Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Summary Two peptides derived from the surface loop 4 of class 1 Outer Membrane Protein (OMP) ofNeisseria meningitidis were synthesized on solid phase using the Boc/Bzl strategy: one containing the entire loop 4 cyclized and the other representing the polymerized cyclic loop 4. To test a more efficient cyclic peptide presentation, in the present study a strategy was developed to obtain polymers of cyclic peptides. In order to obtain the polymeric cyclic peptide, two protecting groups for cysteine were used — Acm and Mob. The Cys(Acm)-protected cyclic peptide was obtained after removing the Mob group. The polymerization reaction was carried out by simultaneous deprotection/oxidation ofS-Acm with iodine. Analysis of the polymeric cyclic peptide in Tris-tricine-SDS-PAGE showed different bands with molecular weights higher than expected for the corresponding monomeric cyclic peptide. Both peptides were used in immunization of four different mouse strains. The antisera raised against the peptides were evaluated by ELISA and Western blotting vs. OMP preparation ofN. meningitidis. The titers raised against the polymerized cyclic peptide were higher than the ones raised against the cyclic peptide. The antisera elicited did not show bactericidal activity. Nevertheless, the antisera elicited against the polymeric cyclic peptide in the CBA/J mouse strain showed opsonic activity. The antibodies raised against the polymeric cyclic peptide were successfully used as probes in Western blotting experiments to verify the display of loop 4 peptide on the surface of filamentous phage M13.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Two peptides derived from the surface loop 4 of class1 Outer Membrane Protein (OMP) of Neisseriameningitidis were synthesized on solid phase usingthe Boc/Bzl strategy: one containing the entire loop4 cyclized and the other representing the polymerizedcyclic loop 4. To test a more efficient cyclic peptidepresentation, in the present study astrategy was developed to obtain polymers of cyclic peptides. Inorder to obtain the polymeric cyclic peptide, twoprotecting groups for cysteine were used – Acm andMob. The Cys(Acm)-protected cyclic peptide wasobtained after removing the Mob group. Thepolymerization reaction was carried out bysimultaneous deprotection/oxidation of S-Acmwith iodine. Analysis of the polymeric cyclic peptidein Tris-tricine-SDS-PAGE showed different bandswith molecular weights higher than expected for thecorresponding monomeric cyclic peptide. Both peptideswere used in immunization of four different mouse strains.The antisera raised against the peptides wereevaluated by ELISA and Western blotting vs. OMPpreparation of N. meningitidis. The titersraised against the polymerized cyclic peptide werehigher than the ones raised against the cyclicpeptide. The antisera elicited did not showbactericidal activity. Nevertheless, the antiseraelicited against the polymeric cyclic peptide in theCBA/J mouse strain showed opsonic activity. Theantibodies raised against the polymeric cyclic peptidewere successfully used as probes in Western blottingexperiments to verify the display of loop 4 peptide onthe surface of filamentous phage M13.     

A type-II beta-turn, proline-containing, cyclic pentapeptide as a building block for the construction of models of the cleavage site of pro-oxytocin.

Previous studies have indicated that proteolytic activation of pro-hormones and pro-proteins occurs most frequently at the level of basic amino acids arranged in doublets and that the dibasic sites are situated in or next to beta-turns. Investigations utilizing synthetic peptides reproducing the N-terminal processing domain of pro-oxytocin-neurophysin have suggested a close relationship between the secondary structure of the cleavage locus and enzyme recognition, the minimal recognized sequence being the -Pro-Leu-Gly-Gly-Lys-Arg-Ala-Val-Leu- segment of the native precursor. NMR investigations and energy minimization studies have demonstrated that this sequence is organized in two type-II beta-turns involving the -Pro-Leu-Gly-Gly- and -Lys-Arg-Ala-Val- sequences. To further strengthen the above reported hypothesis and to study the role of turn subtypes, a new proline containing cyclic substrate of the processing enzyme, in which the N-terminal side that comes before the Lys-Arg pair is constrained to adopt a type-lI beta-turn, has been synthesized. The presence of a type-II beta-turn structure in this cyclic peptide model has been demonstrated by a combined NMR, CD and FT-IR absorption investigation. A preliminary study shows that PC1 is able to recognize and process our constrained substrate.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Design and synthesis of biologically active peptides: a 'tail' of amino acids can modulate activity of synthetic cyclic peptides

In earlier work, we synthesized a cyclic 9-amino acid peptide (AFPep, cyclo[EKTOVNOGN]) and showed it to be useful for prevention and therapy of breast cancer. In an effort to explore the structure–function relationships of AFPep, we have designed analogs that bear a short ‘tail’ (one or two amino acids) attached to the cyclic peptide distal to its pharmacophore. Analogs that bore a tail of either one or two amino acids, either of which had a hydrophilic moiety in the side chain (e.g., cyclo[EKTOVNOGN]FS) exhibited greatly diminished biological activity (inhibition of estrogen-stimulated uterine growth) relative to AFPep. Analogs that bore a tail of either one or two amino acids which had hydrophobic (aliphatic or aromatic) side chains (e.g., cyclo[EKTOVNOGN]FI) retained (or had enhanced) growth inhibition activity. Combining in the same biological assay a hydrophilic-tailed analog with either AFPep or a hydrophobic-tailed analog resulted in decreased activity relative to that for AFPep or for the hydrophobic-tailed analog alone, suggesting that hydrophilic-tailed analogs are binding to a biologically active receptor. An analog with a disrupted pharmacophore (cyclo[EKTOVGOGN]) exhibited little or no growth inhibition activity. An analog with a hydrophilic tail and a disrupted pharmacophore (cyclo[EKTOVGOGN]FS) exhibited no growth inhibition activity of its own and did not affect the activity of a hydrophobic-tailed analog, but enhanced the growth inhibition activity of AFPep. These results are discussed in the context of a two-receptor model for binding of AFPep and ring-and-tail analogs. We suggest that tails on cyclic peptides may comprise a useful method to enhance diversity of peptide design and specificity of ligand–receptor interactions.     

重组环状胸腺五肽结构类似物Cyclo-（Cys- Arg-Lys –Asp-Val-Tyr）的制备、鉴定和活性检测

为了实现蛋白内含肽(Intein)介导的重组环状胸腺五肽结构类似物［cyclo-（Cys -Arg-Lys –Asp-Val-Tyr）,cTP］的高效制备,设计并合成编码6个氨基酸的cTP基因,克隆到表达载体pTWIN1,重组表达质粒pTW-cTp转化E.coli ER2566构建工程菌,IPTG诱导由几丁质结合域纯化标签（chitin binding domain,CBD）、2个蛋白内含肽和目的多肽组成的“多元”融合蛋白(CBD-intein1-cTP-intein2-CBD)的高效表达.几丁质柱亲合层析纯化融合蛋白后,改变pH值和温度诱导intein1 C端切割,硫醇MESNA诱导intein2 N端切割,释放N端为Cys,C端为硫酯的重组cTP线性前体,通过非保护多肽硫酯环合法实现环肽生成.激光飞行质谱结果显示,纯化产物的分子量为764.4,与环肽的理论值相符.免疫活性检测结果显示,环肽cTP较线性多肽TP-5具有更显著的促进巨噬细胞吞噬能力的活性（P<0.01）和促进B细胞抗体生成的活性（P<0.01）.     

Peptide oligomers from ultra-short peptides using sortase

Sortase A catalyzed ligation of ultra-short peptides leads to inter/intra-molecular transpeptidation to form either linear or cyclic oligomers dependent upon the peptide length. Cyclic peptides were the main products for peptides with more than 15aa. However, for ultra-short (<15aa) peptides, cyclic oligomers became predominant in prolonged reactions. Peptides with 1–3 aminoglycines were equally active but peptide oligomers from peptide containing more than one aminoglycine were prone to hydrolysis.     

Solid-phase synthesis of tailed cyclic RGD peptides using glutamic acid: unexpected glutarimide formation.

To provide multiple conjugating sites on cyclic peptides for their increasing biomedical applications, a tailed cyclic RGD peptide, c[RGDfE(GGGKK-NH(2))] was designed with c(RGDfE) linked through Glu to a tail consisting of a spacer of three Gly residues and a linker of two Lys residues. The spacer is used to increase the mobility and binding ability of the c(RGDfE) ligand, and the linker is used to proved multiple active sites for conjugating other molecules or biomaterials. We found that the sequence of Glu(Gly)-OAll leads to glutarimide formation, which disrupts the formation of cyclic RGD peptides. However, our results show that glutarimide formation is sequence dependent and can be inhibited by incorporating an amino acid like Lys(Boc) with steric hindrance from the protecting group. To prevent glutarimide formation, Ser(tBu) was used to replace the glycine in the GGG spacer adjacent to the residue of Glu, and a tailed cyclic RGD peptide, c[RGDfE(SGGKK-NH(2))] was successfully obtained.     

Rational design of cell-permeable cyclic peptides containing a d-Pro-l-Pro motif

Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a d-Pro-l-Pro motif, cyclo(AFΦrpPRRFQ) (where Φ is l-naphthylalanine, r is d-arginine, and p is d-proline). The structural constraints provided by cyclization and the d-Pro-l-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(Y^pVNFΦrpPRR) (where Y^p is l-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant β-hairpin character surrounding the d-Pro-l-Pro motif. These results demonstrate cyclo(AFΦrpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).     

Peptide nanotube aligning side chains onto one side

A novel pseudo cyclic penta‐β‐peptide composed of a β‐naphthylalanine, two β‐alanines, and a sequence of ethylenediamine‐succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod‐ or needle‐shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri‐β‐peptide composed of a β‐naphthylalanine and two β‐alanines. Insertion of a sequence of ethylenediamine‐succinic acid into β‐amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.     

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence–function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX₁HNGRHX₂C, cCX₁HNGRAX₂C, and cCX₁ANGRHX₂C) are related with the position of His residues and cationic amino acids.     

Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.     

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch

Kaposi’s sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi’s sarcoma (KS). When KSHV infects host’s cells, one of the virus’s proteins, latency-associated nuclear antigen 1 (LANA), binds to the host’s nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA–nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

Cross Talk Among Cyclic AMP, Cyclic GMP, and Ca2+ -Dependent Intracellular Signalling Mechanisms in Brain Capillary Endothelial Cells

Abstract: C-type natriuretic peptide and sodium nitroprusside, a nitric oxide donor molecule, induced large increases in cyclic GMP formation in cultured rat brain capillary endothelial cells. Isoproterenol, a potent agonist of adenylate cyclase, potentiated the actions of C-type natriuretic peptide and of sodium nitroprusside. These actions were not observed in the presence of isobutylmethylxanthine and were mimicked by forskolin. Endothelin-1 had no action on basal cyclic GMP levels. It reduced cyclic GMP formation induced by C-type natriuretic peptide and sodium nitroprusside by about 50%. These actions involved an ET_A receptor subtype and a Ca²⁺-dependent and protein kinase C-independent mechanism. Finally, increasing cyclic GMP slightly prolonged intracellular Ca²⁺ transients induced by endothelin-1. The results suggest the presence of extensive cross talk among cyclic AMP, cyclic GMP, and Ca²⁺-dependent mechanisms in endothelial cells of brain microvessels. The relevance of the results to the regulation of the blood-brain barrier permeability is discussed.     

Structure‐antigenicity of the V3 region of SIVmac envelope glycoprotein

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313–Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV‐1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti‐peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti‐V3 peptides from HIV‐1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of a cyclic peptide library based on the somatostatin sequence using the backbone amide linker approach

Herein, we report on the synthesis of a library of cyclic peptides targeted at the somatostatin receptor using the backbone amide linker strategy. After optimising head-to-tail cyclisation and cleavage conditions, a library of discrete cyclic peptides was assembled in high purity and good overall yield.     

Adrenocortical Steroids Modify Neurotransmitter-Stimulated Cyclic AMP Accumulation in the Hippocampus and Limbic Brain of the Rat

Glucocorticoid hormones are known to affect limbic system structures that have high levels of specific receptors for glucocorticoids, especially the hippocampus (HIPP). To understand how glucocorticoids may affect synaptic transmission, we have tested the effects of adrenal removal and glucocorticoid replacement on neurotransmitter-stimulated cyclic AMP accumulation in brain slices from the rat limbic system. Adrenalectomy (ADX) caused an enhancement of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in HIPP, amygdala (AMYG), and septum (SEP). In HIPP, ADX increased the cyclic AMP response to isoproterenol (ISOP) and decreased the response to histamine (HIST). In the AMYG and SEP, ADX did not affect significantly the action of ISOP, but ADX did decrease the response to HIST in AMYG. Administration of dexamethasone or corticosterone reversed the effects of ADX on cyclic AMP accumulation in the HIPP. The dexamethasone action on VIP-stimulated cyclic AMP accumulation takes place within 48 h and is most apparent in the mid-range of the VIP dose-response curve. These results demonstrate that glucocorticoids regulate neurotransmitter-stimulated cyclic AMP generation in a fashion that is specific, both for the neurotransmitter involved and for the brain regions affected.     

Synthesis of a cyclic peptide library based on the somatostatin sequence using the backbone amide linker approach

Summary Herein, we report on the synthesis of a library of cyclic peptides targeted at the somatostatin receptor using the backbone amide linker strategy. After optimising head-to-tail cyclisation and cleavage conditions, a library of discrete cyclic peptides was assembled in high purity and good overall yield.