Antagonistic effects of two novel GIP analogs, (Hyp(3))GIP and (Hyp(3)) GIPLys(16)PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme- resistant, NH2- terminally modified GIP analog ( Hyp(3)) GIP and its fatty acid- derivatized analog ( Hyp(3)) GIPLys(16)PAL. Acute effects are compared with the established GIP receptor antagonist ( Pro(3)) GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor- transfected fibroblasts and in clonal pancreatic BRIN- BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulinreleasing effects of native GIP. Administration of once daily injections of ( Hyp(3)) GIP or ( Hyp(3)) GIPLys(16)PAL for 14 days resulted in significantly lower plasma glucose levels ( P <0.05) after ( Hyp3) GIP on days 12 and 14 and enhanced glucose tolerance ( P <0.05) and insulin sensitivity ( P <0.05 to P <0.001) in both groups by day 14. Both ( Hyp(3)) GIP and ( Hyp(3)) GIPLys(16)PAL treatment also reduced pancreatic insulin ( P <0.05 to P <0.01) without affecting islet number. These data indicate that ( Hyp3) GIP and ( Hyp(3)) GIPLys(16)PAL function as GIP receptor antagonists with potential for ameliorating obesity- related diabetes. Acylation of ( Hyp(3)) GIP to extend bioactivity does not appear to be of any additional benefit.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Synthesis of A83586C analogs with potent anticancer and beta-catenin/ TCF4/osteopontin inhibitory effects and insights into how A83586C modulates E2Fs and pRb.

The synthesis of three potent new antitumor agents is described: the A83586C-citropeptin hybrid (1), the A83586C-GE3 hybrid (2), and l-Pro-A83586C (3). Significantly, compounds 1 and 2 function as highly potent inhibitors of ß-catenin/TCF4 signaling within cancer cells, while simultaneously downregulating osteopontin (Opn) expression. A83586C antitumor cyclodepsipeptides also inhibit E2F-mediated transcription by downregulating E2F1 expression and inducing dephosphorylation of the oncogenic hyperphosphorylated retinoblastoma protein (pRb).

Amolopkinins W1 and W2 – novel bradykinin-related peptides (BRPs) from the skin of the Chinese torrent frog, Amolops wuyiensis: antagonists of bradykinin-induced smooth muscle contraction of the rat ileum.

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog (Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu1, Thr6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu1, Thr6, Trp8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster (Heloderma suspectum) venom: Evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle

Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster (Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1–4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1–3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

Facile biocatalytic reduction of the carbon-carbon double bond of 5-benzylidenethiazolidine-2,4-diones. Synthesis of ( )-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione (BRL 49653), its (R)-(+)-enantiomer and analogs.

Cantello, Barrier C. C.; Eggleston, Drake S.; Haigh, David; Haltiwanger, R. Curtis; Heath, Catherine M.; Hindley, Richard M.; Jennings, Keith R.; Sime, John T.; Woroniecki, Stefan R. SmithKline Beecham Pharmaceuticals, Surrey, UK. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1994), (22), 3319-24. Publisher: Royal Society of Chemistry, CODEN: JCPRB4 ISSN: 0300-922X. Journal written in English. CAN 122:105736 AN 1995:237497 CAPLUS (Copyright (C) 2009 ACS on SciFinder (R)) Abstract A novel biotransformation system for the redn. of carbon-carbon double bonds in 5-benzylidenethiazolidine-2,4-diones to give the corresponding 5-benzylthiazolidine-1,4-diones, using whole cells of red yeasts, is described. These reduced compds., which are recovered in good yield, are of potential use in the treatment of non-insulin dependent diabetes mellitus. The mild reaction conditions developed allow redn. of 5-benzylidenethiazolidine-2,4-diones contg. other functionalities which are not compatible with alternative redn. methods. The biocatalytic redn. is enantioselective and the synthesis of R-(+)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione by Rhodotorula rubra CBS 6469 and structure confirmation by X-ray crystallog. is detailed. Optimization of reaction conditions (including immobilization) for these whole cell redn. system is described.

A fish bradykinin (Arg0; Trp5; Leu8-bradykinin) from the defensive skin secretion of the European edible frog; Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val1, Thr6)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes - common predators of these frogs. Here, we report the presence of (Arg0, Trp5, Leu8)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg0, Trp5, Leu8)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg0, Trp5, Leu8)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.