摘要:

AbstractTargeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three‐dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non‐ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented. Copyright © 2005 European Peptide Society and John Wiley&Sons,

ISSN:1075-2617    

DOI:10.1002/psc.708

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractThe neurohypophyseal hormone oxytocin (CYIQNCPLG‐NH2, OT) is involved in the control of labor, secretion of milk and many social and behavioral functionsviainteraction with its receptors (OTR) located in the uterus, mammary glands and peripheral tissues, respectively. In this paper we propose the interactions responsible for OT binding and selectivity to OTR versus vasopressin ([F3,R8]OT, AVP) receptors: V1aR and V2R, all three belonging to the Class A G protein‐coupled receptors (GPCRs). Three‐dimensional models of the activated receptors were constructed using a multiple sequence alignment and the activated rhodopsin–transducin (MII–Gt) prototype [Ślusarz and Ciarkowski, 2004]as a template. The 1 ns unconstrained molecular dynamics (MD) of three pairs of receptor–OT complexes (two complexes per each receptor) immersed in the fully hydrated 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER7.0 force field. The relaxed models of ligand–receptor complexes were used to identify the putative binding sites of OT. The stabilizing interactions with conserved Gln residues in all complexes were identified. The nonconserved hydrophobic residues were proposed as responsible for OTR–OT selectivity and ligand recognition. These results provide guidelines for experimental site‐directed mutagenesis and if confirmed, they may be helpful in designing new selective OT analogs with both agonistic or antagonistic properties. Copyright © 2005 European Peptide

ISSN:1075-2617    

DOI:10.1002/psc.713

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractVasopressin (CYFQNCPRG‐NH2, AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G‐protein‐coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptorsversusthe closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three‐dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with GtαC‐terminal peptide of transducin MII‐Gt(338‐350) prototype as a template. AVP was docked into the receptor‐Gαsystems. The three lowest‐energy pairs of receptor‐AVP‐Gα(two complexes per each receptor) were selected. The 1‐ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER7.0 force field. Six relaxed receptor‐AVP‐Gαmodels were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed. Copyright © 2005 Eur

ISSN:1075-2617    

DOI:10.1002/psc.714

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractArginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G‐protein coupled receptors, termed V1a(vascular), V1b(pituitary) and V2(renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V1breceptor, we recently reported the human V1b, V1a, V2and OT receptor affinities of the following position 4 substituted analogues of [deamino‐Cys1] arginine vasopressin (dAVP)—(1) d[Leu4]AVP, (2) d[Orn4]AVP, (3) d[Lys4]AVP, (4) d[Har4]AVP, (5) d[Arg4]AVP, (6) d[Val4]AVP, (7) d[Ala4]AVP, (8) d[Abu4]AVP, (9) d[Nva4]AVP, (10) d[Nle4]AVP, (11) d[Ile4]AVP, (12) d[Phe4]AVP, (13) d[Asn4]AVP, (14) d[Thr4]AVP: (15) d[Dap4]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V1breceptor. Furthermore, peptides 1–4 exhibit high selectivities for the human V1breceptor with respect to the V1a, V2and OT receptors and, with d[Cha4]AVP, in functional tests, are the first high affinity selective agonists for the human V1breceptor (Cheng LLet al.,J. Med. Chem.47: 2375–2388, 2004). We report here the pharmacological properties of peptides 1–4, 5 (from a resynthesis), 7, 9–13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro: no Mg++) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V1b, V1a, V2and OT receptor affinities of peptides 1–5 and the rat V2receptor affinities for peptides: 7–15.The antidiuretic activities in units/mg of peptides 1–15, are: 1 = 378; 2 = 260; 3 = 35; 4 = 505; 5 = 748; 6 = 1150; 7 = 841; 8 = 1020; 9 = 877; 10 = 1141; 11 = 819, 12 = 110; 13 = 996; 14 = 758; 15 = 1053. Peptides 1–4 exhibit respectively the following rat and human (in brackets) V2receptor affinities: 1 = 3.1 nm (245 nm); 2 = 3.4 nm (1125 nm); 3 = 24.6 nm (11,170 nm); 4 = 0.6 nm (1386 nm). Their rat V1breceptor affinities are 1 = 0.02 nm; 2 = 0.45 nm; 3 = 9.8 nm; 4 = 0.32 nm. Their rat V1areceptor affinities are 1 = 1252 nm; 2 = 900 nm; 3 = 1478 nm; 4 = 32 nm. Their rat oxytocin (OT) receptor affinities are 1 = 481 nm; 2 = 997 nm; 3 = 5042 nm; 4 = 2996 nm. All four peptides have high affinities and selectivities for the rat V1breceptor with respect to the rat V1aand OT receptors. However, in contrast to their high selectivity for the human V1breceptor with respect to the human V2receptor, they are not selective for the V1breceptor with respect to the V2receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V2receptors. Peptides 1–4 are promising leads to the design of the first high affinity selective agonists for the rat V1breceptor. Copyright © 2005 European Peptide Society

ISSN:1075-2617    

DOI:10.1002/psc.710

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractSynthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine‐based europium(III) chelate designed for solution phase protein labeling is demonstrated. Copyright © 2005 European Peptide Society and John Wiley&Sons, L

ISSN:1075-2617    

DOI:10.1002/psc.697

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractIntegrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD‐based peptides have been used to block integrin‐binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate‐immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD‐containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD‐containing molecules and tested their ability to inhibit or to promote integrin‐dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by α‐helical coiled coil peptides fused at their amino‐terminal ends with an RGD‐containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin αVβ3‐dependent cell adhesion, but when used in solution, they blocked αVβ3‐dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10‐fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100‐fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that α‐helical coiled coil peptides carrying an amino‐terminal RGD motif can be used as soluble antagonists or surface‐immobilized agonists to efficiently inhibit or promote integrin αVβ3‐mediated cell adhesion, respectively. Copyright © 2005 Europea

ISSN:1075-2617    

DOI:10.1002/psc.707

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractPeptides containingNα‐methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of manyNα‐methylamino acids. An efficient and practical three‐step procedure for selectiveN‐methylation of peptides on solid support is described. The procedure was based on the well known solid‐phaseN‐methylation ofNα‐arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by ano‐nitrobenzenesulfonyl group, selectiveN‐methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three‐step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc‐Phe‐MeXaa‐Leu‐OH) containingN‐methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis ofNα‐methylamino acid containing peptides in a very short time using Fmoc solid‐phase peptide synthesis. Copyright © 2005 European Pe

ISSN:1075-2617    

DOI:10.1002/psc.711

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractThe coupling of the techniques, high‐performance liquid chromatography (HPLC), orthogonal acceleration time‐of‐flight mass spectrometry (OATOF‐MS) and inductively coupled plasma mass spectrometry (ICP‐MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK‐(5,9)‐fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4–5 and 7–8. We also demonstrate the equivalent participation of NEP and angiotensin‐converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme. Copyright © 2005 European Peptide Society and

ISSN:1075-2617    

DOI:10.1002/psc.712

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractDecomposition of the resin linkers during TFA cleavage of the peptides in the Fmoc strategy leads to alkylation of sensitive amino acids. TheC‐terminal amide alkylation, reported for the first time, is shown to be a major problem in peptide amides synthesized on the Rink amide resin. This side reaction occurs as a result of the Rink amide linker decomposition under TFA treatment of the peptide resin. The use of 1,3‐dimethoxybenzene in a cleavage cocktail prevents almost quantitatively formation ofC‐terminalN‐alkylated peptide amides. Oxidized by‐product in the tested Cys‐ and Met‐containing peptides were not observed, even if thiols were not used in the cleavage mixture. Copyright © 2005 European Peptide Society and John W

ISSN:1075-2617    

DOI:10.1002/psc.706

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY

摘要:

AbstractThe binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self‐association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range ∼50–500 µM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide–peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both α‐helical and β‐turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the α‐helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the β‐type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation. Copyright © 2005 European Peptide Society and

ISSN:1075-2617    

DOI:10.1002/psc.715

出版商:John Wiley&Sons, Ltd.    

年代:2006

数据来源: WILEY