Structural requirements of the N-terminal region of GLP-1-[7-37]-NH2 for receptor interaction and cAMP production.

A series of GLP-1-[7-36]-NH(2) (tGLP-1) and GLP-1-[7-37] analogs modified in position 7, 8, 9 and 36, have been designed and evaluated on murine GLP-1 receptors expressed in RIN T3 cells for both their affinity and activity. Ten of the synthesized peptides were found full agonists with activities superior or at least equal to that of the native hormone. Five of them were investigated for their plasmatic stability and the most stable, [a(8)-desR(36)]GLP-1-[7-37]- NH(2) (Compound 8), evaluated in vivo in a glucose tolerance test which confirmed a clearly longer activity than that of the native hormone. We also performed circular dichroism study and propose a hypothetical structural model explaining the most part of observed activities of GLP-1 analogs on RIN T3 cells.

[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.

Guanylin and its lysine-containing analogue in the isolated perfused rat kidney: interaction with chymotrypsin inhibitor.

Guanylin and uroguanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP. Their natriuretic and kaliuretic activities are well documented. Since guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of guanylin. Using the isolated perfused rat kidney, guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-guanylin (0.1-2.5 microg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 microg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 microg/ml), guanylin (0.3 microg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 microg/ml of lys-guanylin (from 73.25+/-2.29 to 54.97+/-0.10, P<0.05). Lys-guanylin (1.0 microg/ml) also decreased fractional K+ reabsorption (%TK+) from 59.26+/-3.93 to 30.75+/-0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, guanylin lowered %TNa+ (from 81.2+/-1.86 to 72.6+/-2.45, P<0.05) and %TK+ (from 69.4+/-4.12 to 65.8+/-2.81, P<0.05). At this subthreshold concentration, guanylin alone lacks effects in %TNa+ or %TK+. Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, guanylin induced intestinal secretion in this assay. In marked contrast, lys-guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.

Synthesis of analogues of peptide YY with modified N-terminal regions: relationships of amphiphilic secondary structures and activity in rat vas deferens.

Peptide YY (PYY) not only has distinct sequence homology with neuropeptide Y (NPY) and avian pancreatic polypeptide (APP), but it also exhibits both NPY- and APP-like biological activities. We synthesized two analogues of PYY, A1 and A2, with modified N-terminal regions, and compared their chemical and biological properties to those of PYY and the C-terminal fragment of PYY, (13-36)PYY. This study shows that there is a good correlation between the stability of amphiphilic alpha-helical structure and the biological activity of these peptides. A CD study of (13-36)PYY in mixed H2O and trifluoroethanol (TFE) solutions indicated a significant increase in alpha-helical segments (26-79%) with increasing TFE proportions. Since the fragment (13-36)PYY had potent activity in the rat vas deferens (RVD) assay, the secondary structure is possibly required on the RVD cell surface receptors. The analogues, A1 and A2, were designed to increase the stability of the alpha-helical structures by incorporation of modified N-terminal regions. The CD studies and the RVD assays of A1 and A2, suggest that the amphiphilic alpha-helical structures are stabilized by intramolecular hydrophobic interactions with the N-terminal regions and/or by intermolecular hydrophobic interactions in the self-association process, and subsequently potentiate the activities of the peptides compared to those of PYY and (13-36)PYY.

Isolation and primary structure of human peptide YY.

The isolation, primary structure and chemical synthesis of human peptide YY (PYY) are described. The peptide was purified from human colonic extracts using a chemical method which detected the C-terminal tyrosine amide structure of PYY. Human PYY consists of 36 amino acid residues and the complete amino acid sequence is: Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu- Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. The differences between the structures of porcine and human PYY are at positions 3 (Ala/Ile replacement) and 18 (Ser/Asn). Synthetic human PYY prepared using a solid-phase synthetic technique was found to be structurally identical to the natural peptide.

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides.

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like carboxyl terminal decapeptide sequence studied to date have similar biological activities.

Current contributions of peptide synthesis to studies on brain-gut-skin triangle peptides.

The usefulness of a strong acid, such as MSA or TFMSA/TFA, as a deprotecting reagent in peptide synthesis was examined. By synthesizing several structurally related brain-gut-skin triangle peptides, a number of advantageous features of the thioanisole-mediated deprotecting procedure were demonstrated. New amino acid derivatives, Arg(Mts), Trp(Mts) and Asp(OChp), were introduced to improve the synthetic methodology of complex peptides and the superior properties of Cys(Ad) were evaluated.

Synthesis of the C-terminal octapeptide of pig oxyntomodulin. Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala: a potent inhibitor of pentagastrin-induced acid secretion.

The synthesis of Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala representing the C-terminal octapeptide of oxyntomodulin isolated from pig intestine is described. Its structure was confirmed by its 360-MHz 1H NMR spectra. The octapeptide was tested for its ability to inhibit pentagastrin-induced acid secretion, in the anaesthetized rat, in the conscious rat with chronic gastric fistula, and in the conscious cat with gastric chronic fistula. The octapeptide inhibits pentagastrin-induced acid secretion in all three models. Compared to oxyntomodulin, the parent hormone, the synthetic peptide was approximately 150 times less potent but has the same efficacy. Biological data are presented and discussed.

Solid-phase synthesis and biological activities of gastrointestinal hormones: secretin and motilin.

Many successful solid-phase syntheses of peptide chains in the region of 20-40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparation high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as power lyophilized from dilute acetic acid.

Solid-phase synthesis of chicken vasoactive intestinal peptide by a mild procedure using N alpha-9-fluorenylmethyloxycarbonyl amino acids.

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive peptide (VIP) was assembled on a p-benzyloxybenzylamine resin support using the base-labile 9-fluorenylmethyloxycarbonyl as N alpha-protecting group, cleaved by mild acid treatment, and purified by gel-filtration and ion-exchange chromatography. The symmetrical anhydride coupling was employed and monitored by two independent methods, and acetic anhydride termination was incorporated to minimize formation of deletion peptides. The homogeneity of the final product, obtained in 18% yield, was assessed by t.l.c., disc electrophoresis, amino-terminal amino acid analysis, and amino acid analyses of acid and enzyme hydrolysates. The purified chicken VIP was shown to be active on gastric acid secretion and on pancreatic blood flow. Previously reported ring closure of the Asp-Asn unit seemed to be at a minimum, owing to the mild basic and acid treatments.

Studies on peptides. LXXXVII. Synthesis of an octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive intestinal polypeptide (VIP).

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken VIP was synthesized in a conventional manner, using a new arginine derivative, NG-mesitylene-2-sulfonylarginine, Arg(Mts). Treatment of a fragment, Z(OMe)-Thr-Asp-Asn-Tyr-NHNH2 with methanesulfonic acid or HBr was found to give a product with a low recovery of Asp, after aminopeptidase digestion. Ring closure of the Asp-Asn unit seemed to be responsible for this phenomenon. Deprotection with HF or TFA exhibited definitely less such a tendency. In the final step of the synthesis, all protecting groups, including the Mts group, were removed by HF in the presence of m-cresol and the deprotected peptide was purified by ion-exchange chromatography on CM-cellulose followed by isoelectric focusing in Ampholine pH 9--1. Synthetic peptide exhibited the identical Rf value with that of natural chicken VIP and was active as the natural peptide.

Solid-phase synthesis of porcine vasoactive intestinal peptide.

The 28-residue sequence of porcine vasoactive peptide (VIP) was assembled on a benzhydrylamine resin support, cleaved by HF treatment, and purified by ion-exchange and partition chromatography. In addition to the normal criteria, the homogeneity of the final material, obtained in 16% yield, was assessed by reverse-phase high performance liquid chromatography and the isolation and examination of cyanogen bromide cleavage fragments by the same technique. The purified VIP possessed characteristic inhibitory effects on pentagastrin-induced gastric acid release in the dog. Upon storage, even as the lyophilized powder in vacuo, HPLC revealed the slow formation of a contaminant possibly representing deamidated peptide.

Synthesis and pharmacological properties of the N-terminal decapeptide of the vasoactive intestinal peptide (VIP).

The decapeptide derivative, L-histidyl-L-seryl-L-aspartyl-L-alanyl-L-valyl-L-phenylalanyl-L-threonyl-L-aspartyl-L-asparaginyl-L-tyrosine methyl ester, corresponding to the N-terminal sequence of both porcine and chicken VIP was synthesized in solution, by the stepwise strategy. Its pharmacological properties resemble those of VIP itself, but with a much lower potency, comparable to that of peptides with C-terminal sequences. The presence of two independent sequences carrying similar instructions was recognized in VIP.