Dehydro-dermorphins. II. Synthesis and biological activity of unsaturated dermorphin hexa and heptapeptides

The Phe3 and/or Tyr5 residues in dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and its N-terminal hexapeptide-amide were replaced by delta-Phe or by Phe5 in order to examine the effect on opioid activity. On GPI preparation, the substitution of Phe5 for Tyr5 was well tolerated, whereas the hexa and heptapeptides containing delta Phe in position 3 and/or 5 displayed low potency. The unsaturation at position 3 alone or at positions 3 and 5 was particularly detrimental to mu activity. In the tail flick test, the influence of unsaturation or substitution at positions 3 and 5 generally matched the results of the in vitro assay. Dehydropeptides showed comparatively low antinociceptive effects and [Phe5] analogues displayed about 50% of the analgesic potency of the original peptides.     

Opioid receptor binding profile of selected dermorphin-like peptides

The receptor binding profile of a selected group of dermorphin-like peptides was determined and correlated with the results of the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and with the currently used antinociception tests in the rat. For the peptides with the characteristic dermorphin D-Ala2-Phe3-Gly4 sequence, a linear negative correlation was found between the reciprocal of sodium shift and relative affinity for the mu-type opioid receptor. For the same peptides, a positive correlation was evidenced between relative potency on GPI and MVD and relative affinity for mu- and delta-type receptors, respectively.     

Opioid binding properties in bovine retina

Saturable binding sites for tritiated dihydromorphine ([³H]DHM), D-Ala²-D-Leu⁵-enkephalin ([³H]DADL) and etorphine were found in a crude synaptosomal preparation of bovine retina. Scatchard analysis of saturation binding curves of each ligand was curvilinear and the presence of two independent binding sites inferred. The density of binding sites of [³H]etorphine was similar to that reported in brain crude synaptosomal preparations, and the affinity for the high affinity binding site to each ligand was similar to values determined in brain. Moreover, the regulation of the binding sites by GTP and sodium was also similar to that observed in brain. Selective binding sites for [³H]DADL (δ-sites) were not detectable, although binding sites similar in nature to μ-binding sites were detected.     

Opioid peptides and primary biliary cirrhosis.

Patients with liver disease have increased plasma concentrations of the endogenous opioid peptides methionine enkephalin and leucine enkephalin. As an initial investigation to determine whether opioid peptides contribute to any of the clinical manifestations of hepatic disease nalmefene, a specific opioid antagonist devoid of agonist activity, was given to 11 patients with cirrhosis. They all experienced a severe opioid withdrawal reaction on starting the drug. In the nine patients with primary biliary cirrhosis pruritus was greatly alleviated, fatigue seemed to improve, and plasma bilirubin concentration, which had been rising, showed a modest fall in all except one patient. These results indicate that blocking opioid receptors has an effect on some of the metabolic abnormalities of liver disease.     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.     

Fungicidal and binding properties of three plant peptides

The fungicidal properties of plant seed peptides from Heuchera sanginea (Hs-AFP₁), Raphanus sativus (EA-AFP₂), and Impatiens balsamina (Ib-AMP₃) were determined for the nongerminated and germinated conidia of Aspergillus flavus and Fusarium moniliforme . These peptides were weakly lethal for germinated but not for nongerminated conidia of A. flavus . Both nongerminated and germinated conidia of F. moniliforme were susceptible to these peptides. Overall, F. moniliforme was more susceptible than A. flavus to the peptides. The peptides bound strongly to chitin, mannan, galactocerebrosides, and sphingomyelin. Binding results varied for ergosterol, cholesterol, and β1,3-glucan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analogs of viroidin. Synthesis of four virotoxin-like F-actin binding heptapeptides with one less hydroxyl group in the dihydroxy-proline ring

The vitroxins, toxic cyclic heptapeptides from Amanita virosa fungi, contain a 3,4-dihydroxy-L-proline, an imino acid scarcely accessible in greater amounts. Therefore in the syntheses of the analogs described this imino acid was replaced by 4-cis-hydroxy-L-proline, a component of the analogously toxic phallotoxins. The following syntheses are described: 1a = cyclo (L-alanyl-D-threonyl-D-seryl-L-4-cis-hydroxy-L-prolyl-L-alanyl-2- methylthio-L-tryptophyl-L-leucyl), 1b = 1a but with 2-methylsulfonyl-L-tryptophan in position 6, 2a = cyclic (L-valyl-D-threonyl-D-seryl-4-cis-hydroxy-L-prolyl-L-alanyl-2- methylthio-L-tryptophyl-hydroxyl-L-leucyl), and 2b = 2a, but with 2-methylsulfonyl-L-tryptophan in position 6. In a test for binding strength to rabbit muscle F-actin 2b and 2a exhibited about 40% of that of demethylphalloin. Analogs 1a and 2b have not been tested.     

Synthesis and physico-chemical properties of peptides in soil humic substances.

Soil humic substances (HS) are heterologous, polydispersive, and multi-functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with 13C-glucose or 15N-nitrate for 8.5 months. Evidence for random-coil peptidic structures in the labeled HS was obtained from 2-D nuclear magnetic resonance (NMR), pyrolysis gas chromatography-mass spectrometry (pyro-GC-MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid-state NMR data. Detailed 13C and 15N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC-MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre-existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

Guanylin and related peptides.

Guanylin and uroguanylin are short peptides homologous to heat-stable enterotoxins of Escherichia coli and other enteric bacteria. Guanylin and uroguanylin are synthetized from the respective prepropeptides mainly in gastrointestinal mucosa and are secreted both into intestinal lumen and into the blood. Luminally secreted peptides stimulate chloride and bicarbonate secretion in the intestine through the mechanism involving guanylate cyclase C receptor, cyclic GMP, protein kinase G and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Bacterial enterotoxins, which have greater potency than endogenous peptides, induce excessive fluid secretion into intestinal lumen leading to secretory diarhea. Uroguanylin is expressed mainly in enterochromaffin cells of duodenum and proximal small intestine whereas guanylin is abundant in goblet cells of colonic epithelium. Uroguanylin and guanylin increase urinary sodium and potassium excretion both as circulating hormones and as paracrine mediators produced within the kidney. Uroguanylin functions as "intestinal natriuretic hormone" which is secreted in response to oral sodium loading and maintains sodium balance during postprandial period. Plasma and urinary concentrations of guanylin and uroguanylin increase in renal failure and heart failure. Guanylin peptides possess antiproliferative activity in intestinal cells culture and their expression decreases in colonic carcinoma indicating that their deficiency may contribute to the pathogenesis of this disease.     

Beta-Endorphin. Synthesis and properties of double-headed and related analogs

A double-headed analog of human beta-endorphin (betah-EP), N,N'-bis (beta-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with betah-EP-Cys(CH2CONH2)-OH (I) and [Tyr31]-betah-EP (III). Their relative potencies in a radioreceptor-binding assay were: betah-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: betah-EP, 100; II, 86; I, 93; and III, 116.     

Opioid peptides derived from food proteins. The exorphins.

Peptides with opioid activity are found in pepsin hydrolysates of wheat gluten and alpha-casein. The opioid activity of these peptides was demonstrated by use of the following bioassays: 1) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma X-glioma hybrid cells; 2) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens; 3) displacement of [3H]dihydromorphine and [3H-Tyr, dAla2]met-enkephalin amide from rat brain membranes. Substances which stimulate adenylate cyclase and increase the contractions of the mouse vas deferens but do not bind to opiate receptors are also isolated from gluten hydrolysates. It is suggested that peptides derived from some food proteins may be of physiological importance.     

Synthesis of cholecystokinin-related peptides and their biological properties

Two tyrosine-O-sulfate containing cholecystokinin-related peptides were synthetized, i.e. the undeca- and the dodecapeptide amide corresponding to the C-terminus of this gut hormone; in the undecapeptide the N-terminal serine was O-substituted with the 2-deoxy-alpha-D-galactosyl group. Within the limits of error of the biological assay systems the two CCK peptides exhibited potencies in stimulating amylase secretion from dispersed rat pancreatic acini comparable to those of the CCK deca-, nona- and octapeptide.