Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues

Neurotensin (NT), is a linear tetradecapeptide (pGlu¹-Leu²-Tyr³-Glu⁴-Asn⁵-Lys⁶-Pro⁷-Arg⁸-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. A C-terminal hexapeptide analogue [N^x-Arg-Lys-Pro-Trp-Tle-Leu, (1) Tle =tert-leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure-activity and conformational properties of the dipeptide, Pro-Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D-amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro¹⁰ position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp¹¹ position accepted extra steric bulk more readily than conformational constraints.     

Effects of Cα-methyl substitution on the conformation of linear GnRH antagonist analogs

We have examined the effect of C^α-methyl groups on the conformational ensemble of GnRH analog peptides by comparing ¹H 2D NMR data from two analogs, Ac-D-Nal¹-D-4-Cl-C^α-Me-Phe²-D-Pal³-Ser⁴-Tyr⁵-D-Arg⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂(1)andAc-D-Nal¹-D-4-CI-C^α-Me-Phe²-D-Pal³-Ser⁴-C^α-Me-Tyr⁵-D-Arg⁶-Leu⁷-C^α-Me-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂ (2). The two additional C^α-methyl groups in residues 5 and 8 of 2 do not influence significantly the pattern of the observable main chain NOE intensities, or of the backbone HN proton chemical shifts, which indicates that they do not produce global changes in the conformational ensemble of the peptide. A local change induced by the substitution was observed in the conformation at d -Arg⁸-Pro⁹.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

New proctolin analogues modified by d-amino acids in the peptide chain and their high cardioexcitatory effect on Tenebrio molitor

The object of our studies was the synthesis and conformational and biological evaluation of the series of 14 analogues of the insect neuropeptide, proctolin. The analogues were obtained by replacement of the native l -amino acids by their d -isomers in one, two, and all positions. Biological effects of the peptides were examined by cardioexcitatory test on the heart of yellow mealworm, Tenebrio molitor, in vitro. In biotest performed on insects, d -Arg-d -Tyr-d -Leu-d -Pro-d -Thr. [d -Arg(N-G-nitro)¹,d -Leu³]-, [d -Arg^l,d -Leu3]-, [d -Tyr²,d -Thr⁵]- and [d -Arg¹,d -Pro⁴]-proctolin exert high agonistic activity of proctolin on the heart of insects at 10^?11-10^?10 M concentrations. The proctolin analogue containing only d -amino acid residues in the peptide chain unexpectedly shows a much higher cardioexcitatory effect than the native peptide. Moreover, preliminary CD and NMR conformational studies show that proctolin analogues investigated here seem to prefer rather ordered structures. although their conformations differ in some cases. © Munksgaard 1996.     

Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK-2 antagonists

The conformational analysis of three cyclic hexapeptides is presented. Cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-d -Met¹¹-) (1) and cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-) (2) are NK-2 antagonists in the hamster trachea assay, whereas cyclo-(-Gln⁶-Trp⁷-Phe⁸-(R)-Gly⁹-[ANC-2]Leu¹⁰-Met¹¹-) (3), where Gly⁹[ANC-2]Leu¹⁰ represents (2S)-2-((3R)-3-amino-2-oxo-1-pyrrolidinyl)-4-methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β- and γ-turns, we find a γⁿ-turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γⁱ-turn at Phe⁸, and βI′- or βII-turns with Gly⁹ and Leu¹⁰ at the corner positions, these β-turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.     

NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH)

The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-c(Asp⁵-D-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( I ), Ac-D-Phe(p-Cl⁾l-D-Phe(p-Cl)²-D-Trp³-Ser⁴-c(Glu⁵-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( II ) and their linear analogues, Ac-D-Phe(p-Cl)¹-Phe(p-C1)²-Trp³-Ser⁴-Asp⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( III ) and Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-Glu⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( IV ), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II′β-turn involving residues 3–6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a β-turn between residues 5 and 8. © Munksgaurd 1995.     

Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme)

Previous NMR reports indicated that Tyr⁹⁸, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg²³, Arg⁷⁴, Arg⁷⁷, and Arg⁹⁷, appear to be conserved in all species checked thus far. We prepared the des-Tyr⁹⁸ and des-Arg⁹⁷-Tyr⁹⁸ derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr⁹⁸ was found to be catalytically less effective than the native one, whereas the des-Arg⁹⁷-Tyr⁹⁸ acylphosphatase was completely inactive. This evidence suggests that Arg⁹⁷ participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg⁹⁷-Tyr⁹⁸ enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg⁹⁷ to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.     

Evidence for the metabolic activation of non-steroidal antioestrogens: a study of structure-activity relationships

1 The stimulatory effects of neurotensin (NT) and several NT fragments were evaluated in two pharmacological preparations: rat stomach strips and isolated spontaneously beating atria of guinea-pigs.

2 In rat stomach strips, NT elicited a dose-dependent contractile effect in concentrations varying between 1.3 × 10^-9 and 5.4 × 10^-7 M.

3 The contractile effect of NT (1.3 and 5.4 × 10^-8 M) in this tissue was not modified by atropine (3.4 × 10^-7 M), methysergide (2.0 × 10^-6 M), a mixture of cimetidine (8.0 × 10^-6 M) and diphenhydramine (7.8 × 10^-6 M), indomethacin (1.4 × 10^-5 M), 8-Leu-angiotensin II (1.0 × 10^-6 M), glucagon (2.0 × 10^-6 M) or somatostatin (3.0 × 10^-7 M).

4 Rat stomach strips desensitized by bradykinin (6.1 × 10^-6 M) or substance P (7.4 × 10^-6 M) maintained their sensitivities to NT (1.3 and 5.4 × 10^-8 M).

5 In guinea-pig atria, NT produced a dose-dependent positive inotropic action in concentrations varying between 5.4 × 10^-10 and 2.7 × 10^-7 M.

6 The inotropic effect of NT (2.7 × 10^-9 M) was not influenced by methysergide (2.8 × 10^-6 M), atropine (3.4 × 10^-7 M), practolol (1.5 × 10^-5 M), 8-Leu-angiotensin II (1.0 × 10^-6 M), or indomethacin (1.4 × 10^-5 M), but it was reduced by 37% by cimetidine (4.0 × 10^-5 and 2.0 × 10^-4 M). A combination of cimetidine (4.0 × 10^-5 M) and diphenhydramine (3.9 × 10^-6 M) did not produce a greater inhibition of NT than cimetidine alone.

7 Atria desensitized by bradykinin (6.1 × 10^-6 M) or glucagon (2.0 × 10^-6 M) maintained their sensitivities to NT (2.7 × 10^-9 M). Substance P was inactive both as an agonist or antagonist of NT.

8 These results suggest the existence of specific NT receptors in rat stomach strips and guinea-pig atria.

9 The data derived from our structure-activity study suggest that the minimum structure required for the full stimulation of NT receptors in these two preparations is H-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³-OH. The sequence PyroGlu¹-Leu²-Tyr³-Glu⁴-Asn⁵-Lys⁶-Pro⁷-Arg⁸- and the amino acids Ile¹² and Leu¹³ appear to contribute mainly to the affinity or binding of NT to its receptor. The chemical groups responsible for the full activation (intrinsic activity) of NT receptors seem to be located in the sequence -Arg⁹-Pro¹⁰-Tyr¹¹.

     

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: A comparison with dermenkephalin

Deltorphin-I, Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂ and dermenkephalin, Tyr-d -Met-Phe-His-Leu-Met-Asp-NH₂, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the δ-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala²-Phe³-Asp⁴ and Val⁵-Val⁶-Gly⁷ triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at Ψ Asp⁴ backbone dihedral angle, the backbone structures at segment 1–4 are similar and spatial arrangements of Tyr¹ (t) and Phe³ (g^–) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr¹ (t) and Phe3 (g^–) underlie their ability to bind with high selectivity to the δ-opioid receptor.     

Solution conformations of the peptide backbone for DPDPE and its β-MePhe4-substituted analogs

The solution structures of DPDPE, a conformationally restricted pentapeptide with the sequence H-Tyr¹-d -Pen²-Gly³-Phe⁴-d -Pen⁵-OH, and its four β-MePhe⁴-substituted analogs were examined by a combined approach including the NMR measurements in DMSO and water as well as independent energy calculations. It was concluded that several low energy conformers of DPDPE backbone satisfy the NMR data obtained in this study as well as in previous studies by other authors. These possible solution conformers of DPDPE in both DMSO and water share virtually the same type of cyclic backbone structure, with the Gly³ residue in a conformation close to a γ-turn, and the Phe⁴ residue in a conformation close to α-helical torsion angles. They differ in the space arrangements of the flexible Tyr¹ moiety. The solution structures of the β-MePhe⁴-substituted analogs of DPDPE are interesting. For analogs with an S-configuration at the C^α atom in the Phe⁴ residue, the cyclic backbone conformations resemble those of DPDPE itself, whereas for analogs with an R-configuration at the C^α atom, the backbone conformation is somewhat different. This observation is in line with the high biological potencies and selectivities displayed by the former compounds but not by the latter ones. It was noted also that as far as the peptide backbone conformers are concerned, some of the possible DPDPE conformers in water are similar to the previously suggested model for the δ-receptor-bound conformation of DPDPE, becoming virtually identical to this conformation by rotating the side chains of the Tyr¹ and the Phe⁴ residues.     

Pathways and Kinetics of Deslorelin Degradation in an Airway Epithelial Cell Line (Calu-1)

Purpose. The objective of this study is to investigate the pathways and kinetics of degradation of deslorelin, pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-D-Trp⁶-Leu⁷-Arg⁸-ProNHEt⁹ (Des1-9), in a human airway epithelial cell line (Calu-1). Methods. The degradation of deslorelin in membrane and cytosolic fractions of Calu-1 cells was studied at 37°C up to 24 h. The degradation products were separated using HPLC and identified by amino acid analysis, sequencing, and mass spectrometry. The rate constants for deslorelin degradation and the formation of degradation products were determined by fitting the concentration vs. time data to pharmacokinetic models using WinNonlin^TM. The effect of enzyme inhibitors, captopril, phosphoramidon, and disodium EDTA on deslorelin degradation was also assessed. Results. Des1-3, Des4-9, and Des5-9 were the deslorelin fragments detected in the membrane fraction. Apart from these degradation products, Des5-7 was also detected in the cytosolic fraction. The deslorelin degradation was 8.5 times faster in the cytosolic fraction compared to the membrane fraction. The disappearance of deslorelin and the kinetics of degradation products could be explained by simple 2 compartment iv bolus model and 1 compartment absorption model, respectively. EDTA and captopril decreased deslorelin degradation in the membrane and cytosolic fractions. Conclusions. Deslorelin is initially cleaved at the 3-4 bond in the membrane and cytosolic fractions, possibly by a metalloendopeptidase and/or angiotensin converting enzyme, with the degradation being more rapid in the cytosol.     

Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψ[CS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Conformational energy calculations on Substance P

The conformational and spacial configurations of the biologically active undecapeptide, Substance P, were studied using conformational energy calculations. Low energy conformers of residues 1–5 and 6–11 were found by energy minimization and the two fragments were then combined to find low-energy structures for Substance P. Several configurational classes were found with different backbone conformations. A comparison of the final low-energy structures with data from biological tests on analogs of Substance P gives some insight into the conformation required for interaction at the biological receptor.     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two‐dimensional NMR and theoretical conformational analysis

Two analogues of Scyliorhinin I (ScyI), a tachykinin with N‐MeLeu in position 8 and a 1,5‐disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid‐phase method. Conformational studies in water and DMSO‐d₆ were performed on these peptides using a combination of the two‐dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low‐energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least‐squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three‐dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu⁸]ScyI, the C‐terminal 6–10 fragment adopts more rigid structure than the N‐terminal one. In the case of the analogue with the tetrazole ring in DMSO‐d₆ the three‐diemnsional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 Å) in the 6–9 fragments. All conformers calculated in both solvents superimpose in their C‐terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the ScyI molecule rigidifies its structure significantly more than that of MeLeu.     

Evaluation of the Biological Properties and the Enzymatic Stability of Glycosylated Luteinizing Hormone-Releasing Hormone Analogs

The enzymatic stability, antitumor activity, and gonadotropin stimulatory effects of glycosylated luteinizing hormone-releasing hormone (LHRH) analogs were investigated in this study. Conjugation of carbohydrate units, including lactose (Lac), glucose (GS), and galactose (Gal) to LHRH peptide protected the peptide from proteolytic degradation and increased the peptides’ half-lives in human plasma, rat kidney membrane enzymes, and liver homogenate markedly. Among all seven modified analogs, compound 1 (Lac-[Q¹][w⁶]LHRH) and compound 6 (GS⁴-[w⁶]LHRH) were stable in human plasma during 4 h of experiment. The half-lives of compounds 1 and 6 improved significantly in kidney membrane enzymes (from 3 min for LHRH to 68 and 103 min, respectively). The major cleavage sites for most of the glycosylated compounds were found to be at Trp³-Ser⁴ and Ser⁴-Tyr⁵ in compounds 1–5. Compound 6 was hydrolyzed at Ser⁴-Tyr⁵ and the sugar conjugation site. The antiproliferative activity of the glycopeptides was evaluated on LHRH receptor-positive prostate cancer cells. The glycosylated LHRH derivatives had a significant growth inhibitory effect on the LNCaP cells after a 48-h treatment. It was demonstrated that compound 1 significantly increased the release of luteinizing hormone (LH) at 5 and 10 nM concentrations and compound 5 (GS-[Q¹]LHRH) stimulated the release of follicle-stimulating hormone (FSH) at 5 nM concentration in dispersed rat pituitary cells (p < 0.05). In our studies, compound 1-bearing lactose and d-Trp was the most stable and active and is a promising candidate for future preclinical investigations in terms of in vitro biological activity and metabolic stability.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9769-x) contains supplementary material, which is available to authorized users.KEY WORDS: antiproliferative activity, carbohydrate conjugation, LH and FSH release, LHRH, peptide     

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

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr¹-Cys²-Gly³ Phe⁴-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-Tyr^l-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.     

Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin,cyclo[-β-OH-d-Leu-d–Ile–(NMe)Ala (NMe)Leu-Leu-(NMe)Ala-d–(NMe)Ala-]

The crystal structures of two solvated forms of ternatin, cyclo[-β-OH-d -Leu-d -Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-d -(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2₁2₁2₁, a = 11.563(1), b = 21.863(2), c = 36.330(4) Å. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2₁2₁2₁, a = 14.067(2), b = 16.695(1), c = 36.824(6) Å. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of d -Ile², have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala³ and (NMe)Leu⁴, (ii) a type II β-bend, involving residues Leu⁵-(NMe)Ala⁶-d -(NMe)Ala⁷-β-OH-d -Leu², stabilized by two H-bonds, N1′05 and N5′01, between Leu⁵ and β-OH-d -Leu¹ residues, (iii) a third intramolecular H-bond, observed in each of the four molecules, between the hydroxyl group of β-OH-d -Leu¹ and the carbonyl oxygen of d -Ile².     

NMR and molecular modeling characterization of RGD containing peptides

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d₆ of the cyclic pentapeptides, Ac-Cys-Arg-Gly-Asp-Cys-OH (CRGDC), a potent fibrinogen receptor antagonist, and Ac-Cys-Arg-Gly-D-Asp-Cys-OH (CRGdC), a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. ¹H-¹H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from ³JNH-h α coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the μ- and §-types. They included [D-Cys², L-Cys⁵]- and [D-Cys², D-Cys⁵]-enkephalinamides showing moderate preference for preceptors, the selective compounds [D-Pen², L-pen⁵] and [D-Pen², D-Pen⁵]-enkephalins and Tyr-D-Lys-Gly-Phe analogue possessing very high affinity to receptors of the μ-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.