Proton NMR studies of partially modified retro–inverso peptides

High-resolution ¹H-nmr analyses are presented for partially modified retro-inverso derivatives of peptides with emphasis on enkephalins. Studies in DMSO-d₆ reveal the unique as well as common characteristics of the non-amino acid residues incorporated into these modified peptides. The complete assignment of 1,1-diaminoalkyl and malonyl, as well as of amino acid, residues provides the basis for the exploration of the conformational features induced in peptides by such topochemical modifications.     

Computer simulations of cyclic enkephalin analogues

Molecular dynamics simulations and energy minimization studies of cyclic enkephalin analogues incorporating retro-inverso modifications have been carried out. The dynamic trajectories are analyzed in terms of the relative mobility of the 14-membered rings, conformational transitions among equilibrium states, and hydrogen-bonding patterns. The cyclization of the molecules reduces the motion of the ring structures substantially. Time-correlated conformational transitions resulting in the reorientation of peptide units are observed. Hydrogen bonds form principally C7 structures. Because of the incorporation of retro-inverso residues, C6 and C8 structures are also formed. Starting conformations for energy minimizations were obtained from the molecular dynamics simulations and from a systematic search of the conformational space available to the molecules. Several minimum energy backbone and side-chain conformations were found for each analogue. The effect of retro-inverso residues on hydrogen-bonding patterns and backbone conformations is discussed.     

Syntheses and biological activities of sandostatin analogs containing stereochemical changes in positions 6 or 8

In a continuation of our research efforts on the design and synthesis of novel peptidomimetic structures, we have synthesized a series of sandostatin amide analogs in which stereoisomers of threonine and beta-hydroxyvaline(beta-Hyv) are employed. The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) explore the effects on biological activity of stereochemical modifications and beta-methylation at positions 6 or 8. By these modifications, we examine the role of the two residues in binding to somatostatin receptors. We describe the synthesis and biological activity of these analogs. In combination with the results of the conformational analysis, this study provides new insights into the structural requirements for the binding affinity of somatostatin amide analogs to somatostatin receptors [Mattern et al., Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8].     

Effect of Sequence and Stereochemistry Reversal on p53 Peptide Mimicry

Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15–29) using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15–29) result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.     

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.     

Deltorphin analogs restricted via a urea bridge: structure and opioid activity.

Eight cyclic heptapeptides related to the full sequence of deltorphin 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. Depending on protection procedures, the N-protected peptide-resins or N-protected peptide amides with free amino groups in the side chains were obtained, which were subsequently treated with bis-(4-nitrophenyl)carbonate to form a urea unit. Opioid activities of the peptides were determined in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Several compounds showed high delta opioid agonist potency and high selectivity for delta receptors. The results were compared with those obtained earlier for respective 1-4 deltorphin analogs. The conformations of these peptides have been studied using 2D-NMR in H2O/D2O and molecular dynamics. We observed that the backbone rings had well defined conformations, while the Tyr and Phe side chains and the C-terminal tail had significant conformational freedom. The bioassay data and conformational parameters of these peptides were compared with those of previously described, corresponding 1-4 deltorphin analogs. This comparison permitted an assessment of the role of the C-terminal peptide segment in defining the conformation and receptor interaction of the N-terminal portion and provided insight into the relationship between the putative bioactive conformations and bioactivity.     

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S,3R)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Peptide conformation. 48. Conformation and biological activity of proline containing cyclic retro-analogues of somatostatin

Six cyclic retro-analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(-Xaa1-Phe2-Thr3-Lys4-Ybb5-Phe6-) and cyclo(-Phe1-Xaa2-Thr3-Lys4-Ybb5-Phe6-) with Xaa = D- or L-Pro and Ybb = D- or L-Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO-d6 solution were determined by a number of homo- and heteronuclear two-dimensional n.m.r.-techniques including 2D rotating frame NOE-spectroscopy. Two-step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all-trans cyclopeptides consists of a beta-turn containing the Pro residue in the position i + 1. These retro-analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.     

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro nor the retro-inverso S peptides could do so.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

Peptide Scaffolds: Flexible Molecular Structures With Diverse Therapeutic Potentials

Peptide scaffolds are diverse chemical structures providing a major base for drug development. Nature modifies a premature peptide with respect to a basic scaffold structure to create a mature and active peptide. Mimicking the natural scaffolds with desirable modifications i.e., scaffold-hopping will decrease the enormous efforts of chemical syntheses and testing for drug development. We have surveyed the scaffold-based compounds being used for anticancer, antiinfective, antiinflammatory and antidiabetic activities. Synthetic peptidomimetics like aptamers, dendrimers, arylamide foldamers, β peptides, d peptides etc. provide an anticipative picture for the therapeutic use of scaffold structures. Free energy based conformational analysis of peptidomimetics provides details of their structure–activity relationships. Diverse forms of such peptidomimetics with respect to their structure and applications are discussed alongwith the mimetics which reached clinical trials. The review gives an insight into the future panoramas of drug development and identifies few peptide scaffolds having diverse potential with chemical modifications.     

Conformational analyses by 1H NMR and computer simulations of cyclic hexapeptides related to somatostatin containing acidic and basic peptoid residues.

We report the conformational analysis by 1H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c-[Phe11-Pro6-Phe7-D-Trp8-Lys9-Thr10]. The analogs c-[Phe11-Nasp6-Phe7-D-Trp8-Lys9-Thr10](1), c-[Phe11-Ndab6Phe7-D-Trp8-Lys9-Thr10] (2) and c-[Phen11-Nlys6-Phe7-D-Trp8-Lys9-Thr10](3) where Nasp denotes N-(2-carboxyethyl) glycine, Ndab N-(2-aminoethyl) glycine and Nlys N-(4-aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp6 analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp6 compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors.     

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.     

Synthesis and biologic activity of conformationally constrained analogs of L-363,301.

We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L-363,301, c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11] (numbering as in the native hormone somatostatin-14). The d-Trp in position 8 was replaced with (2R,3S)- and (2R,3R)-beta-MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d-Trp upon binding to human somatostatin receptors hsst1-5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3. Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

Enkephalin-degrading dipeptidyl carboxypeptidase in guinea pig serum: its properties and action on bioactive peptides

A dipeptidyl carboxypeptidase, which cleaved the Gly3-Phe4 bond of enkephalins, was purified from guinea pig serum 420-fold. The optimum pH of the enzyme was in the neutral range (pH 7.25), and the molecular weight was estimated to be approx. 280,000. The enzyme hydrolyzed Met- and Leu-enkephalin with Km values of 0.30 and 0.50 mM, respectively. The enzyme was inhibited by metal chelators and p-chloro-mercuribenzoate. Captopril showed high inhibitory potency, while phosphoramidon and Phe-Ala showed no effect on the enzyme activity. Therefore, the obtained enzyme can be classified as an angiotensin-converting enzyme (EC 3.4.15.1). Among the bioactive peptides examined, bradykinin and angiotensin I were hydrolyzed by the enzyme. Angiotensin III showed a stronger inhibitory effect than that of angiotensin II. Substance P, gastrin I, and secretin were also inhibitory toward the enzyme activity. On high-performance liquid chromatography analysis, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6 were cleaved sequentially at the second peptide bond of the C terminus. Thus, the dipeptidyl carboxypeptidase in guinea pig serum may play a role not only in the angiotensin-bradykinin system but also in the metabolism of circulating enkephalins and other bioactive peptides.     

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.     

Conformational studies of antimetastatic laminin-1 derived peptides in different solvent systems, using solution NMR spectroscopy.

Due to its critical role in cancer progression, interactions between laminin-1 and the 67 kDa Laminin-Binding Protein (the 67 kDa LBP) have been the focus of a number of structural and biological studies. As laminin-1 is such a large and complex molecule, research interests have turned to the investigation of bioactive peptides derived from binding domains of laminin-1. Two peptides of interest, CDPGYIGSR (peptide 11) and YIGSR, both derived from the beta1 chain of laminin-1, have been shown to block invasion of basement membranes by tumor cells. Substituting the C-terminal arginine to lysine, a conservative substitution, results in a loss of peptide antimetastatic activity. This difference in bioactivity has been attributed, based on numerous modeling studies of free peptide conformations, to structural differences between YIGSR and YIGSK. Yet the nature of the 'active' free peptide backbone conformation has been a matter of debate and controversy. In order to test the validity of the structural modeling claims, we have undertaken detailed conformational studies of the two laminin-1 derived peptides YIGSR and CDPGYIGSR along with the biologically inactive YIGSK analog by two-dimensional solution 1H NMR spectroscopy in three different solvent systems. Herein we report that although both the active (YIGSR, CDPGYIGSR) and the inactive (YIGSK) peptides can adopt several closely related conformations in solution, the two peptides share similar conformational preferences, and there are no significant structural differences between the active and inactive peptides, contrary to previously reported modeling data. We conclude that the basis of the peptide biological activity, in contrast to published models, cannot be attributed to well-defined structural preferences of the free peptides. We infer that the difference in bioactivity observed between YIGSR and YIGSK originates primarily from the chemical nature of the arginine versus lysine sidechain substitution, rather than being due to a structural change in the free peptide conformations.