Dipeptide backbone conformation and antibody recognition of a viral octapeptide epitope.

The peptide YKGTMDSG (Tyr-Lys-Gly-Thr-Met-Asp-Ser-Gly) represents an important antigenic determinant from the glycoprotein G2 of the pathogenic Rift Valley fever virus. By preparing a series of single-residue substitution peptides, the importance to antigenicity of individual residues within this octapeptide has been determined. Here, we investigated a simple and rapid computational analysis to test for correlations between the observed antigenicity of the substitution analogue peptides and the calculated conformational preferences in local regions of the peptides. Conformational energy analyses were carried out on all dipeptide combinations represented in the wild-type octapeptide and in the single-residue substitution analogue peptides. Conformational similarities and differences between wild-type and substitution dipeptide pairs were determined. The results of these computational analyses were then compared with the data on the relative antigenicity of the wild-type octapeptide and the substitution analogues. This comparison revealed a positive correlation. Substitution peptides showing changes in antigenicity possessed significant changes in the calculated backbone conformation relative to wild type in the dipeptides encompassing the residue substitution. Substitution peptides showing no change in antigenicity similarly showed no significant changes in dipeptide conformation. The potential utility of dipeptide conformational energy analyses and this preliminary structure-activity correlation are discussed.     

Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin.

We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.     

Structure-function analysis of tritrypticin, an antibacterial peptide of innate immune origin.

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.     

Synthesis, conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(But)-Ala-Thr(But)-Thr(But)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO2)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO2)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Enhanced antitumor activity and selectivity of lactoferrin-derived peptides.

A number of peptide analogs derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT-29 and MT-1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure-activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.     

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment.

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

Peptide synthesis on a phenolic resin support

Protected peptides assembled on a phenolic resin support were cleared by peroxide-catalysed hydrolysis. In genenal peptide phenyl ester resins were more labile to nucleophiles than were corresponding Merrifield resin derivatives; transesterification with dimethylaminoethanol providing on alternative cleavage method for peroxide-sensitive peptides. Losses of radiolabelled peptide from both Merrifield and phenolic resins were determined during acid deprotection, base wash and coupling steps in the synthesis of a tetrapeptide. Using 40% (v/v) trifluoroacetic acid in dichloromethane for Boc-deprotection the phenolic resin gave improved results compared to the Merrifield resin. The merits of the procedure for the preparation of protected peptide acids suitable for subsequent condensation reactions were exemplified by the synthesis of an octapeptide sequence of a modified lysozyme.     

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure-activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.     

Elucidation of discontinuous linear determinants in peptides.

Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.     

Conformation and inhibitory properties of peptides based on the tissue kallikrein-aprotinin complex.

A series of peptides encompassing the primary binding segment (residues 12-19) of aprotinin has been synthesized and tested for their ability to inhibit porcine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12-16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a Ki-value of 1.2 x 10(-4) M. The solution structure of the octapeptide was studied by one- and two-dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12-19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150,000-fold increase in Ki of the octapeptide compared to intact aprotinin.     

Synthesis of peptides by the solid-phase method. III. Bradykinin: fragments and analogs

The natural sequence of bradykinin (BK) and 55 fragments or analogs of this peptide were perpared via the solid-phase method. The peptides were purified using ion-exchange (O-carboxymethyl(CM) and partition (Sephadex G-25) chromatography. The purity of each peptide was established by paper and thin-layer chromatography, paper electrophoresis, amino acid analysis, and biological assays. The compounds were tested in anesthetized rats (tested in vivo) and in two smooth-muscle preparations (rabbit aorta strip, cat ileum strip) in which BK produces contraction by stimulating specific receptors of different types. Some of the new peptides are interesting in that they either resist pulmonary inactivation, or are more potent than BK itself, or antagonize the myotropic effect of BK in rabbit aorta strips.     

A proposed bioactive form of peptide T and the design of peptidomimetics

Summary Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp 120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo β-turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure-activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

Solid-phase synthesis of neurokinin A antagonists. Comparison of the Boc and Fmoc methods.

During the preparation of the NK-2 selective tachykinin antagonist MEN 10208 (Thr-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH2) and its analogs by the solid-phase method employing the Boc strategy routinely used in our laboratory, we encountered difficulties in the coupling of hydrophobic amino acids D-Trp and Val. To study the coupling problems several syntheses of MEN 10208 and analogs were carried out with different activation strategies. These syntheses yielded considerable amounts of deletion sequences even though a negative Kaiser test was obtained after each coupling. Inaccessibility of the free amino group of the growing peptide due to steric hindrance of the hydrophobic residues during coupling, and for the ninhydrin complex during the Kaiser test, may account, at least in part, for the unsatisfactory synthetics results and for the false-negative ninhydrin tests. Repetition of each synthesis with the Fmoc strategy on a newly developed DOD resin for peptide amides using the DCC/HOBt chemistry gave superior results in terms of the yield and purity of the crude peptides. Therefore, the Fmoc strategy appears to offer advantages over the Boc method for the preparation of these peptides containing hydrophobic amino acids.     

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.     

Synthesis, metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line.     

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated β1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.     

Synthesis of novel basic head-to-side-chain cyclic dynorphin A analogs: strategies and side reactions

Novel N-terminus-to-side-chain cyclic analogs of the opioid peptide dynorphin (Dyn) A-(1-11)NH(2) were prepared that retain the basicity of the N-terminal amine and restrict the backbone conformation around the important Tyr(1) residue. Cyclic peptides were synthesized in which the N-terminal amine and the N(epsilon)-amine of a Lys at position 3 or 5 were attached to the alpha-carbon and carbonyl of an acetyl group, respectively. Several synthetic strategies were explored with detailed analysis of the side reactions in order to obtain the desired cyclic peptides. One of the side reactions observed involved premature loss of the N-terminal 9-fluorenylmethoxycarbonyl (Fmoc) group during the neutralization step following deprotection of the Mtt (4-methyltrityl) protecting group from the side chain of Lys. The successful strategy involved the synthesis of the linear peptide up through Gly(2) and functionalization through the N(epsilon)-amine of Lys. A linear N-terminal alkylated analog was prepared by alkylation of the peptide on the resin with an equimolar amount of bromoacetamide, followed by treatment of the peptide with Fmoc-OSu prior to cleavage from the resin to facilitate separation by reversed phase high performance liquid chromatography of unreacted peptide from the desired alkylated product. The novel N-terminal cyclic Dyn A analogs and the linear analog were evaluated for their opioid receptor affinities. These peptides exhibited large losses in affinity for opioid receptors; the low affinity of the linear N-terminal alkylated peptide suggested that the alpha-acetamide group on the N-terminal amine resulted in unfavorable interactions with opioid receptors.