Raw News | Boulder Peptide Symposium

February 24, 2014

Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domain in vitro

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347–361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Studying the interactions of Bin1 SH3 with peptides derived from the HCV protein NS5A and c-Myc revealed that NS5A can displace c-Myc.

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February 20, 2014

Novel cleavable cell-penetrating peptide–drug conjugates: synthesis and characterization

We report the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide. Furthermore, we compare two different doxorubicin cell-penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline-rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N-terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C-13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug-release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF-7 and HT-29 cell lines. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The synthesis and characterization of novel cleavable cell-penetrating peptide drug conjugates are reported. A new heterobifunctional crosslinker was applied to join the N-terminal cysteine of the peptide to the aliphatic ketone of doxorubicin. The two conjugates can be efficiently cleaved by glutathione within a very short period and deliver the toxic freight into the nucleus.

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February 17, 2014

The use of versatile plant antimicrobial peptides in agribusiness and human health

16 February 2014 Source:Peptides

Plant immune responses involve a wide diversity of physiological reactions that are induced by the recognition of pathogens, such as hypersensitive responses, cell wall modifications, and the synthesis of antimicrobial molecules including antimicrobial peptides (AMPs). Read more...

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February 13, 2014

Insulin-releasing and cytotoxic properties of the frog skin peptide, tigerinin-1R: a structure-activity study

Publication date: Available online 12 February 2014 Source:Peptides

Author(s): Dinesh Srinivasan , Opeolu O. Ojo , Yasser H.A. Abdel-Wahab , Peter R. Flatt , Laure Guilhaudis , J. Michael Conlon

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH2) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by L-arginine, L-lysine and L-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P<0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01nM compared with 0.1nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P<0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3μM indicating that plasma membrane integrity had been preserved. [A5W]tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC50 =265±16μM) and inhibited growth of Escherichia coli (MIC=500μM) and Staphylococcus aureus (MIC=250μM). The circular dichroism spectra of tigerinin-1R and [A5W]tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R]tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P<0.05) enhanced insulin release and improved glucose tolerance over a 60min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.

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February 12, 2014

NMR structures of fusion peptide from influenza hemagglutinin H3 subtype and its mutants

The influenza fusion peptide located at the N-terminus of the hemagglutinin HA2 subunit initiates the fusing process of the viral membrane with the host cell endosomal membrane. It had been reported that the structure of a 20-residue H3 subtype fusion peptide (H3-HAfp20) was significantly different with that of a H1 subtype 23-residue one (H1-HAfp23). The sequential difference between the 12th and 15th residues of H1 and H3 subtypes could not fully explain the conformational variation. The first and last three amino acids of H3-HAfp23 involved in formation of hydrogen bonds may play an important role in fusion process. To confirm this hypothesis, we investigate the structures of H3-HAfp23 peptide and its mutants, G1S and G1V, in dodecylphosphatidyl choline micelles by using heteronuclear NMR technology. The results demonstrate that, similar to H1-HAfp23 but significantly different with H3-HAfp20, H3-HAfp23 also has tight helical hairpin structure with the N- and C-terminuses linked together because of the hydrogen bonds between Gly¹ and the last three amino acids, Trp²¹―Tyr²²―Gly²³. Although the ‘hemifusion’ G1S and lethal G1V mutants have hairpin-like helical structures, the distances between the N- and C-terminuses are increased as shortage of the hydrogen bonds and the larger kink angle between the antiparallel helices. The paramagnetic ion titration experiments show that the terminuses are inserted into the dodecylphosphatidyl choline micelles used as solving media. These may imply that the tight helical hairpin structure, especially the closed conformation at terminus, plays an important role in fusion activity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

In this work, the structure of a 23-residue fusion peptide from influenza hemagglutinin H3-subtype was determined at pH 5.0 in DPC micelles. The peptide adopts a tight hairpin-fold conformation, while its two mutants G1S and G1V show partially opened structures. Our results identify that the tight helical hairpin structure, especially the closed conformation at terminus, plays an important role in fusion activity.

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February 12, 2014

Synthesis and activity of isoxazoline vinyl ester pseudopeptides as proteasome inhibitors

The ubiquitin–proteasome pathway (UPP) influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses. The main proteolytic component of the UPP is the 26S proteasome, which is responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered. We report herein the synthesis, proteasome inhibition and modelling studies of novel C-terminal isoxazoline vinyl ester pseudopeptides. Some new compounds that contain a C-terminal extended conjugation inhibit β1 and especially β5 proteasomal catalytic subunits with IC₅₀ values ranging from 10 to 100 µm. These results will permit further optimization based on these structural moieties to develop more active and selective molecules. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We report the synthesis, proteasome inhibition and modelling studies of novel C-terminal isoxazoline vinyl ester pseudopeptides. Some new compounds inhibit proteasomal catalytic subunits with an IC₅₀ around the micromolar concentration.

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February 11, 2014

Cyclic pentapeptide analogs based on endomorphin-2 structure: Cyclization studies using liquid chromatography combined with on-line mass spectrometry and tandem mass spectrometry

Publication date: Available online 10 February 2014 Source:Peptides

Author(s): Justyna Piekielna , Alicja Kluczyk , Renata Perlikowska , Anna Janecka

The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-D-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-D-Cys-Phe-Phe-Cys-NH2 (where Dmt=2′,6’dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides were obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.

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February 8, 2014

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana

Publication date: Available online 8 February 2014 Source:Peptides

Author(s): Doori Park , Je Won Jung , Mi Ok Lee , Si Young Lee , Boyun Kim , Hye Jun Jin , Jiyoung Kim , Young-Joon Ahn , Ki Won Lee , Yong Sang Song , Seunghun Hong , James E. Womack , Hyung Wook Kwon

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

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February 8, 2014

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana

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February 6, 2014

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2-amino acid with either two 2-naphthyl-methylene or two para-CF₃-benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5-fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9-fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC-5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide-based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Detection of structure–activity relationship of anticancer potency and selectivity for linear and cyclic tetrapeptides containing an achiral β^2,2-amino acid are reported. Pharmacokinetic investigation of the interaction with the major transporter protein human serum albumin shows binding to drug site II is exclusively through the hydrophobic parts of the peptides and is in the micromolar range. In vitro testing indicates in vivo implication for the potency of these peptides when human serum albumin is present.

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