yawning. In the case of the des-tyrosine derivatives, we can assume a dopaminergic modulation, too. An increase in serotonergic activity might be supposed for [des-Tyr-D-Pro4]CM.     

Linear and cyclic beta-casomorphin analogues with high analgesic activity.

We investigated the antinociceptive efficacy of casomorphin (CM) derivatives using the vocalization test. Male Wistar rats received chronic microcannulae into the right lateral ventricle. One week later we examined the analgesic effect of CM derivatives 10, 30, 60, and 90 min after intraventricular injection (5 microliters). The analgesic effect was calculated as the individual percent increase in the pain threshold and was compared to controls (saline treatment). The substitution of D-lysine and D-ornithine in position 2 in connection with a cyclization through ring closure of the 2 position side chain amino group to the C-terminal glycine-COOH group resulted in high analgesic potency. The substitution of D-Pro4 was without any effect in the ineffective linear derivatives and decreased the effectiveness in the highly effective cyclic derivatives. The cyclic [D-Orn2]CM-5 and the cyclic [D-Lys2]CM-5 are the CM derivatives with the highest antinociceptive activity. The cyclic [D-Orn2]CM-5 is greater than 1000 times more effective than morphine. We conclude, on the basis of studies of receptor binding and in vitro investigations, that mu receptor activity alone is not responsible for the analgesic activity. The delta receptor and possibly also the kappa receptor could modulate the nociceptive effectiveness.     

Effect on brain monoamines in the rat of substituted and protected analogues of the oxytocin fragment, prolyl-leucyl-glycinamide following N-terminal substitution by D- and L-pipecolic acid

The effect of modified and substituted analogues of prolyl-leucyl-glycinamide (PLG, MIF-I) was investigated on the steady-state level of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in various brain regions. Proline was replaced by D- or L-pipecolic acid (D- or L-Pip), which analogues in turn were protected by benzoxy-carbonyl (Z) group. Substitution by D- or L-pipecolic acid caused opposite changes in the DA level of the dorsal hippocampus. These effects were absent it the N-terminal of either analogues was protected by Z-group. Following the above mentioned N-terminal modification, the amino group of the C-terminal glycine was also substituted by methyl-esther (Gly-OMe), Z-D-Pip-Leu-Gly-OMe decreased the mesencephalic DA level, while Z-L-Pip-Leu-Gly-OMe increased the 5-HT content of the mesencephalon and striatum. In general, N-terminal substitution by D-pipecolic acid decreased, whereas that by L-pipecolic acid increased the monoamine level in the brain.     

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.     

Effect of substitutions in position 12 of bombesin on antagonist activity

Recent studies show that substitutions for the His in position 12 of bombesin (Bn) are important in determining antagonist activity. The present study was designed to investigate the chemical properties of the substitution in position 12 of Bn that determined antagonist activity and affinity. Nine [Leu14]Bn analogues with a single amino acid substitution and two analogues with multiple substitutions in addition to position 12 were synthesized. Replacing His12 with Phe12 resulted in an agonist with 100-fold decrease in potency and as reported previously, replacement with D-Phe12 resulted in an antagonist, but with a 10,000-fold decrease in affinity. Substitution of D-beta-naphthylalanine (D-Nal12), a larger and more hydrophobic group than D-Phe, produced a complete loss of antagonist activity, whereas substitution of D-pyridylalanine (D-Pal12), a group more hydrophilic and similar in size to D-Phe, converted the analogue to a very weak agonist with 300-fold lower affinity than the D-Phe analogue. Antagonist activity depended on the nature of the aromatic moiety, with a D-Trp12 resulting in an inactive analogue, and with a D-Tyr12 resulting in a weak antagonist being 100-fold less potent than the D-Phe12 substitution. The addition of an electron withdrawing group to the D-Phe substitution (D-Cpa12) resulted in a minimal decrease in antagonist activity, whereas the addition of an electron donating group (p-hydroxy in D-Tyr12) resulted in a 30-fold decrease in antagonist activity. The addition of a basic group (D-Arg12 or D-Pal12) resulted in weak agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

Structure-activity studies with cholecystokinin on gastric secretion in the cat

The influence of the position of the sulphate group in CCK on its gastric acid and pepsin stimulating activities was investigated in conscious cats with gastric fistulae. In Boc-CCK7, substitution of tyrosine-SO₃H by ε-hydroxynorleucine-SO₃H, an aliphatic amino acid approximating the length of tyrosine, enhanced acid secretory potency, whilst similar substitution by serine-SO₃H reduced potency, possibly due to the serine residue holding the sulphate group closer to the peptide backbone. Desulphation of Ser-CCK6 reduced acid secretory potency indicating that the known loss of potency upon desulphation of CCK-like peptides is not wholly dependent upon the presence of tyrosine residue in position 7. Sulphated CCK-like peptides are partial agonists of pepsin secretion, and desulphation confers full agonist activity. Analogues of CCK with serine or ε-hydroxynorleucine substituting for tyrosine, whether sulphated or not, showed full agonist activity in stimulating pepsin secretion. These data suggest the presence of the aromatic tyrosine residue, as well as sulphation, to be a necessary prerequisite for pepsin partial agonist activity in CCK-like peptides.     

Desmethionine alkylamide bombesin analogues: a new class of bombesin receptor antagonists with potent antisecretory activity in pancreatic acini and antimitotic activity in Swiss 3T3 cells.

Bombesin-related peptides have a large number of physiological functions as well as having an autocrine growth mechanism for the regulation of small cell lung cancer cells. In the present study we have synthesized 21 des-Met amide or alkylamide analogues of bombesin and compared their abilities to function as bombesin receptor antagonists in guinea pig pancreatic acini and Swiss 3T3 cells with those of the previously most potent antagonist described, [Leu13 psi(CH2NH)Leu14]bombesin (analogue I). All des-Met analogues functioned as antagonists. Bn(1-13)NH2 was approximately equipotent to I (Ki = 60-80 nM) whereas Bn(6-13)NH2 was 30-fold less potent (Ki = 1800 nM). Formation of an ethylamide, Bn(6-13)ethylamide, increased the potency 30-fold such that this octapeptide was equipotent to I. The addition of a D-Phe6 moiety to I did not change potency but caused a 30-fold increase in potency of Bn(6-13)NH2 and a 8-fold increase in the potency of Bn(6-13)ethylamide (Ki = 16 nM). Additional studies of both NH2- and COOH-terminal alterations in Bn(6-13)NH2 demonstrated that the most potent antagonist was [D-Phe6]Bn(6-13)propylamide (PA), having IC50's of 1.6 nM and 0.8 nM for bombesin-stimulated amylase release and Swiss 3T3 cell growth, respectively. Detailed studies of the most potent amide analogue, [D-Phe6]Bn(6-13)NH2, and alkylamide analogue, [D-Phe6]Bn(6-13)PA, demonstrated that these analogues functioned as competitive antagonists and that their action was selective for the bombesin receptor. These results demonstrate that, as with CCK- and gastrin-related peptides, the C-terminal amino acid is important for initiating a biologic response but not essential for determining receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical requirements for receptor recognition of the mu-opioid peptide endomorphin-1

A series of diastereoisomers of endomorphin-1 (EM1, Tyr(1)-Pro(2)-Trp(3)-Phe(4)-NH(2)) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe(4)]EM1 possessed 1/10 the potency of EM1, while potencies of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 were 50- and 100-fold lower, respectively. Drastic loss of activity occurred in the [D-Pro(2)]EM1 peptide. The structural determinants for the inactivity and reduced potency of the diastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro(2)]EM1 using NOE-derived distance constraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D-Pro(2)]EM1 was explained by structural comparison with EM1 (, FEBS Lett. 439:13-20). The two peptides showed an opposite orientation of the Trp(3) residue with respect to Tyr(1), thus suggesting a role of Pro(2) as a stereochemical spacer in orienting Trp(3) and Phe(4) toward regions suitable for mu-receptor interaction. The agonist activity of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 was attributed to their ability to adopt low-energy conformations that mimic those of EM1. The requirements for mu-receptor activation were examined further by comparing EM1 with the mu-peptide [D-Ala(2), MePhe(4), Gly-ol]-enkephalin (DAMGO). Conformations of DAMGO with a Tyr(1)-MePhe(4) phenyl ring separation of approximately 12 A were found to mimic Tyr(1)-Phe(4) of EM1, thus suggesting overlapping binding modes between these two peptides.     

Bombesin, bombesin analogues, and related peptides: effects on thermoregulation

The synthesis and biological evaluation on thermoregulation of 39 peptides related to bombesin (structural analogues or other naturally occurring peptides) are described. The bioassay system reported measures the ability of peptides injected intracisternally to lower body temperature of cold (4 degrees C) exposed rats. The most potent analogues of bombesin were those in which positions one to five (not included) were altered, indicating that the decapeptide C terminal was sufficient for full potency. Gln at the seventh position and Gly at the 11th position could be replaced by D-Gln and D-Ala (but not D-Pro or D-Phe), respectively, without any change in potency. Methionine at the 14 position could be replaced with its D isomer with retention of 10% biological activity. Any other alteration of the C terminus (deletions or free acid with the exception of the N-methylamide) drastically reduced the biological potency of those peptides. Among other naturally occurring peptides, alytesin was found to have 100% of bombesin potency whereas litorin, neurotensin, xenopsin, substance P, physalaemin, and eledoisin were found to be in the order of 10(4) times less potent. The shortest peptide found to have full biological activity is the octapeptide des-Glp-Gln-Arg-Leu-Gly-Asn[D-Glp7, D-Ala11]-bombesin.     

Effects of substance P analogues on spinal dorsal horn neurons

The effects of iontophoretically applied (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP on the spontaneous and evoked activity of functionally identified cat spinal dorsal horn neurons have been investigated in vivo by means of extracellular single unit recording technique. In addition, the rat spinal cord slice preparation has been used to study the actions of (D-Pro2, D-Trp7,9)-SP and (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP on the resting membrane potential of dorsal horn neurons and also on their responses to dorsal root stimulation and exogenous SP application. We have observed that both (D-Pro2, D-Phe7, D-Trp9)-SP and (D-Pro2, D-Trp7,9)-SP produced an excitation of about 15% of all neurons tested and had a weak antagonistic effect against SP in the cat spinal cord. (D-Pro2, D-Trp7,9)-SP suppressed the SP-induced excitation in 63% of examined cells. In addition, depression of the glutamate-induced excitation and spontaneous activity was evident in 10% and 19% of the cat dorsal horn neurons tested, respectively. In the spinal cord slice preparation (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP proved to be a more potent antagonist of the SP-induced depolarization and the dorsal root-elicited slow depolarization, if compared with (D-Pro2, D-Trp7,9)-SP.     

Synthetic glucagon antagonists and partial agonists.

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)