bluuebunny/crossref_metadata_2025 huggingface dataset:
Design and synthesis of highly selectivein vitroandin vivouterine receptor antagonists of oxytocin: comparisons with Atosiban
Authors: MANNING M. , MITEVA K. , PANCHEVA S. , STOEV S. , WO N.C. , CHAN W.Y.
Published: August 2010

We report the solid phase synthesis and some pharmacological properties of seven position two analogues (peptides 1–7) of one of our lead oxytocin antagonists. des‐9‐glycinamide[1‐(β‐mercapto‐β, β‐pentamethylenepropionic acid), 2‐O‐methyltyrosine, 4‐threonine]ornithinevasotocin(desGly‐NH2,d(CH2)5‐ [Tyr(Me)2,Thr4]OVT) (A). Peptides 1–7 have the following substituents at position two (1)d‐Tyr(Me); (2)l‐Tyr(Et); (3)d‐Tyr(Et); (4)l‐Tyr; (5)d‐Tyr; (6)d‐Phe and (7)d‐Trp. These were evaluated for agonistic and antagonistic activities inin vitroandin vitroOT assays,in vivovasopressor (V1a‐receptor) assays andin vivoantidiuretic (V2‐receptor) assays. None of the seven peptides exhibits oxytocic or vasopressor agonism. Peptides 1,2,4,6 and 7 are extremely weak V2agonists (V2activities range from 0.001 to 0.02 U/mg). Peptides 3 and 5 exhibit weak V2antagonism (pA2>6.0 and >5.5, respectively). Peptides 1–7 exhibit potentin vitro(no Mg2+) OT antagonism (anti‐OT pA2values range from 7.66 to 8.03). Peptides 1 and 4–7 exhibit potentin vivoOT antagonism. Estimated in vivo anti‐OT pA2values range from 7.06 to 7.79 (peptides 2 and 3 were not tested). With anti‐VlapA2values of 5.17‐6.25 all seven peptides exhibit reduced anti‐V1a, potencies relative to the parent peptide (A) (anti‐V1a. pA2= 6.46). Four of these peptides (4‐7) exhibit striking gains inin vitroandin vivoanti‐OT/anti‐V1a. selectivities compared to (A) which has anin vitroselectivity of 30 and anin vivoselectivity of 18. Thed‐Tyr2(5),d‐Trp2(7),d‐Phe2(6) andl‐Tyr2(4) analogues of (A) exhibit anti‐OT (in vitro)/anti‐V1aselectivities = 240, 390, 404 and 540, respectively. Thel‐Tyr2(4),d‐Trp2(7),d‐Phe2(6)andd‐Tyr2(5) analogues exhibited anti‐OT (in vivo)/anti‐V1aselectivities of 72, 80, 88 and 95, respectively. Peptides 4–7 appear to be the most selective peptide OT antagonists reported to date. In this regard it may be noted that they appear to be as or more potent and much more selective than the closely related OT antagonist 1‐deamino[D‐Tyr(Et)2,Thr1]OVT (Atosiban) which is currently undergoing clinical trial as a potential therapeutic agent for the prevention of premature labor. Atosiban (peptide 8) was resynthesized and pharmacologically evaluated in our laboratories. Atosiban exhibits the following antagonistic potencies. Anti‐OT (in vitro, no Mg2) pA2= 7.71; anti‐OTin vivopA2= 7.05; anti‐V1pA2= 6.14 and anti‐V2pA2≅ 5.9. Its anti‐OT (in vivo)/anti‐V1aselectivity is 8. Some of these antagonists may be suitable candidates for evaluation as potential tocolytic agents for use in the treatment of pre‐term labor. They could also serve as useful new pharmacological tools for studies on the physiological roles of oxytocin. Finally, the findings presented here provide useful clues for the design of more potent and more selective OT antagonists.
Design, synthesis, and characterization of HIV‐1 enhancer‐binding polypeptides derived from bacteriophage 434 repressor
Authors: STÄDLER KURT , LIU NIANKUN , TROTMAN LLOYD , HILTPOLD ANDRÉ , CADERAS GEORG , KLAUSER STEPHAN , HEHLGANS THOMAS , GUTTE BERND
Published: August 2010

We have designed and synthesized HIV‐1 enhancer‐binding polypeptides that were derived from bacteriophage 434 repressor. These peptides were 39–54 residues long and contained either the recognition helix or the entire helix‐turn‐helix motif of the DNA‐binding domain of 434 repressor. The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70‐bp HIV enhancer DNA was ca. 10−8 M. The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF‐κB from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I. R42 analogues with mutated recognition helix had lower DNA binding specificity. It remains to be investigated whether our artificial HIV enhancer‐binding polypeptides are active in vivo.
The 2,4‐dinitrophenyl group for protection of hydroxyl function of tyrosine during solid‐phase peptide synthesis
Authors: PHILOSOF‐OPPENHEIMER RACHEL , PECHT ISRAEL , FRIDKIN MATI
Published: August 2010

The facile thiolytic cleavage of the O‐2,4‐dinitrophenyl (Dnp) tyrosine bond was applied to the solid‐phase synthesis of the 22‐amino acid residue peptide H‐Asp‐Ala‐Val‐Tyr‐Thr‐Gly‐Leu‐Asn‐Thr‐Arg‐Asn‐Gln‐Glu‐Thr‐Tyr‐Glu‐Thr‐Leu‐Lys‐His‐Glu‐Lys‐OH, corresponding to positions 62‐83 in the chain of the type 1 receptor for Fcε, domains expressed on the rat mucosal‐type mast cells (line RBL‐2H3). A method for the spectrophotometric determination of insoluble O‐Dnp as well as of unprotected phenolic moieties of tyrosine was developed. It is based on monitoring S‐Dnp‐2‐mercaptoethanol, produced upon O‐Dnp thiolysis by 2‐mercaptoethanol. © Munksgaard 1995.

Dedicated to the memory of Dr. Susumu Funakoshi, a dear friend and a leader in peptide chemistry.
Mechanism of solvent‐induced thermal stabilization of α‐amylase from Bacillus amyloliquefaciens
Authors: RAJENDRAN S. , RADHA C. , PRAKASH V.
Published: August 2010

The transition temperature of irreversible thermal inactivation of α‐amylase from Bacillus amyloliquefaciens was estimated to be 60°C. At this temperature, the enzyme inactivation followed first‐order kinetics, having a half‐life (t1/2) of 12 min with a rate constant (k) of 0.06 min−l. Conformational change was a prerequisite for this thermal inactivation. This is governed by stepwise temperature‐dependent phenomena. Among the solvent stabilizers tested, the enzyme was thermally stable in presence of DMSO and PEG 300 and the stabilizing efficiency of these cosolvents was concentration‐dependent. The enzyme was partially stabilized by 5.0 M DMSO and 1.9 M PEG 300 up to 78°C. However, above 78°C the enzyme was inactivated in these cosolvents also. The mechanism of stabilization has been explained by preferential hydration of the enzyme in these structure stabilizing solvents by exclusion from the protein surface and interface by measurement of partial specific volume in these cosolvents. The data suggest a high value of preferential interaction parameter, (δg3/δg2)T, μ1,μ3 being ‐0.606 g/g in 40% DMSO and a low value of ‐0.025 g/g in 5% glycerol. The preferential interaction parameters in sucrose and glycerol suggests that (δg3/δg2)T, μ1,μ3 is highest of ‐0.420 g/g in 10% glycerol than any other cosolvent. © Munksgaard 1995.
Peptide synthesis using novel S‐sulfocysteine derivatives
Authors: MAUGRAS ISABELLE , GOSTELI JACQUES , RAPP ELKE , NYFELER ROLF
Published: August 2010

New cysteine S‐sulfonate derivatives, Boc‐Cys(SO3Na)‐ONa 2 and Fmoc‐Cys(SO3Na)‐ONa 3, were prepared and their utility for peptide synthesis examined. The Fmoc derivative 3 was used in the solid‐phase peptide synthesis of Arg8‐vasopressin 9 via the Bunte salt 7. Satisfactory S‐sulfonate stability was observed when p‐cresol scavenged the cleavage from the resin. The intermediate 7 was purified by ion‐exchange chromatography prior to S‐sulfonate cleavage with tributylphosphine. © Munksgaard 1995.
Disulfide bond‐forming reaction using a dimethyl sulfoxide/aqueous HCl system and its application to regioselective two disulfide bond formation
Authors: TAMAMURA HIROKAZU , OTAKA AKIRA , NAKAMURA JUNKO , OKUBO KAORI , KOIDE TAKAKI , IKEDA KYOKO , IBLKA TOSHIRO , FUJII NOBCTAKA
Published: August 2010

Disulfide bond formation in S‐acetamidomethyl (Acm) cysteine‐containing peptides by successive treatments with silver trifluoromethanesulfonate (AgOTf) and dimethyl sulfoxide (DMSO)/aqueous HCl is described. An S‐Acm cysteine was found to be quantitatively converted into cystine by deprotection of the Acm group with AgOTf followed by DMSO/aqueous HCl treatment. Under these reaction conditions, no significant side reactions were observed with oxidation‐sensitive amino acids such as Met, Tyr and Trp. Oxytocin and a Trp‐containing peptide, urotensin II, were prepared by this method. Furthermore, regioselective two disulfide bond formation was found to be feasible by the combination of air oxidation and the AgOTf‐DMSO/HCl system. This strategy has been successfully applied to the syntheses of tachyplesin I and endothelin I, which have two disulfide bonds and a Trp residue in the molecule.
Design and synthesis of amphipathic antimicrobial peptides
Authors: ZHONG LINGXIU , PUTNAM REBECCA J. , JOHNSON W. CURTIS , RAO A. GURURAJ
Published: August 2010

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic α‐helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no α‐helicity in buffer or liposomes, exhibits a modest degree of α‐helical structure in the presence of the a‐helical inducer, 2,2,2‐trifluoroethanol. However, rational modifications result in a derivative which assumes an α‐helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.
End‐group modified retro‐inverso isomers of tripeptide oxytocin analogues: binding to neurophysin II and enhancement of its self‐association properties
Authors: RUVO MENOTTI , FASSINA GIORGIO
Published: August 2010

The importance of peptide backbone structure in peptide/protein recognition events has been tested evaluating the binding properties of end‐group modified retro‐inverso isomers of MYF and LYF amides, tripeptides able to mimic oxytocin in binding neurophysin II and in potentiating its self‐association. The isomers, topochemically related to their parent peptides, have been prepared respectively from all‐D N‐acetyl‐FYM and N‐acetyl‐FYL amides via the Hofmann‐type rearrangement mediated by iodobenzene bis‐trifluoroacetate. Retro‐inverso isomers recognised neurophysin II with similar affinity as the parent peptides, as determined by analytical affinity chromatography on columns prepared immobilising neurophysin II on preactivated supports. In addition, their effect on neurophysin II self‐association was similar to the tripeptide oxytocin analogues, potentiating neurophysin II dimerization to the same extent, as evaluated by solid‐phase binding assays on microtiter plates coated with neurophysin II. Recognition specificity of retro‐inverso isomers was further demonstrated by their inhibitory effect on the interaction between neurophysin II and oxytocin tripeptide analogues. Results suggest that only the proper orientation of the α‐amino group and of the side chains plays a dominant role in the binding of tripeptide analogues to neurophysin II and potentiation of its self‐association, while the peptide backbone topology has little influence on the recognition process.
Conformational studies on model dipeptides of Gly, L ‐Ala and their Cα‐substituted analogs
Authors: RAMNARAYAN KALYANARAMAN , CHAN MING FAI , BALAJI VITUKUDI N. , PROFETA SALVATORE , RAO SHASHIDHAR N.
Published: August 2010

As a part of the development of conformational guidelines for the design of metabolically altered peptidomimetics, we present conformational energy calculations on model dipeptide compounds with glycine (Gly), L‐alanine (Ala), α‐aminoisobutyric acid (Aib), L‐tert‐butylglycine (Tle), L‐phenylglycine (Phg), (α,α)‐diphenylglycine (Dφg), L‐2‐aminobutyric acid (Abu), 2‐amino‐2‐ethylbutync acid (Deg), L‐2‐amino‐2‐vinylacetic acid (Ava) and (α,α)‐divinylglycine (Dvg). The energy calculations have been made using molecular mechanics methods with a force field derived from MM2. The salient features are expressed in terms of conformational energy plots, drawn as a function of the backbone torsion angles φ(Ci‐1′‐Ni‐Ciα‐Ci′) and ψ(Ni‐ Ciα‐Ni + 1). The low‐energy structures of these compounds are qualitatively consistent with the X‐ray crystal structure analyses of peptides and peptidomimetics. They are also in agreement with the results of the solution‐phase studies carried out by NMR and IR techniques. The results obtained have important implications in the design of conformationally restricted peptidomimetics.
Synthesis and characterization of indolicidin, a tryptophan‐rich antimicrobial peptide from bovine neutrophils *
Authors: ABEL ROBERT J. VAN , TANG YI‐QUAN , RAO V.S.V. , DOBBS CRAIG H. , TRAN DAT , BARANY GEORGE , SELSTED MICHAEL E.
Published: August 2010

Indolicidin, a novel tryptophan‐rich microbicidal tridecapeptide amide isolated originally from granules of bovine neutrophils, has been prepared by optimized manual and automated protocols of stepwise solid‐phase synthesis with Nα‐9‐fluorenylmethyloxycarbonyl (Fmoc) amino acid derivatives. Both standard polystyrene (PS) and polyethylene glycol‐polystyrene (PEG‐PS) graft supports were used in combination with handles that provide C‐terminal peptide amides: 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid (PAL) or 5‐(9‐Fmoc‐aminoxanthen‐2‐oxy)valeric acid (XAL). Final deprotection/cleavage was carried out with reagent K, trifluoroacetic acid–phenol–water–thioanisole–1,2‐ethanedithiol (82.5:5:5:5:2.5), or reagent B, trifluoroacetic acid–phenol–water–tri(isopropyl)silane (88:5:5:2), and related cocktails. Initial purities as high as 93% were obtained immediately following cleavage. In the largest‐scale synthesis carried out, 0.8 g of HPLC‐purified indolicidin (> 99% pure) was obtained, representing a 39% overall yield based on C‐terminal Arg(Pmc) anchored to PAL‐PS‐resin. The main synthetic product, and some by‐products, were characterized by analytical high‐performance liquid chromatography (HPLC), sequencing, and fast atom bombardment mass spectrometry (FABMS). The antimicrobial potencies of natural and synthetic indolicidin, as determined by in vitro antibacterial and antifungal assays, were identical. Further, the reactivities of natural and synthetic peptides with anti‐indolicidin antibody were indistinguishable. © Munksgaard 1995.
Development of a radioimmunoassay for a pseudononapeptide bombesin/GRP antagonist with antitumor activity
Authors: GROOT KATE , HORVÀTH JUDIT E. , CAI REN‐ZHI , SCHALLY ANDREW V.
Published: August 2010

Bombesin‐like and GRP‐like peptides may act as autocrine growth factors in the proliferation of some cancers. A pseudononapeptide bombesin antagonist, [d‐Tpi6,Leu13φ(CH2NH)‐Le14]bombesin(6–14), and related analogs synthesized in our laboratory significantly inhibit tumor growth in various cancer models. A radioimmunoassay (RIA), suitable for determination of RC‐3095 and its congeners in unextracted serum, was developed in order to facilitate further experimental and clinical evaluation of this bombesin/GKP receptor antagonist for the treatment of various tumors. Antibodies were generated against RC‐3095 and Des‐Tpi1‐RC‐3095, conjugated to bovine serum albumin with glutaraldehyde. Antiserum JH‐631b was selected for further experiments based on the antibody characterization. At an antiserum dilution of 1:189000, this antibody bound approximately 50% of 7 fmol of added radiolabeled Tyr1‐RC‐3095. The antibody cross‐reacted with C‐terminal fragments of RC‐3095. Fragments without the C‐terminus and naturally existing peptides of the bombesin family or structurally unrelated peptides did not cross‐react. The minimum detectable dose of RC‐3095 was 0.4 pg/tube. Intra‐ and interassay coefficients of variation ranged from 3.2 to 4.4% and from 5.6 to 12.8%, respectively. The RIA is suitable for direct determination of RC‐3095 in serum. The RIA should be of value for monitoring levels of this analog in serum during long‐term therapy. © Munksgaard 1995.
Chemical synthesis of phosphopeptides using the arylthio group for protection of phosphate: application to identification of cdc2 kinase phosphorylation sites
Authors: UENO YOSHIHITO , MAKINO SHINGO , KITAGAWA MASATOSHI , NISHIMURA SUSUMU , TAYA YOICHI , HATA TSUJIAKI
Published: August 2010

Synthesis of two O‐phosphorylated dipeptides, N‐Boc‐O[S,S‐bis(p‐methoxyphenyl)phosphorodithioyl]‐serylproline and N‐Boc‐O‐[S,S‐bis(p‐methoxyphenyl)phosphorodithioyl]threonylproline, as new O‐phosphorylated dipeptide building blocks for the synthesis of O‐phosphorylated peptides, is described. Peptides containing phosphoserine or phosphothreonine were prepared by use of these building blocks in the Boc mode of liquid‐phase peptide synthesis. Sites phosphorylated by cdc2 kinase were easily identified using these chemically synthesized phosphopeptides.
Synthesis of the basic‐helix‐loop‐helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties
Authors: BISHOP PATRICIA , JONES CORY , GHOSH INDRANEEL , CHMIELEWSKI JEAN
Published: August 2010

The basic‐helix‐loop‐helix (bHLH) region of the immunoglobulin enhancer binding protein E47 (IEB E47) was prepared in high yield by a solid‐phase peptide synthesis methodology. Size‐exclusion chromatography, sedimentation equilibrium and cross‐linking data showed that the synthetic bHLH protein, 1, was dimeric, and higher‐order aggregates of trimer and tetramer were also observed. The circular dichroism spectrum of 1 showed a high helical content, which increased upon addition of DNA containing the κE2 sequence. Gel mobility shift experiments showed that protein 1 bound sequence specifically to the κE2 sequence with a binding constant of 10−10 M2, and had an affinity for other E box sequences as well. Comparisons between the co‐crystal structure of IEB E47 with DNA and structural studies in solution showed lower helical contents in solution as would have been predicted from the crystal structure. © Munksgaard 1995.
Synthesis of a metal binding protein designed on the α/β scaffold of charybdotoxin*
Authors: PIERRET BRIGITTE , VIRELIZIER HENRI , VITA CLAUDIO
Published: August 2010

The α/β scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid‐phase synthesis and HBTU (2‐(1H‐benzotriazol‐l‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate) coupling of Fmoc‐protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post‐synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2°, yield. Circular dichroism analysis showed that the newly designed protein is folded in a α/β structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu2+ and Zn2+ ions bind with high affinity to the newly engineered protein. These results demonstrate that the α/β fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins. © Munksgaard 1995.
A model of the active site of dipeptidyl peptidase IV predicted by comparative molecular field analysis and molecular modelling simulations
Authors: BRANDT W. , LEHMANN T. , THONDORF I. , BORN I. , SCHUTKOWSKI M. , RAHFELD J.‐U. , NEUBERT K. , BARTH A.
Published: August 2010

A molecular model of the active site of the serine protease dipeptidyl peptidase IV (DPP IV or CD26) has been developed on the basis of comparative molecular field analysis (CoMFA) of competitive inhibitors and by force field calculations. By application of CoMFA experimentally obtained inhibition constantsKihave been successfully predicted. The resulting steric and electrostatic coefficients of CoMFA were used for the development of the molecular model. The main assumptions of the model are the recognition of substrates or inhibitors by the side chains of a tyrosine (S1‐position) and a tryptophan residue (S2‐position). The model helps us to understand a multitude of experimental data regarding the substrate specificity of this enzyme as well as results obtained by genetic engineering experiments by other authors. General conclusions concerning a new family of serine proteases are drawn and discussed. © Munksgaard 1995.
Proposals for the angiotensin II receptor‐bound conformation by comparative computer modeling of AII and cyclic analogs
Authors: JOSEPH M.‐P. , MAIGRET B. , SCHERAGA H. A.
Published: August 2010

A conformational search using high‐temperature molecular dynamics on angiotensin II (AII) and on two cyclic S‐S bridged analogs, namely [Hcy3,5]AII and [Cys3,5]AII, In conjunction with a cluster analysis based on the similarities of the three‐dimensional patterns of the binding and activation elements, has led to putative AII receptor‐bound conformations. These conformations are characterized by a compact folded shape of the peptide backbone, and by particular relative positions of the four pharmacophore groups, namely the aromatic moieties of the Tyr4, His6 and Phe8 residues, and the C‐terminal carboxyl group. This compact folded shape, arising from attractive electrostatic interactions between the desolvated N‐ and C‐terminal groups, is similar to the crystallographically determined conformation of AII bound to the antibody Fab receptor. © Munksgaard 1995.
Correlations between biological activity and structural properties for two short homologous sequences in thymosin β4 and gelsolin
Authors: FEINBERG JEANNE , MERY JEAN , HEITZ FRÉDÉRIC , BENYAMIN YVES , ROUSTAN CLAUDE
Published: August 2010

Gelsolin and thymosin β4 appear to be two important actin‐associated proteins involved in the regulation of actin polymerization. It has been widely demonstrated that thymosin is the major cellular actin‐sequestering factor shifting the polymerization equilibriuni of actin towards a monomeric state. At the same time gelsolin, a Ca2+ and inositol phosphate sensitive protein, regulates actin filament length. The interactions of these two proteins with actin are rather complex and require the participation of several complementary peptide sequences. We have identified a common motif, (I. V)EKFD, in the two proteins in the functional sequences so far examined. Gelsolin‐ and thymosin β4‐related peptides including the common motif were synthesized and their structural and functional properties studied. These two sequences exert a major inhibitory effect on salt‐induced actin polymerization. We used circular dichroism and Fourier‐transform infrared spectroscopy to show that the two synthetic peptides present some secondary structure in solution. As far as the peptide derived from the thymosin sequence was concerned, α‐helical structure was induced by trifluoroethanol as observed with the full‐length molecule. These experiments underscore the importance of the conformational state of peptide fragments in their biological activities. ELISA and fluorescence measurements have been used to identify the binding regions of these fragments to a C‐terminal region (subdomain 1) of the actin sequence. Our results also emphasize the relationship between the propensity of small sequences to form secondary structures and their propensity for biological activity as related to actin interaction and inhibition of actin polymerization. ©Munksgaard 1996.
Dynorphin‐phospholipid membrane interactions: Role of phospholipid head‐group and cholesterol
Authors: ALFORD DENNIS R. , RENUGOPALAKRISHNAN V. , DÜZGÜNES NEJAT
Published: August 2010

The interaction of the κ‐opioid receptor‐selective heptadecapeptide dynorphin A(1‐17) (Tyr1‐Gly‐Gly‐Phe‐Leu5‐Arg‐Arg‐Ile‐Arg‐Pro10‐Lys‐Leu‐Lys‐Trp‐Asp15‐Asn‐Glu) with phospholipid membranes has been investigated by monitoring the leakage of the internal aqueous contents of liposomes, the changes in the tryptophan emission spectrum, and the collisional quenching of tryptophan fluorescence by brominated lipids. The peptide induces more extensive leakage of contents from phosphatidylserine than from phosphatidyl‐choline vesicles, and experiences a blue shift of the Trp fluorescence emission maximum in the presence of phosphatidylserine vesicles. In the presence of phosphatidylcholine vesicles, however, the Trp fluorescence intensity is reduced without a blue shift. In phosphatidylserine membranes containing 10 mol% phosphatidylcholine, the intensity of the blue‐shifted fluorescence is enhanced. This avid interaction of dynorphin A(1‐17) with phosphatidylserine membranes is likely to be mediated by the positively charged Arg and Lys groups. It is proposed that. while the N‐terminus of the peptide may be embedded in the bilayer in analogy with dynorphin (1–13), the C‐terminal region of dynorphin A (1‐17) bends back onto the bilayer/water interphase, and that the Trp14, residue is stabilized in a hydrophobic pocked near the interphase by the interaction of the neighboring charged amino acids with the phosphate, carboxyl and amino groups on phosphatidylserine. ©Munksgaard 1996.
β‐Endorphin1–31 in the rat pituitary Discrepancy between the molecular masses of the DNA‐deduced peptide and of the mass spectrometric‐measured endogenous peptide
Authors: GRIGORIANTS OLGA O. , DESIDERIO DOMINIC M.
Published: August 2010

Mass spectrometry was used to determine the molecular mass of rat pituitary β‐endorphin1–31 (BErat, 1–31 The measured molecular mass (3435 ± 1 Da, n= 5) of endogenous BErat, 1–31 differed from the molecular mass of commercially available synthetic BErat, 1–31 (3465 ± 1 Da, n= 9), but corresponded to the molecular mass of synthetic BEbovine, 1–31 (3436 ± 3 Da, n= 3). Based on the combination of these ESIMS molecular mass measurements, HPLC retention time data, LSIMS measurement of the molecular mass of selected tryptic fragments, and consideration of codon sequences, we suggest that the amino‐acid sequence of endogenous BErat, 1–31differs from the DNA‐deduced sequence of BErat, 1–31, and that endogenous BErat, 1–31 contains Ala instead of Val in position 26. ©Munksgaard 1996.
Fourier‐transform infrared conformational study of bovine insulin in surfactant solutions
Authors: VECCHIO GIUSEPPE , BOSSI ALESSANDRA , PASTA PIERO , CARREA GIACOMO
Published: August 2010

To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier‐transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23‐30)octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600–1700 cm−1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a β‐sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (≥ 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar. © Munksgaard 1996
Investigation of the 35–62 conserved helix and disulfide loop of bovine prothrombin using an anti‐peptide antibody
Authors: CULBERTSON ANNE A. , KEVERLINE KATHRYN I. , KLAPPER DAVID G. , HISKEY RICHARD G.
Published: August 2010

A 28‐residue peptide corresponding to the 35–62 region of bovine prothrombin fragment 1 (BF1) was synthesized by solid‐phase methods. In BF1 this region consists of three conserved aromatic residues within an α‐helical region followed by a disulfide loop. This synthetic peptide was used to produce murine monoclonal antibodies (MAbs) that would recognize and bind native BFI. Antibody AH.Ab.E3, an IgGl antibody that was isolated and cloned, recognized and bound to both the synthetic peptide and the BF1 molecule. Residues 55–59 (REKLN) were shown to be critical for antibody binding. This MAb was subsequently used to study the 48‐62 disulfide loop region of BFl. MAb AH.Ab.E3, which has been shown to bind the BF1 calcium‐dependent conformation (BFl:Ca), does not appear to perturb the binding interaction between BFI:Ca and phospholipid (PL) vesicles as studied by light scattering methods. © Munksgaard 1996.
Synthesis of peptide p‐nitroanilides mimicking fibrinogen‐ and hirudin‐binding to thrombin Design of slow reacting thrombin substrates
Authors: RIJKERS DIRK T.S. , HEMKER H. COENRAAD , TESSER GODEFRIDUS I.
Published: August 2010

The design and synthesis of 20 peptide p‐nitroanilides is described. The nitroanilides are used as thrombin substrates that share uncommon properties. These substrates are tested for their applicability to measure thrombin generation in activated plasma. This technique requires substrates that must slowly but selectively be hydrolyzed by thrombin. To ensure selectivity, thrombin's natural substrate and its most potent inhibitor were used as lead compounds. Eighteen peptide p‐nitroanilides were synthesized using human fibrinogen Aα[7‐16] decapeptide as lead structure since this fragment constitutes the minimal sequence which binds to thrombin with high affinity. Two other chromogenic substrates were designed using 5‐amino‐2‐nitrobenzoic acid as the chromophore. A peptide giving subsite interactions with the fibrinogen recognition exosite was coupled to the carboxylic function. As the peptide segment, the carboxyl terminal part of hirudin (residues 50‐65) was chosen to ensure highly specific thrombin recognition. From the 20 nitroanilides synthesized. We indeed obtained a number of compounds which can be used as substrate in the thrombin generation assay. © Munksgaard 1996.
Three‐dimensional structure of the RGD‐containing snake toxin albolabrin in solution, based on 1H NMR spectroscopy and simulated annealing calculations
Authors: SMITH K. JOHN , JASEJA MAHESH , LU XINJIE , WILLIAMS JANICE A. , HYDE EVA I. , TRAYER IAN P.
Published: August 2010

Albolabrin is a snake toxin that contains a RGD‐(Arg‐Gly‐Asp) sequence motif and competes with fibrinogen to bind to the integrin αIibβ3 (GpIIb‐IIla) on platelets. It thus inhibits platelet aggregation and cell‐cell adhesion. It shows a high sequence similarity to other disintegrins, yet the reported disulfide bonding pattern for this peptide differs from that of others in this family. Recently we reported the assignment of the 1H‐NMR spectrum of albolabrin and a preliminary description of its secondary structure [Jaseja, M., Smith, K.J., Lu, X., Williams, J.A., Trayer, H., Trayer, I.P. & Hyde, E.I. (1993) Eur. J. Biohem.218, 853‐8601, Here we present a more detailed description of the secondary and the tertiary structure, based on the 1H NMR results and simulated annealing methods.

The structure of albolabrin in solution was calculated using 318 distance and 18 dihedral angle restraints. The average atomic RMS deviation between 12 refined structures and the mean structure was 3.1 Å for the backbone. The protein appears to be highly mobile. Its structure is dominated by a series of turns and by three hairpins. each with a short region of distorted antiparallel β‐pleated sheet, held together by six disulfide bridges. The most well defined area is the hydrophobic core, residues 21‐47 and 57‐67, which is clustered around F40 and has a backbone atomic RMS deviation of only 1.3 Å from the mean structure. The RGD adhesion sequence is found at the highly mobile tip of one of the β‐hairpins, protruding from the body of the protein.

Many of these structural features are similar to those of other disintegrins, and differences in the disulfide bonding pattern of the disintegrins can be acconiodated without significant energy penalty. Comparison of this structure with other proteins of similar function suggests that it is the RGD‐loop, rather than the precise topology of the proteins. that is important to antagonist activity. © Munksgaard 1996.
Synthesis and conformational analysis of cyclic pentapeptide endothelin antagonists
Authors: LIN MENGFEN , CHAN MING FAI , BALAJI V.N. , CASTILLO ROSARIO S. , LARIVE CYNTHIA K.
Published: August 2010

Two endothelin antagonists cyclo(d‐Leu‐d‐Val‐Pro‐d‐Asp‐Trp) (IPI‐147), and cyclo(d‐Trp‐d‐Asp‐Ac3c‐d‐Val‐Leu) (IPI‐725) have been synthesized. Their solution conformations have been studied in aqueous solution by NMR spectroscopy and dynamics simulation. Activity studies show that IPI‐725 is a strong ETA antagonist, while IPI‐147 is a weak ETA antagonist. Comparison of the solution conformations of these two ETA antagonists suggests that the difference in their activities results from their structural differences. IPI‐147 contains a type II β‐turn with a hydrogen bond between NH of d‐Val and the C = O of d‐Asp. IPI‐725, on the other hand, contains two turns, a type II β‐turn with a hydrogen bond between NH of d‐Asp and C=O of d‐Val, as well as a γ‐turn with a hydrogen bond formed between d‐Val NH and d‐Asp carbonyl group. Therefore IPI‐147 appears to be more flexible than IPI‐725. Although both γ‐turns contain the same residues, their orders in the turn are reversed. The β‐turn in IPI‐725 is formed with d‐Val:Leu:d‐Trp:d‐Asp, while in IPI‐147, the β‐turn is formed with d‐Asp:Trp:d‐Leu:d‐val. The activities and solution conformations of IPI‐147 and IPI‐725 were also compared with BQ‐123 [Cyclo(d‐Trp‐d‐Asp‐Pro‐d‐Val‐Leu)], a well characterized, highly potent endothelin antagonist. © Munksgaard 1996.
Structure–biological activity relationships of 11‐residue highly basic peptide segment of bovine lactoferrin
Authors: KANG JOO HYUN , LEE MYUNG KYU , KIM KIL LYONG , HAHM KYUNG‐SOO
Published: August 2010

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure‐antimicrobial activity relationships, the basic amino acid‐rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue‐long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N‐termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 MM SDS were correlated with their antimicrobial activities. These results suggested that the 11‐residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity. © Munksgaard 1996.
Addition of the (28–38) peptide sequence of PACAP to the VIP sequence modifies peptide selectivity and efficacy
Authors: GOURLET PHILIPPE , VANDERMEERS ANDRÉ , VANDERMEERS‐PIRET MARIE‐CLAIRE , DE NEEF PHILIPPE , ROBBERECHT PATRICK
Published: August 2010

Chimeric peptides were synthesized by adding the C‐terminal extension 28‐38 of the pituitary adenylate cyclase activating polypeptide (PACAP) to the sequences (1–27), (2–27), (3–27) and (6–27) of VIP. The capacity of these peptides to occupy the selective PACAP‐ and the non‐selective PACAP‐VIP receptors and to stimulate adenylate cyclase activity was studied in Chinese hamster ovary (CHO) cells expressing the recombinant receptors. The results were compared to those obtained with VIP and the corresponding VIP fragments.

The presence of the (28–38) PACAP extension increased at least 100‐fold the VIP‐ or VIP fragment affinities for the selective PACAP receptor but not for the non‐selective PACAP‐VIP receptors. Furthermore, on both receptors, the extension increased peptide intrinsic activity: VIP(3–28) was a partial agonist; while VIP(3–27)/PACAP(28–38) was as potent as VIP and was apparently a full agonist; VIP(6–28) had no intrinsic activity, but VIP(6–27)/PACAP(28–38) was a partial agonist.

These results suggest: (1) the presence of a specific domain for the (28–38) PACAP sequence on the selective PACAP receptor; and (2) a stabilizing effect of the (28–38) PACAP sequence on the structure ofN‐terminally truncated VIP. © Munksgaard 1996.
Combined use of NMR, distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)‐Cys2, D(L)‐Cys5]enkephalin
Authors: KOLP BRUNHILDE , ANDREAE FRITZ , FABIAN WALTER M.F. , STERK HEINZ
Published: August 2010

The solution structures of a series of conformationally restricted pentapeptides with a sequence H‐Tyr1‐Cys2‐Gly3Phe4‐Cys5‐OH cyclic (2‐5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H‐Tyr1‐Cys2‐Gly3‐Phe4‐Cys5‐OH cyclic (2‐5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H‐Tyr1‐D‐Cys2‐Gly3‐Phe4‐Cys5‐OH cyclic (2‐5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H‐Tyrl‐Cys2‐Gly3‐Phe4‐Cys5‐OH cyclic (2‐5) disulfide, which contains solely L‐Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.
Folded conformations of the δ‐selective opioid dermenkephalin with head‐to‐tail interactions. A simulated annealing study through NMR restraints
Authors: NAIM MOHAMMED , NICOLAS PIERRE , BARON DANIEL
Published: August 2010

Despite similar tripeptideN‐termini, dermorphin (Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) and dermenkephalin (Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ‐ and δ‐opioid receptors, respectively. Structure‐activity relationship studies have shown that theN‐terminal tripeptide, Tyr‐D‐Xaa‐Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ‐ and δ‐receptors while the nature and/or the conformation of theC‐terminus His‐Leu‐Met‐Asp‐NH2of dermenkephalin are responsible for addressing the peptide to the δ‐receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ‐receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two‐dimensional1H‐NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty‐four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues,S‐shaped structures, mean planes ofN‐terminal andC‐terminal moieties almost at right angles to each other, aC‐terminus region above the plane of theN‐terminal region andg−as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (torg−) and proximity between Tyr and Asp, or Tyr and theC‐terminus. In contrast to previous models, practically no β‐turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ‐turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ‐opioid receptor selectivity is discussed. © Munksgaard 1996.
Conformational differences of ovine and human corticotrophin releasing hormone A CD, IR, NMR and dynamic light scattering study
Authors: DATHE MARGITTA , FABIAN HEINZ , GAST KLAUS , ZIRWER DIETRICH , WINTER RÜDIGER , BEYERMANN MICHAEL , SCHÜMANN MICHAEL , BIENERT MICHAEL
Published: August 2010

The differences in the conformational properties of ovine (o) and human (h) CRH in aqueous solution, structure‐inducing TFE and in the presence of detergent micelles and lipid vesicles have been investigated by circular dichroism, Fourier transform infrared spectroscopy, NMR and dynamic light scattering. o‐CRH was found to exist as a monomer with little regular structure in dilute aqueous solution. Association at concentrations higher than 10−3mol/L results predominantly in dimers. The induction of a substantial amount of intermolecular β‐structure seems to be the result of interactions of theC‐terminal hexapeptide and theN‐terminal region 6‐12 of o‐CRH chains in antiparallel orientation. In contrast, h‐CRH exhibits a high tendency of association which is highly sensitive to the pH. The formation of tetramers at millimolar peptide concentration is related to a helical content of ca. 50%. The potentially helical, highly hydrophobic region 6‐20 enlarged by more hydrophobic residues in position 23 and 25 is proposed to stabilize the h‐CRH associates. In the presence of structure inducing TFE (<40%v) both CRH peptides exist as monomers. o‐CRH reveals about 72% helicity, in h‐CRH the formation of about 85% helix is observed. The differences in helicity of the two CRH molecules are located in theC‐terminal heptapeptide, as concluded on the basis of NMR studies. Both peptides bind to detergent micelles at pH 4 as well as 7.4 associated with an increase in the α‐helical content. Interaction of the two peptides with DMPC vesicles was found exclusively at pH 4. Above the phase transition temperature of DMPC the α‐helical content in h‐CRH increases slightly; however, o‐CRH reveals a substantial amount of β‐type structure. The intramolecular type of β‐structure is associated with a deeper insertion of the o‐CRH region 6‐12 into the hydrophobic region of the lipid bilayer, whereas the corresponding region of h‐CRH is kept in the bilayer surface. The higher helicity of h‐CRH might explain to some extent its higher affinity to the CRH receptor, CRH antibodies and the CRH binding protein. © Munksgaard 1996.
Gramicidin S is active against both gram‐positive and gram‐negative bacteria
Authors: KONDEJEWSKI LESLIE H. , FARMER SUSAN W. , WISHART DAVID S. , HANCOCK ROBERT E.W. , HODGES ROBERT S.
Published: August 2010

Four linear and four cyclic analogs of gramicidin S (GS) in which d‐Phe was replaced with either d‐His, d‐Ser, d‐Tyr or D‐Asn have been prepared by solid‐phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram‐positive as well as gram‐negative bacteria. GS showed MICs ranging from 3 to 12.5 μg/mL for gram‐negative bacteria, compared to MICs of 3 μg/mL for gram‐positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution‐based assays showing greater activity against gram‐negative bacteria than agar‐based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the d‐Phe to d‐Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N‐phenyl‐1‐naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad‐spectrum antibiotics and should be reevaluated for such purposes. © Munksgaard 1996.
Structural comparison of alanine‐substituted analogues of the calcitonin gene‐related peptide 8‐37 Importance of theC‐terminal segment for antagonistic activity
Authors: BOULANGER Y. , KHIAT A. , LAROCQUE A. , FOURNIER A. , ST‐PIERRE S.
Published: August 2010

Replacement of specific residues of the antagonistic fragment human calcitonin gene‐related peptide 8–37 (hCGRP 8–37) by alanine residues produces good antagonists to CGRP1 receptors when the replacement is made at positions 17 and 20 but a poor antagonist when the replacement is made at position 21. The solution structures of hCGRP 8‐37 and of the three alanine analogues have been determined by two‐dimensional1H NMR spectroscopy and molecular modeling. Following the complete assignment of the NMR spectra, a comparison of the chemical shifts and of the temperature dependence of the amide chemical shifts showed that these parameters differed for [Ala17]‐hCGRP 8–37 and [Ala20]‐hCGRP 8–37 relative to hCGRP 8–37 in theN‐terminal and central segments but not in theC‐terminal segment (residues 31–37). In the case of [Ala21]‐hCGRP 8–37, differences were observed all along the chain. Molecular modeling calculations were performed by distance geometry, simulated annealing and energy minimization using NOE distance constraints. Molecular models showed a structural homology between [Ala17]‐hCGRP 8–37, [Ala20]‐hCGRP 8–37 and hCGRP 8–37 in theC‐terminal segment Asn31‐Phe37as well as hydrogen bonding between Val28and Asn31. These structural similarities are not observed with [Ala21]‐hCGRP 8–37. Therefore, the structure of theC‐terminal segment of hCGRP 8–37 appears to be critical for antagonistic activity at CGRP1 receptors.
Specific cleavage of immunoglobulin G by copper ions
Authors: SMITH MARJORIE A , EASTON MARK , EVERETT PETER , LEWIS GARNET , PAYNE MICHAEL , RIVEROS‐MORENO VALENTINA , ALLEN GEOFFREY
Published: August 2010

The hinge region of a recombinant‐DNA‐produced human IgG1 (Campath 1H) is specifically cleavable at a single copper‐sensitive peptide bond, yielding a distinct fragment resolved by size‐exclusion high‐performance liquid chromatography. This novel metal ion‐catalysed cleavage at slightly alkaline pH is inhibited by EDTA and its rate is reduced at slightly acidic conditions (pH5‐6) and accelerated by increasing concentrations of cupric ion and higher temperature. Complete cleavage was observed after incubation at pH 8 for 24 h with 1 mM CuC12. Sequence analysis determined the cleavage site to be the Lys226‐Thr227 bond in the hinge‐region sequence DKTHT. Cleavage of other IgGs was observed to varying degrees, and specific cleavage of synthetic peptides containing this pentapeptide sequence was also observed. © Munksgaard 1996.