Design, synthesis, and characterization of peptide nanostructures having ion channel activity

We report the synthesis and the functional studies of multiple crown alpha-helical peptides designed to form artificial ion channels. The approach combines the versatility of solid phase peptide synthesis, the conformational predictability of peptidic molecules, and the solution synthesis of crown ethers with engineerable ion-binding abilities. Several biophysical methods were employed to characterize the activity and the mode of action of these crown peptide nanostructures. The 21 residue peptides bearing six 21-EC-7 turned out to facilitate the translocation of ions in a similar fashion to natural ion channels.     

Design, synthesis, and biological activity of potent and selective inhibitors of mast cell tryptase

A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydrophobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excellent selectivity against other serine proteases.     

Design,synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa inhibitors with potent anticoagulant activity

A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl, and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC₅₀ (FXa) value of 0.14 μM and 98% inhibition rate, warranted further investigation as an FXa inhibitor.     

Calcitonin receptor-stimulating peptide,a new member of the calcitonin gene-related peptide family. Its isolation from porcine brain,structure, tissue distribution,and biological activity

We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLC-PK(1). Determination of the amino acid sequence and cDNA analysis encoding a CRSP precursor showed that this peptide has approximately 60% identity in the amino acid sequence with human calcitonin gene-related peptide type-alpha (alphaCGRP), type-beta (betaCGRP), and porcine CGRP. Northern blot analysis and radioimmunoassay demonstrated that CRSP is expressed mainly in the thyroid gland and the central nervous system, in which the calcitonin receptor was abundantly expressed. Synthetic CRSP elicited a potent stimulatory effect on the cAMP production in LLC-PK(1) cells. Although it shows significant sequence similarity with CGRPs, this peptide did not elicit cAMP elevation in cells that endogenously expressed a CGRP receptor or an adrenomedullin receptor or were transfected with either of these recombinant receptors. Administration of CRSP into anesthetized rats did not alter the blood pressure but induced a transient decrease in the plasma calcium concentration. In fact, this peptide potently increased the intracellular cAMP concentration in COS-7 cells that expressed the recombinant calcitonin receptor. These unique properties indicate that CRSP is not a porcine counterpart of betaCGRP and probably elicits its biological effects via the calcitonin receptor.     

beta-endorphin: synthesis and biological activity of shortened peptide chains

Three analogs of beta-endorphin have been synthesized by the solid-phase method: betac-endorphin-(1--5)-(28--31), betac-endorphin-(6--31) and betah-endorphin-(1--5)-(16--31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than beta-endorphin. These data suggest that the entire beta-endorphin molecule is necessary for full in vivo analgesic activity.     

A new chitosan-thymine conjugate: synthesis, characterization and biological activity

Conjugation of chitosan with nucleobases is expected to expand its not only antimicrobial activity but also anti-cancer activity. Here, we report the synthesis of a novel chitosan-thymine conjugate by the reaction between chitosan and thymine-1-yl-acetic acid followed by acylation. The synthesized conjugate was characterized by FTIR, XRD, (1)H NMR, TGA and SEM. The microbiological screening results demonstrated the antimicrobial activity of the conjugate against bacteria viz., Escherichia coli, Staphylococcus aureus, and fungi viz., Aspergillus niger. The chitosan-thymine conjugate also inhibited (p<0.05) the proliferation of human liver cancer cells (HepG2) in a dose-dependent manner but had no cellular toxicity in non-cancerous mouse embryonal fibroblast cells (NIH 3T3). Thus, the chitosan-nucleobase conjugate may open a new perspective in biomedical applications.     

Polysialylated insulin: synthesis,characterization and biological activity in vivo

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.     

Design,synthesis and structure activity relationships of spirocyclic compounds as potent CCR1 antagonists

A series of CCR1 antagonists based upon spirocyclic compounds 1b and 2b were synthesised in which substituted aniline moiety was replaced with substituted benzamides. In vitro data revealed that CCR1 potency could be retained in such compounds.     

Polysialylated insulin: synthesis,characterization and biological activity in vivo

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96–99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12–48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60–1.74 for the 22-kDa CA and 2.37–2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.     

Chemical synthesis of bovine transforming growth factor-alpha: synthesis, characterization and biological activity

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.     

Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity.

A synthetic, 26-residue peptide having a strong helix forming potential in the protonated state was designed to interact with lipid bilayers in a pH-dependent way. On the basis of this concept a cluster of four glutamic acid residues was inserted in the central region of the amphipathic peptide to promote helix destabilization by mutual charge repulsion at neutral pH. Protonation of these residues might then bring about both a pH-mediated change in hydrophobicity and conformation forming a membrane-active amphiphilic helix. The sequence GLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategy induced pH-triggered lysis of human erythrocytes. A molecular model correlating the lytic activity to the formation of transmembrane pores which were detected by electron microscopy in erythrocyte membranes is discussed. Circular dichroism studies indicated a self-association of the monomeric random coil form with increasing peptide concentration leading to the apparent induction of strong alpha-helix formation (approximately 100% helicity) in the fully aggregated state. However, no pH-dependent helix-random coil transition was observed, implying that interhelical hydrophobic and ionic interactions not only govern the self-association but also decisively influence the conformational stability of the peptide.     

Calcitonin receptor-stimulating peptide: Its evolutionary and functional relationship with calcitonin/calcitonin gene-related peptide based on gene structure

This review focuses on the evolutionary and functional relationship of calcitonin receptor-stimulating peptide (CRSP) with calcitonin (CT)/calcitonin gene-related peptide (CGRP) in mammals. CRSP shows high sequence identity with CGRP, but distinct biological properties. CRSP genes (CRSPs) have been identified in mammals such as pigs and dogs of the Laurasiatheria, but not in primates and rodents of the Euarchontoglires or in non-placental mammals. CRSPs have genomic organizations highly similar to those of CT/CGRP genes (CT/CGRPs), which are located along with CGRPs in a locus between CYP2R1 and INSC, while the other members of the CGRP superfamily, adrenomedullin and amylin, show genomic organizations and locations distinct from CT, CGRP, and CRSP. Thus, we categorized these three peptides into the CT/CGRP/CRSP family. Non-placental mammals having one and placental mammals having multiple CT/CGRP/CRSP family genes suggests that multiplicity of CT/CGRP started at an early stage of mammalian evolution. In the placental mammals, Laurasiatheria generally possesses multiple CRSPs and only one CT/CGRP, while Euarchontoglires possesses CT/CGRP and CGRPβ but no CRSP, indicating an increase in the diversity and multiplicity of this family of genes in mammalian evolution. Phylogenetic analysis suggests that some CRSPs have been generated very recently in mammalian evolution. Taken together, the increase in the number and complexity of the CT/CGRP/CRSP family genes may have due to evolutionary pressure to facilitate adaptation during mammalian evolution. In this regard, it is important to elucidate the physiological roles of CT, CGRP and CRSP from the viewpoint of the CT/CGRP/CRSP family even in Euarchontoglires.     

Enterococcal peptide sex pheromones: synthesis and control of biological activity

The enterococcal pheromone-inducible plasmids such as pCF10 represent a unique class of mobile genetic elements whose transfer functions are induced by peptide sex pheromones. These pheromones are excreted by potential recipient cells and detected by plasmid-containing donor cells at the cell surface, where the pheromone is imported and signals induction of the plasmid transfer system. Pheromone is processed from a chromosomally encoded lipoprotein and excreted by both the donor and recipient cells, but a carefully controlled detection system prevents a response to self-pheromone while still allowing an extremely sensitive response to exogenous pheromone.     

Novel tricyclic poly (ADP-ribose) polymerase-1/2 inhibitors with potent anticancer chemopotentiating activity: Design,synthesis and biological evaluation

8,9-Dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones were designed and synthesized as a new class of PARP-1/2 inhibitors. The compounds displayed a variable pattern of PARP-1/2 enzymes inhibition profile that, in part, paralleled the antiproliferative activity in cell lines. Among them, compound 9e exhibited not only the significant IC₅₀ value of 28 nM in the PARP-1 and 7.7 nM in PARP-2 enzyme assay, but also a profound synergic efficacy combined with temozolomide with PF₅₀ values of 2.6, 2.5, and 6.5 against MDA-MB-468, SW-620 and A549 and cell line, respectively.     

Solution structure and biological activity of recombinant salmon calcitonin S-sulfonated analog

Salmon calcitonin S-sulfonated analog (abbreviated as [S-SO(3)(-)]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500IU/mg, which is about 30% higher than that (4500IU/mg) of the wild type. The solution conformation of [S-SO(3)(-)]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic alpha-helix in the region of residues 4-22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO(3)(-)-Lys11(+)-Glu15(-)-Lys18(+)). The multiple salt bridges resulted in the stabilization of the longer amphipathic alpha-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic alpha-helix.     

Design, synthesis and characterisation of a peptide with oxaloacetate decarboxylase activity

The synthesis of Oxaldie-3, a synthetic 31-residue peptide with oxaloacetate decarboxylase activity, is described. Biophysical characterisation by gel filtration, CD and NMR spectroscopy indicated that the peptide adopted a folded structure in solution. Oxaldie-3 was an efficient catalyst at concentrations as low as 2 microM, 100-fold lower than the previously described Oxaldie-2, which relied on aggregating alpha-helices for activity. Oxaldie-3 speeded decarboxylation by more than three orders of magnitude relative to simple amines.