Conformational modifications of cyclic hexapeptide somatostatin analogs

A model for the bioactive conformation of the highly active cyclic hexapeptide somatostatin analog cyclo-(Pro-Phe-D-Trp-Lys-Thr-Phe) has been proposed. As a test of this model, several compounds containing lactam and N-Me amino acid conformational modifications in the Thr-Phe-Pro-Phe beta turn were synthesized. The N-Me alanine and sarcosine substitutions for proline gave highly active analogs, while lactam dipeptides in place of Phe-Pro decreased potency. 1H n.m.r. and CD spectra of these analogs illustrate the conformational effects in solution of these modifications. The results provide additional support for the proposed conformational model.     

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.     

Conformations of a model cyclic hexapeptide,CYIQNC: 1H-NMR and molecular dynamics studies

Solution conformation of the cyclic hexapeptide sequence, [cyclo-S-Cys-Tyr-Ile-Gln-Asn-Cys-S] (CYIQNC) – a disulfide-linked fragment of a neurohypophyseal peptide hormone oxytocin (OT) – has been investigated by high-field one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic methods and compared with the results obtained from computer simulation studies. ¹H-NMR results based on temperature dependence of amide proton chemical shifts and nuclear Overhauser effect indicate that peptide in solution populates different conformations, characterized by two fused β-turns. The segment Ile³-Gln⁴-Asn⁵-Cys⁶ yields a preferred type-III β-turn at residues 4, 5 (HB, 3HN → 6CO), while the segment Cys⁶, Cys¹-Tyr²-Ile³ exhibits inherently weaker, flexible β-turn either of type I/II’/III/half-turn at residues 1, 2 (HB, 6HN → 3CO). The computer simulation studies using a mixed protocol of distance geometry-simulated annealing followed by constrained minimization, restrained molecular dynamics, and energy minimization showed the possibility of existence of additional conformations with the hydrogen bonds, (a) 5HN → 3CO and (b) 2HN → 6CO. These results, therefore, indicate that the additional conformations obtained from both NMR and simulation studies can also be possible to the peptide. These additional conformations might have very small population in the solution and did not show their signatures in these conditions. These findings will be helpful in designing more analogs with modifications in the cyclic moiety of OT.     

Conformational mimicry: Synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II′ β turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala⁶-Tyr⁷-D -Trp⁸-Lys⁹-Val¹⁰-Phe¹¹-Ψ[CN₄]), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain χ₁ and χ₂ were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected βII′ turn at D -Trp⁸-Lys⁹ and αβVIa turn in the Phe¹¹-Ψ[CN₄]-Ala⁶ portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution. © 1995 John Wiley & Sons, Inc.     

Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment

The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.     

Conformational analysis of a cyclic thymopoietin-analogue by 1H n.m.r. spectroscopy and restrained molecular dynamics simulations

The internuclear distances of the cyclic thymopoietin derivative c[D-Val-Tyr-Arg-Lys-Glu] have been determined using two-dimensional nuclear Overhauser n.m.r. spectroscopy. These distances are used as constraints for a restrained Molecular Dynamics (MD) simulation. The two starting structures used for the calculations consist of a beta and gamma turn for model 1 and two gamma turns for model 2. The rms difference in atomic positions of the two conformations is 0.242 nm. They converge during the restrained MD simulation to the same final structure. The positional rms difference of the time averaged (5-14 ps) conformations is 0.011 nm. The hydrogen bond pattern is similar to that of model 1, but in addition we find three more gamma turns. The vicinal NH-C alpha H couplings agree well with those calculated from the time averaged structures.     

Conformational investigation of a cyclic enterobacterial common antigen employing NMR spectroscopy and molecular dynamics simulations

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.     

Conformational changes of alpha-chymotrypsin in a fibrillation-promoting condition: a molecular dynamics study

Amyloid nanofibril formation appears to be a generic property of polypeptide chains. α-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended β-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.     

Synthesis and hydrolysis by pepsin and trypsin of a cyclic hexapeptide containing lysine and phenylalanine.

1. A cyclic hexapeptide, cyclo(-Gly2-Phe2-Gly-Lys-), and the corresponding open-chain hexapeptides, Gly2-Phe2-Gly-Lys and Phe-Gly-Lys-Gly2-Phe, have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined. 2. The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2-Phe2-Gly-Lys, the value of the Michaelis constant for this reaction being Km equals 0.00022 M. 3. The cyclic peptide was not cleaved by pepsin at all, but Gly2-Phe2-Gly-Lys was hydrolyzed rapidly at a Phe-Phe bond; Km equals 0.0091 M. 4. The cyclic peptide inhibits the hydrolysis of Gly2-Phe2-Gly-Lys by pepsin in a linear non-competitive manner, the value of the inhibition constant being Ki equals 0.004 M.     

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.     

Inhibition of neurotensin release by a cyclic hexapeptide analog of somatostatin

Studies were performed to determine whether the cyclic hexapeptide analog of somatostatin, cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe) II, could alter circulating levels of neurotensin (NT) and inhibit the release of NT from small intestine following the intraluminal perfusion of lipid and ETOH. The small intestine of anesthetized rats was perfused with 0.9% NaCl, 1mM ETOH, 100 mM ETOH or 1 mM oleic acid with and without the intravenous infusion of the somatostatin analog. Plasma samples collected from the superior mesenteric vein were extracted, chromatographed on HPLC and assayed with both C-terminal and N-terminal antisera to NT. The basal circulating levels of chromatographically and immunochemically identified NT observed during the perfusion of the small intestine with 0.9% NaCl were significantly lower (p less than 0.01) during the IV infusion of the somatostatin analog as compared to animals infused IV with saline. The 2-3 fold increase in plasma levels of NT observed with the intestinal perfusion of oleic acid and ETOH did not occur in animals simultaneously infused IV with the somatostatin analog. The somatostatin analog was also effective in decreasing the basal levels of NT metabolite NT(1-8) as well as inhibiting the increase in this metabolite that accompanies the stimulated release of NT.     

Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

A super active cyclic hexapeptide analog of somatostatin

The cyclic hexapeptide, cyclo (Pro-Phe-D-Trp-Lys-Thr-Phe), I, has been shown to have the biological properties of somatostatin. We now report structure-activity studies which optimize the potency of this cyclic hexapeptide series with the synthesis of cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), II, which is 50–100 times more potent than somatostatin for the inhibition of insulin, glucagon and growth hormone release. The hydroxyl group of tyrosine is seen to lend a 10-fold enhancement to the potency. Potency also is found to be correlated with hydrophobicity. II is found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The analog is found to be quite stable in the blood and in the gastrointestinal tract, but the bioavailability after oral administration is only 1–3%. The biological properties and long duration of II should allow clinical evaluation of the inhibition of glucagon release as an adjunct to insulin in the treatment of patients with diabetes.     

Conformational behavior of the linear hexapeptide senktide: a receptor specific tachykinin analog

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N-methylation of the third amino acid residue in the sequence and the negatively charged N-terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded beta-pleated sheet. The observed senktide-receptor specificity is suggested to result from a selection of this or a closely related conformation.     

Synthesis and conformational study of a cyclic hexapeptide analog of somatostatin containing dehydrophenylalanine

A cyclic hexapeptide analog of somatostatin, cyclo-(Pro-delta z-Phe-D-Trp-Lys-Thr-Phe) (II) has been synthesized by a combination of solid phase and solution methodology. It shows a potency for inhibition of growth hormone release in vitro about one-tenth that of the corresponding saturated analog, cyclo-(Pro-Phe-D-Trp-Lys-Thr-Phe) (I). N.m.r. studies indicate comparable backbone conformations for analogs I and II. However, the sum of our findings from biological evaluation and solution physical data suggest that on the receptor the position-7 phenyl ring of I is adopting a conformation which differs from that of one of the major solution conformers defined previously by n.m.r. studies.     

Backbone modifications in cyclic peptides. Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess beta- and gamma-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Pro psi[CH2S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a gamma-turn intramolecular hydrogen bond in the all-amide structure. In CDCl3, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining beta-turn hydrogen bond was weaker according to delta delta/delta TNH measurements. In DMSO-d6, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly interconverted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1-Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Is the sodium ionic channel a cyclic hexapeptide? (author's transl)]

A cyclic hexapeptide affords a model of an ionic channel, permeable to Na+ but not to K+. The selective fixation of tetrodotoxin and saxitoxin could occur through six hydrogen bonds, one with each of carbonyles of the crown exposed at the outside of the membrane.     

Crystal and molecular structure of cyclo(L-prolyl-glycyl)3. A cyclic hexapeptide with a cis peptide bond

The crystal structure of cyclo(L-Pro-Gly)3 was solved using X-ray crystallographic techniques. The backbone of the peptide is asymmetric and is made up of five trans peptide units and one cis peptide. There is a hydrogen bonded water bridge that links the carbonyl oxygens, O1 and O4. The molecules exist as dimers in the crystal lattice. The two molecules of the dimer are related by crystallographic twofold symmetry and are linked by two N-H ... O hydrogen bonds. The crystals are trigonal, space group P3(2)12 with a = 11.379(3), c = 32.93(1) and z = 6. The structure was solved by multisolution methods and refined by least squares technique to an R of 0.083.