Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides.

Ghrelin, a novel peptide purified from the stomach, is the endogenous ligand of the growth hormone secretagogue receptor. The Ser(3) residue of ghrelin is modified with a lipid n-octanoic acid, a modification necessary for hormonal activity. To clarify the role of acyl modification and to identify the active core of ghrelin, we examined the activities of partially digested ghrelin and synthetic ghrelin derivatives. The activities confirmed that the N-terminal portion is the active core. Moreover, synthetic ghrelin derivatives demonstrated that octanoic acid is not the only modification of the Ser(3) side chain to sustain the activity of ghrelin; other acyl acid modifications maintained activity. Amino acid replacement of Ser(3) indicated that an L-configuration of the third residue is critical for ghrelin activity. In addition, more stable ether or thioether bonds are capable of replacing the octanoyl ester bond in ghrelin, advantageous for the generation of pharmaceuticals with longer stability.

Bullfrog ghrelin is modified by n-octanoic acid at its third threonine residue.

We have identified the amphibian ghrelin from the stomach of the bullfrog. We also examined growth hormone (GH)-releasing activity of this novel peptide in both the rat and bullfrog. The three forms of ghrelin identified, each comprised of 27 or 28 amino acids, possessed 29% sequence identity to the mammalian ghrelins. A unique threonine at amino acid position 3 (Thr(3)) in bullfrog ghrelin differs from the serine present in the mammalian ghrelins; this Thr(3) is acylated by either n-octanoic or n-decanoic acid. The frog ghrelin-28 has a complete structure of GLT (O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGNM; the structure of frog ghrelin-27 was determined to be GLT(O-n-octanoyl)FLSPADMQKIAERQSQNKLRHGN; frog ghelin-27-C10 possessed a structure of GLT(O-n-decanoyl)FLSPADMQKIAERQSQNKLRHGN. Northern blot analysis demonstrated that ghrelin mRNA is predominantly expressed in the stomach. Low levels of gene expression were observed in the heart, lung, small intestine, gall bladder, pancreas, and testes, as revealed by reverse transcription polymerase chain reaction analysis. Bullfrog ghrelin stimulated the secretion of both GH and prolactin in dispersed bullfrog pituitary cells with potency 2-3 orders of magnitude greater than that of rat ghrelin. Bullfrog ghrelin, however, was only minimally effective in elevating plasma GH levels following intravenous injection into rats. These results indicate that although the regulatory mechanism of ghrelin to induce GH secretion is evolutionary conserved, the structural changes in the different ghrelins result in species-specific receptor binding.

Structural similarity of ghrelin derivatives to peptidyl growth hormone secretagogues.

Ghrelin is a 28-amino acid residue endogenous growth hormone secretagogue. Intensive investigations revealed that the N-terminus tetrapeptide, having octanoyl group at Ser(3), is the minimum active core. In this study, we further explored the structure-function relationships of the active N-terminus portion of ghrelin using a Ca(2+) mobilization assay. The smallest and most potent ghrelin derivative we have found so far is 5-aminopentanoyl-Ser(Octyl)-Phe-Leu-aminoethylamide, showing comparable activity to the natural molecule. In the process of modifying the active core, the ghrelin-derived short analogues emerged structurally close to peptidyl growth hormone secretagogues. The N-terminus modification suggested that Gly(1)-Ser(2) unit works as a spacer, forming adequate distance between N(alpha)-amino group and n-octanoyl group. Replacement of 3rd and 4th amino acid residues to D-isomer suggested that the N-terminal dipeptide contributes to shape the biologically active geometry by effecting conformation of residues in positions 3 and 4.

Labelling peptides with fluorescent probes for incorporation into degradable polymers.

Two peptides, atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were conjugated with a fluorescent, amine-reactive probe 5-(and 6-)carboxytetramethylrhodamine,-succinimidylester (5-(6)-TAMRA-SE). The labelling reaction was followed by HPLC and found to be complete after 2 h. The labelled peptides were purified by gel filtration chromatography and characterised by [1H]NMR, UV/VIS and fluorescence spectroscopy. NMR-spectra confirmed the conjugation of dye to the peptides. Two absorption maxima between 500 and 600 nm were recorded in the UV/VIS-spectra. The fluorescence spectra were found to be pH-dependent, which allowed the measurement of pH in aqueous solution. The labelled peptides were encapsulated into poly(lactic acid) (PLA) microspheres using a double emulsion technique. Probe attachment permitted location of the peptides in the polymer.

Effect of the spine venom from the crown-of-thorns starfish, Acanthaster planci, on drug-metabolizing enzyme in rat liver.

The effect of spine venom from the crown-of-thorns starfish (Acanthaster planci) on drug-metabolizing enzymes in rat liver was studied. The spine venom was prepared by saturation of spine homogenate with ammonium sulfate and the protein fraction precipitating 50% saturation was used as venom B. Venom A was the protein precipitated between 50 and 100% saturation. When venom B (100-200 mg/kg) was given to rats, liver microsomal GSH S-transferase and cytochrome P450 activities decreased while cytosolic GSH S-transferase activity was not changed. The decrease in these microsomal enzyme activities was seen from 12 hr to 24 hr after giving 100 mg/kg of venom B. Rats given venom A died, suggesting an involvement of the lethal factor in venom A. The data showed that the spine venom B from A. planci depressed microsomal GSH S-transferase and cytochrome P450 activities in rat liver and that this venom was distinct from the lethal factor of the spine venom.

Biodegradable long-circulating polymeric nanospheres.

Injectable nanoparticulate carriers have important potential applications such as site-specific drug delivery or medical imaging. Conventional carriers, however, cannot generally be used because they are eliminated by the reticulo-endothelial system within seconds or minutes after intravenous injection. To address these limitations, monodisperse biodegradable nanospheres were developed from amphiphilic copolymers composed of two biocompatible blocks. The nanospheres exhibited dramatically increased blood circulation times and reduced liver accumulation in mice. Furthermore, they entrapped up to 45 percent by weight of the drug in the dense core in a one-step procedure and could be freeze-dried and easily redispersed without additives in aqueous solutions.

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells.

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.

Characterization of ligand-binding properties and selectivities of three rat tachykinin receptors by transfection and functional expression of their cloned cDNAs in mammalian cells.

We investigated the ligand-binding properties and selectivities of rat substance P, substance K and neuromedin K receptors by transfection and functional expressions of the cDNAs for these receptor subtypes in monkey kidney COS cells. Selective radioligand binding analysis of both substance P and substance K receptors revealed the presence of high-affinity and low-affinity components which are governed primarily by the difference in rates of dissociation of the ligand-receptor complex. The two affinity components were interconvertible by the presence and absence of a guanine nucleotide, suggesting the involvement of a G protein in the two affinity states. The ligand bindings of the three receptors were inhibited in different potencies by naturally occurring tachykinins and a series of carboxyl-terminal fragments containing a common tachykinin sequence, and this comprehensive analysis indicated that a high and selective affinity of each of the tachykinin receptors is governed by interaction with several key amino acids under recognition of the fundamental core sequence of the tachykinin peptides. The potencies and selectivities of several synthetic agonists and an antagonist for the three receptors were also examined, and senktide was found to be a highly selective and potent agonist for neuromedin K receptor. This investigation thus indicates the detailed properties and binding selectivities characteristic of the three tachykinin receptors and also the usefulness of this receptor expression system for examination and development of a new receptor agonist or antagonist.

Structure-activity relationships of alpha-human atrial natriuretic peptide (alpha-hANP) analogs: role of charged groups in alpha-hANP.

To determine whether the addition of a methylene unit in the side chain of the Asp or Arg residue in alpha-human atrial natriuretic peptide (alpha-hANP) influences its biological activity, analogs of alpha-hANP, [Glu13]-alpha-hANP (7-28) (1), [Aad13]-alpha-hANP (7-28) (2), and [Harn]-alpha-hANP(7-28) (where n is any possible combination of 11, 14 and 27) (3-9), where the original Asp or Arg residue was replaced by a homo-amino acid, were synthesized by the solid-phase synthesis method. All the analogs were evaluated for their receptor binding, cyclic guanosine monophosphate (cGMP) accumulation activity in rat vascular smooth muscle cells (VSMC), and for vasorelaxant activity employing rat aorta. 1 and 2 were 0.9 and 0.03 times as potent as alpha-hANP (7-28), respectively, in binding. Har-containing analogs (3-9) were as potent as alpha-hANP (7-28) in binding. Among the Har-containing analogs, [Har11,14]-alpha-hANP (7-28) (6) and [Har11,27]-alpha-hANP (7-28) (7) were remarkably vasorelaxant active, being 4.2 and 5.3 times potent than alpha-hANP (7-28), respectively, in spite of relatively lower cGMP accumulation activity in the case of 7. The roles of the chargeable amino acid residues in biological activity are discussed.

Importance of hydrophobic residues in alpha-human atrial natriuretic peptide (alpha-hANP) for vasorelaxant activity.

To investigate the roles of the hydrophobic residues in the ANP molecule on biological activities, we synthesized a series of analogs containing various phenylalanine-homologs in position 8 or methionine-homologs in position 12. Among the analogs [pCl-Phe8]-alpha-hANP(7-28) was 4.8 times as potent as alpha-hANP(7-28) in cGMP accumulation and 3.5 times as potent in vasorelaxant activity. All the analogs showed nanomolar affinity to the receptor. In contrast, vasorelaxation and cGMP accumulation activity of the analogs ranged widely. These results suggest that these hydrophobic residues in the cyclic core are critical for vasorelaxant activity rather than for the "apparent receptor binding", and that these residues may possibly discriminate the "bioactive receptor" which is coupled to guanylate cyclase from the non-coupled receptor.

Synthesis and biological property of alpha-human atrial natriuretic peptide analogs with a constrained or stereochemically modified cyclic moiety.

Conformationally restricted analogs of alpha-human atrial natriuretic peptide (alpha-hANP) containing L- or D-penicillamine, or D-cysteine in place of cysteine residues at positions 7 and 23 were synthesized by the liquid phase procedure. Their biological properties in the assays of receptor binding and cyclic guanosine monophosphate (cGMP) accumulation employing rat vascular smooth muscle cells (VSMC), vasorelaxant activity using rat isolated aorta were evaluated. We found that the constrained and/or stereochemically altered ring moiety generally did not influence the receptor binding activity, however, cGMP accumulation and vasorelaxant activities were quite sensitive to conformational perturbation. Furthermore, a lack of correlation between cGMP accumulation activity and vasorelaxant activity was observed. Dissociation between these activities was typical in the case of [DPen7,23]-alpha-hANP(7-28), which showed quite weak vasorelaxant activity in spite of its full cGMP accumulation and receptor binding potencies. This result suggests that cGMP accumulation alone is not sufficient to promote ANP-induced vasorelaxation, and that the other second messenger(s) may mediate this activity.

Novel natriuretic peptide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells.

The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atrial natriuretic peptide (alpha-hANP) while CNP was 4- and 16-fold weaker than alpha-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP.

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells.

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.

A molluscan neuropeptide related to the crustacean hormone, RPCH.

A peptide that potentiates twitch contraction of the radula retractor muscle of the prosobranch mollusc Fusinus ferrugineus was isolated from the ganglia of the animal. Its primary structure is H-Ala-Pro-Gly-Trp-NH2 (APGWamide) closely related to the C-terminal tetrapeptide of the crustacean red-pigment-concentrating hormone. APGWamide showed modulatory actions on contractions in various molluscan muscles.

Linear alpha-human atrial natriuretic peptide analogs display receptor binding activity and inhibit alpha-hANP-induced cGMP accumulation.

We have synthesized a series of [Cys(R)7,23]alpha-hANP analogs, in which the two Cys residues were modified with various alkyl groups(R); i.e., R=Acm, Pe, Qe, Cam, Me, Ae, Bzl, Cm, Ocam and sulfo. The Acm-, Cam-, and Me-analogs exhibited binding activity as potent as alpha-hANP in rat vascular smooth muscle cells (VSMC). Binding activity of the analogs decreased progressively as the bulkiness of the R group increased. None of the analogs caused accumulation of cGMP in VSMC and vasorelaxant activity in rat aorta. Acm-, Cam- and Me-analogs substantially antagonized alpha-hANP-induced cGMP accumulation, but did not antagonize vasorelaxation induced by alpha-hANP in vitro.

Human atrial natriuretic polypeptides (hANP): purification, structure synthesis and biological activity.

In the present survey for natriuretic factors in human atrial extract, conducted by using in vitro assay for the relaxant activity of chick rectum, three distinct human atrial natriuretic polypeptides (hANP) (alpha-, beta- and gamma-hANP), differing in molecular weight (alpha, 3000; beta, 6000, gamma, 13 000 daltons) have been purified and sequenced. gamma-hANP, of 13 000 daltons, has been identified as a 126-residue peptide which carries the whole sequence of alpha-hANP of 28-amino acids at the C-terminus, while beta-hANP has been proved to be an antiparallel dimeric form of alpha-hANP. Synthetic alpha-hANP at a dose of 0.2 nmol induces diuresis and natriuresis as well as hypotension, when injected into assay rats [1]. Natriuretic activity of beta- and gamma-hANP was estimated to be about one-fifth or less on molar base than that of alpha-hANP.