Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline     

Cyclic enkephalin and dermorphin analogues containing a carbonyl bridge.

Four cyclic enkephalin analogues and four cyclic dermorphin analogues have been synthesized. Cyclization of linear peptides containing basic amino acid residues of various side chain length in position 2 and 5 (enkephalin analogues) or 2 and 4 (dermorphin analogues) was achieved by treatment with bis-(4-nitrophenyl) carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse activity was observed, depending on the size of the ring and the location of the urea unit. The conformation of two dermorphin analogues has been studied: one of high activity (IC(50) = 4.15 nM in the GPI assay) and a second of low activity (IC(50) = 6700 nM in the GPI assay). The conformational space of these peptides was examined using the EDMC method. Using data from the NMR spectra, each peptide was described as an ensemble of conformers. Biological activity was discussed in light of the structural data.     

Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu–enkephalin

Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu–enkephalin was synthesized with ¹⁵N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu–enkephalin in a DMSO_d6/H₂O mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure–activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. β- and γ-turns) and finally to select only those consistent with the requirements of δ selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new δ selective opioid peptides. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational studies of neurohypophyseal hormones analogues with glycoconjugates by NMR spectroscopy

Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α‐d ‐mannose through the oxygen β atom, whereas peptide II is a lysine‐vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β‐turn characteristic of OT‐like peptides, and vasopressin‐glycosylated analogue (II) exhibits β‐turns typical of vasopressin‐like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT‐like and vasopressin‐like drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Configurationally driven folding of model tetrapeptides containing L- or D-morpholine-3-carboxylic acids as beta-turn nucleators

The conformational analysis by NMR, IR, and molecular modeling of tetrapeptides containing morpholine-3-carboxylic acid (Mor) as a proline surrogate is presented. The relationship between the chirality of the cyclic amino acid at position i+1 and the turn propensity is maintained with respect to the reference proline-containing peptides, although marked differences in the type of folded structures were observed. The conformational profile of morpholine-containing turn peptides as a function of the chirality of the cyclic amino acid indicated that the heterochiral tetrapeptide containing the D-isomer of the cyclic amino acid is more prone to nucleate compact folded structures, although with no resemblance to the beta-turn structures of D-proline-containing peptides. Also, the solvation system proved to influence the organization of folded structures, as in the more interactive CD(3)CN the model peptides showed more compact conformations. The L-Mor-containing peptide displayed two rotamers at the Val-Mor amide bond. The trans isomer did not experience any turn structures, nor any intramolecular hydrogen-bonds, whereas the cis isomer showed a strong preference for a type VI beta-turn structure, thus providing a different conformational asset with respect to the beta-turn structure as reported for the reference L-proline model peptide.     

Conformational solution studies of neuropeptide gamma using CD and NMR spectroscopy.

Neuropeptide gamma is one of the largest members of the tachykinin family of peptides, exhibiting strong agonistic activity towards the NK-2 tachykinin receptor. This peptide was synthesized by the solid-phase method using the Fmoc chemistry. Circular-dichroism spectroscopy (CD) investigations of this peptide were performed in phosphate buffer, in the presence of sodium dodecylsulphate (SDS) micelles and trifluoroethanol (TFE) solutions and in DMSO-d6 using the 2D NMR technique in conjunction with two different theoretical approaches. The first assumes multiconformational equilibrium of the peptide studied characterized by the values of statistical weights of low-energy conformations. These calculations were performed using three different force fields ECEPP/3, AMBER4.1 and CHARMM (implemented in the X-PLOR program). The second method incorporates interproton distance and dihedral angle constraints into the starting conformation using the Simulated Annealing algorithm (X-PLOR program). The CD experiments revealed that although the peptide studied is flexible in polar solvents, a tendency to adopt a helical structure was observed in the hydrophobic environment. The NMR data (NOE effects) indicate a helical or reverse structure in the Ile7-His12 fragment of the peptide studied in DMSO-d6 solution. The results obtained cannot be interpreted in terms of a single conformation. Most of the conformations obtained with the ECEPP/3 force field possess a high content of a helical structure. None of the conformers, obtained with the AMBER4.1 and CHARMM force fields, can be considered as the dominant one. In all conformations several beta-turns were detected and in some cases gamma-turns were also found. But in fact, it is rather difficult to select the position of the secondary element(s) present in the structure of NPgamma in solution. All conformers calculated with the X-PLOR program (with using NMR derived distance and torsion angle constraints) are stabilized by several beta-turns. Common structural motives are a type IV beta-turn in the Gln6-His12 fragment. All conformations obtained using two approaches adopt very similar turn shapes in the middle region of molecule and a random structure on the N- and C-terminal fragments.     

The influence of 1-aminocyclopentane-1-carboxylic acid at position 2 or 3 of AVP and its analogues on their pharmacological properties.

This study describes the synthesis and some pharmacological properties of eight new analogues of arginine vasopressin (AVP) substituted at position 2 or 3 with cycloleucine (1-aminocyclopentane-1-carboxylic acid, Apc). All new peptides were tested for their pressor, antidiuretic and uterotonic in vitro potency. The Apc3 modification resulted in an almost complete loss of potency in all three tests, which is interpreted as a loss of interaction with all three neurohypophyseal hormone receptors. On the other hand, the Apc2 modification resulted in compounds having differently modified activities (high antidiuretic potency, low and graded pressor activity and either no activity or low oxytocin antagonizing activity in the uterotonic in vitro test) thus selectively altering the interaction with the receptors similar to that of 1-aminocyclohexane-1-carboxylic acid (Acc). The results obtained may be helpful for designing new analogues of arginine vasopressin.     

Ureido group containing cyclic dermorphin(1-7) analogues: synthesis, biology and conformation.

Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.     

Conformational analysis of the dipeptide taste ligand L-aspartyl-D-2-aminobutyric acid-(S)-α-ethylbenzylamide and its analogues by NMR spectroscopy,computer simulations and X-ray diffraction studies

A dipeptide taste ligand L -aspartyl-D -2-aminobutyric acid-(S)-α-ethylbenzylamide was found to be about 2000 times more potent than sucrose. To investigate the molecular basis of its potent sweet taste, we carried out conformational analysis of this molecule and several related analogues by NMR spectroscopy, computer simulations and X-ray crystallographic studies. The results of the studies support our earlier model that an ‘L’-shape molecular array is essential for eliciting sweet taste. In addition, we have identified an aromatic group located between the stem and the base of the ‘L-shape’, which is responsible for enhancement of sweetness potency. In this study, we also assessed the optimal size of the essential hydrophobic group (X) and the effects of the chirality of the second residue toward taste. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors. III. Probable binding conformations of mu-agonists with phenylalanine in position 3.

Theoretical conformational analysis was carried out for several tetrapeptide analogues of beta-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the mu-selective peptides [N-Me-Phe3, D-Pro4]-morphiceptin and Tyr-D-Orn-Phe-Asp-NH2 were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the delta-selective peptide Tyr-D-Cys-Phe-D-Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH2. Two pairs of geometrically similar conformations of mu-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the mu-receptor, were selected as two alternative models of probable mu-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear mu-selective peptide Tyr-D-Ala-Phe-Phe-NH2. Putative binding conformations of Tyr1 and Phe3 side chains are also discussed.     

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL).The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL.The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds.The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.     

Structure and Conformational Properties of the HIVThailand gp120 Immunodominant Epitope

NMR data and the previously developed theoretical method were used to determine the three-dimensional structure of the immunodominant epitope (IDE) of the HIV_Thailand protein gp120. The best energy IDE conformers consistent with the theoretical and experimental data were calculated, and their ensemble was shown to give rise to the main chain folds found earlier in examining the HIV_MN IDE structure. The gp120 IDE is supposed to behave as a metastable oligopeptide that, depending on the microenvironment, largely assumes one of the conformations from the ensemble. The results are discussed in the light of literature data on HIV-1 IDE structure.     

The multiple-minima problem in the conformational analysis of polypeptides. III. An Electrostatically Driven Monte Carlo Method: Tests on enkephalin

The three-dimensional conformation of Met-enkephalin, corresponding to the lowest minimum of the empirical potential energy function ECEPP/2 (empirical conformational energy program for peptides), has been determined using a new algorithm, viz. the Electrostatically Driven Monte Carlo Method. This methodology assumes that a polypeptide or protein molecule is driven toward the native structure by the combined action of electrostatic interactions and stochastic conformational changes associated with thermal movements. These features are included in the algorithm that produces a Monte Carlo search in the conformational hyperspace of the polypeptide, using electrostatic predictions and a random sampling technique to locate low-energy conformations. In addition, we have incorporated an alternative mechanism that allows the structure to escape from some conformational regions representing metastable local energy minima and even from regions of the conformational space with great stability. In 33 test calculations on Met-enkephalin, starting from arbitrary or completely random conformations, the structure corresponding to the global energy minimum was found inall the cases analyzed, with a relatively small search of the conformational space. Some of these starting conformations wereright orleft-handed -helices, characterized by good electrostatic interactions involving their backbone peptide dipoles; nevertheless, the procedure was able to convert such locally stable structures to the global-minimum conformation.On leave from the National University of San Luis, Faculty of Sciences and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Matemática Aplicada, San Luis, Ejército de los Andes 950, 5700 San Luis, Argentina.     

Analogues of arginine vasopressin modified in position 2 and 3 with conformationally constrained dipeptide fragments.

This study describes the synthesis and some pharmacological properties of ten new analogues of arginine vasopressin (AVP) containing a conformationally constrained dipeptide fragment in the N-terminal part of their molecules. Amino acid residues in positions 2 and 3 of AVP and some of its agonistic analogues were replaced with -Phe-Phe and D-Phe-D-Phe, dipeptides having a -CH2-CH2- link bridging two nitrogens. All the new peptides were tested for vasopressor and antidiuretic activities. Four peptides with pA2 values ranging from 5.96 to 7.21 turned out to be weak or moderately potent V1a antagonists. The results supplied new information about the structure-activity relationship of AVP analogues. As some of these were unexpected, they point to the need for caution when extrapolating previously known effects of modifications to analogues having conformationally constrained fragments in their molecules.     

寡糖的构象分析

糖在生物体内具有极为重要的生物学功能,其在细胞及分子识别等许多过程中的作用已日益得到重视,糖的结构特征已成为研究识别过程的重要依据.集中介绍了有关寡糖结构的基本概念、理论研究热点及现状,并对有关研究方法进行了评述．寡糖结构理论研究的发展方向之一是研究糖与蛋白质的相互作用以及糖蛋白中糖链的结构与功能.     

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg¹ (TOAC⁰-BK) or replacing Pro³ (TOAC³-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism(CD) approach was used to examine the conformational propertiesof the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionicN-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC³-BK's EPR spectral lineshapes are broader than those of TOAC⁰-BK, indicating amore restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC⁰-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidlyinterconverting forms), while TOAC³-BK's spectra correspondto a more ordered structure. SDS binding induces drastic changesin BK and TOAC⁰-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. Incontrast, TOAC³-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC³-BK to acquire the correct receptor-bound conformation, leading to loss of biological activity. On the other hand, the greater flexibility of TOAC⁰-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Conformational analyses of cyclic hexapeptide analogs of somatostatin containing arylalkyl peptoid and naphthylalanine residues

We report the conformational analysis by ¹H‐NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of peptoid analogs of the cyclic hexapeptide c‐[Phe¹¹‐Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] L‐363,301 (the numbering refers to the positions in native somatostatin). The compounds c‐[Phe¹¹‐Nphe⁶‐Nal⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nphe ⁶ ‐ Nal ⁷ analog 1 ), c‐[Nal¹¹‐Nphe⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nal ¹¹ ‐ Nphe ⁶ analog 2 ) and c‐[Phe¹¹‐Nnal⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nnal ⁶ analog 3 ), where Nphe denotes N‐benzylglycine and Nnal denotes N‐(1‐naphthylmethyl)glycine, are subjected to SAR studies in order to investigate the influence of the bulky naphthyl aromatic ring on the conformation. The Nal ¹¹ ‐ Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs exhibit potent binding to the hsst2, hsst3 and hsst5 receptors, whereas the Nnal ⁶ analog has decreased binding affinity to all receptors but is more selective towards the hsst2 than the other two analogs and L‐363,301. The conformational search employing distance geometry, energy minimization and molecular dynamic simulations gives insight into the conformational flexibility of these analogs. The molecules adopt both cis and trans orientations of the peptide bond between residues 11 and 6. The cis isomers of these analogs adopt type II′ β‐turns with d ‐Trp in the i+1 position and type VIa β‐turns with the cis peptide bond between residues 6 and 11. The results of free and distance restrained molecular dynamics simulations at 300 K indicate that the Nphe ⁶ ‐Nal ⁷ and Nal ¹¹ ‐Nphe ⁶ compounds adopt a preferred backbone conformation which can be described as ‘folded’ about residues 7 and 10. The Nnal ⁶ analog, which binds less effectively to the hsst receptors, has a more flexible backbone structure than the Nal ¹¹ ‐Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs and prefers a ‘flat’ structure with regard to the orientations about Phe⁷ and Thr¹⁰ during molecular dynamics simulations. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.