Modified chemotactic peptides: Synthesis,conformation, and biological activity of for-Thp-Leu-ΔzPhe-OMe

For-Thp-Leu-Δ^zPhe-OMe ( 2 ), an analogue of the chemotactic tripeptide For-Met-Leu-Phe-OMe, containing 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and (Z)-2,3-didehydrophenylalanine (Δ^zPhe) as achiral, conformationally restricted mimics of Met and Phe, respectively, has been synthesized. In the crystal the new formyltripeptide adopts a type I β-turn conformation stabilized by a weak H bond between the formylic oxygen and the Δ^zPhe NH. ¹H-nmr analysis based on NH solvent accessibility and nuclear Overhauser effect experiments suggests that the β-turn is not preferred in CDCl₃ solution where a γ-turn, centered at the Thp residue, prevails. The biological activity of 2 has been determined on human neutrophils and compared to that of previously studied analogues. The tripeptide 2 is practically unable to elicit superoxide anion production and lysozyme release, while slight, but not statistically significant activity was induced in chemotaxis. The role of the orientation of the aromatic ring with respect to the backbone adjacent atoms is discussed. © 1994 John Wiley & Sons, Inc.     

Conformationally constrained chemotactic peptide analogs of high biological activity

The stereochemically constrained chemotactic peptide analogs, formylmethionyl-alpha-aminoisobutyryl-phenylalanine (formyl-Met-Aib-Phe-OH) and formylmethionylcycloleucinylphenylalanine (formyl-Met-Cyl-Phe-OH) are highly effective in inducing lysosomal enzyme release from rabbit neutrophils. NMR studies of the Aib2 analog in (CD3)2SO favor a folded conformation in which the Phe NH group is inaccessible to solvent. Intramolecularly hydrogen-bonded conformations involving a Met-Aib-beta-turn or a gamma-turn centered at Aib2 are considered. The results suggest that folded conformations may allow highly active interactions with the neutrophil formylpeptide receptor.     

Modified chemotactic peptides: Synthesis,conformation, and activity of HCO-Thp-Ac6c-Phe-OMe

HCO-Thp-Ac₆c-Phe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent HCO-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 contains 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 1-aminocyclohexane-1-carboxylic acid (Ac₆c) as achiral, conformationally restricted mimics of Met and Leu, respectively. In the crystal, the formyltripeptide adopts an helical conformation at the Thp and Ac₆c residues, of the type α_R and α_L, respectively, whereas the C-terminal phenylalanine is quasi-extended. A system of two consecutive γ-turns, centered at the first two residues, better explains the nmr data as compared with an alternative β-turn structure. The conformation of the new analogue 3 is compared with those of two related peptides containing Thp as N-terminal residue. The biological activity of 3 has been determined on human neutrophils and compared to that of the previously studied model [Ac₆c²] fMLP-OMe. While the above analogue is highly active in the superoxide anion production, the new tripeptide 3 is practically unable to elicit any of the tested biological activities. © 1996 John Wiley & Sons, Inc.     

Tryptophan rich peptides: influence of indole rings on backbone conformation

Synthetic peptides with defined secondary structure scaffolds, namely hairpins and helices, containing tryptophan residues, have been investigated in this study to probe the influence of a large number of aromatic amino acids on backbone conformations. Solution NMR investigations of Boc-W-L-W-(D)P-G-W-L-W-OMe (peptide 1), designed to form a well-folded hairpin, clearly indicates the influence of flanking aromatic residues at the (D)Pro-Gly region on both turn nucleation and strand propagation. Indole-pyrrolidine interactions in this peptide lead to the formation of the less-frequent type I' turn at the (D)Pro-Gly segment and frayed strand regions, with the strand residues adopting local helical conformations. An analog of peptide 1 with an Aib-Gly turn-nucleated hairpin (Boc-W-L-W-U-G-W-L-W-OMe (peptide 2)) shows a preference for helical structures in solution, in both chloroform and methanol. Peptides with either one (Boc-W-L-W-U-W-L-W-OMe (peptide 3)) or two (Boc-U-W-L-W-U-W-L-W-OMe (peptide 4)) helix-nucleating Aib residues give rise to the well-folded helical conformations in the chloroform solution. The results are indicative of a preference for helical folding in peptides containing a large number of Trp residues. Investigation of a tetrapeptide analog of peptide 2, Boc-W-U-G-W-OMe (peptide 5), in solution and in the crystal state (by X-ray diffraction), also indicates a preference for a helical fold. Additionally, peptide 5 is stabilized in crystals by both aromatic interactions and an array of weak interactions. Examination of Trp-rich sequences in protein structures, however, reveals no secondary structure preference, suggesting that other stabilizing interactions in a well-folded protein may offset the influence of indole rings on backbone conformations.     

A computergraphic determination of the chemotactic peptide preferred conformation

Replacement of leucine in the chemotactic peptide For-Met-Leu-Phe by the sterically constrained amino acids alpha-aminoisobutyric acid and aminocyclohexanecarboxylic acid affords compounds of equal or greater activity than the parent. NMR studies indicate that the parent compound is present as a beta-sheet in solution, whereas the analogues prefer a beta-turn. Application of molecular modelling would indicate that the beta-turn conformer is energetically preferable and thus suggests that it is the orientation adopted by the peptides.     

Modified chemotactic peptides: synthesis,crystal conformation,and activity of For-Hse(Me)-Leu-Phe-OMe

The presence of the sulfur atom of the methionine side chain exerts significant effects at different levels on biochemical behavior of chemotactic N-formylpeptides. In order to acquire more information on this point, the synthesis, the conformation in the crystal, and the activity of For-Hse(Me)-Leu-Phe-OMe (2) —an oxygen analogue of For-Met-Leu-Phe-OMe (f MLP-OMe) containing the O-methyl-L -homoserine in place of the native methionine at position 1—is reported. The new analogue 2 adopts a conformation that is extended at the first two residues and folded at the C-terminal phenylalanine. This conformation is different from that of the parent f MLP-OMe and strikingly similar to that adopted by f MLP-OBu^t. The side-chain spatial orientation of 2 corresponds to that adopted by f MLP-OH when cocrystallized with an immunoglobulin possessing binding properties similar to those of neutrophil receptors. When tested on human neutrophils the formylpeptide 2 is more active than the parent in the stimulation of directed mobility and maintains both the granule enzyme release activity an the superoxide anion production. © 1994 John Wiley & Sons, Inc.     

Mechanism of interaction of the antileukemic drug cytosine arabinoside with aromatic peptides: role of sugar conformation and peptide backbone

Interaction of the antileukemic drugs, cytosine-arabinoside (Ara-C) and adenosine-arabinoside (Ara-A) and a structural analogue, cytidine, with aromatic dipeptides has been studied by fluorescence and NMR spectroscopy. Ara-C and cytidine bind tryptophanyl and histidyl dipeptides but not tyrosyl dipeptides, while Ara-A does not bind to any of them. Both studies indicate association involving stacking of aromatic moieties. NMR spectra also indicate a protonation of the histidine moiety by Ara-C. In case of cytidine, the chemical shifts observed on binding to His-Phe imply that the backbone protons of the dipeptide participate in the binding. The conformation of the sugar and the base seem to play a very important role in the binding phenomenon as three similar molecules, Ara-C, Ara-A and cytidine bind in totally different ways.     

Role of peptide backbone in T cell recognition

T cells recognize self and nonself peptides presented by molecules of the MHC. Amino acid substitutions in the antigenic peptide showed that T cell specificity is highly degenerate. Recently, determination of the crystal structure of several TCR/MHC-peptide complexes suggested that the peptide backbone may significantly contribute to the interaction with the TCR. To directly investigate the role of the peptide backbone in T cell recognition, we performed a methylene-amino scan on the backbone of an antigenic peptide and measured the capacity of such pseudopeptides to bind their cognate MHC molecule, to sensitize target cells for T cell lysis, and to stimulate IL-2 secretion by two T cell hybridomas. For one of these pseudopeptides, we prepared fluorescent tetramers of MHC molecules and compared the staining of two T cell hybridomas. Our results demonstrate that the peptide backbone has an important contribution to TCR binding and suggest that some interactions between the peptide backbone and the TCR may be partially conserved. We discuss this finding in the perspective of TCR plasticity and T cell function.     

Crystal structure, conformation, and potential energy calculations of the chemotactic peptide N-formyl-L-Met-L-Leu-L-Phe-OMe

The tripeptide N-formyl-L-Met-L-Leu-L-Phe-OMe (FMLP-OMe) crystallizes in the orthorhombic system, space group P 2(1)2(1)2(1), with the following unit-cell parameters: a = 21.727, b = 21.836, c = 5.133 A, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I greater than 2 omega (I). The peptide backbone is folded at the Leu residue (phi L = -67.7, psi L = -49.1 degrees) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side-chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP-OMe and the anionic form of N-formyl-L-Met-L-Leu-L-Phe-OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have been carried out in order to compare the energies of the various backbone conformers.     

Manufacturing of peptides exhibiting biological activity

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.     

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.     

Antibiotics and peptides with agonist and antagonist chemotactic activity.

It has been found that the polypeptide antibiotics gramicidin S, tyrocidin and bacitracin, containing Leu-Phe or Ile-Phe sequences, are chemoattractants for neutrophils. Related synthetic pentapeptides containing the sequence Leu-Phe have stronger biological activities, provided the N-terminal residue is acylated. The formylated peptide f-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe is a potent agonist (4 × 10^?9 M) whereas the t-butyloxycarbonyl analog is a good antagonist (8 × 10^?7 M).     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.     

Role of lysine residue at 7th position of wasp chemotactic peptides

Most of chemotactic peptides isolated from various kinds of wasp venom have lysine residue at 7th (or 8th) position, but only a chemotactic peptide from Icaria sp. has no basic amino acid residues in the sequence. The relation of chemotaxis with other biochemical activities such as superoxide generation and lysosomal enzyme release from guinea pig neutrophils was studied by the use of substitution analogs of Icaria chemotactic peptide at 7th position. Findings revealed two distinct ways of chemoattractant signal transduction in neutrophils.     

Neutrophil chemotactic activity of N-terminal peptides from the calpain small subunit

On the basis of previous findings that N-acetyl nonapeptide from the human calpain I large subunit has chemotactic activity for neutrophils, a series of peptides with the N-terminal sequence of the calpain small subunit were synthesized and their chemotactic activity was examined. Potent activity was found in N-acetyl tetra, hepta, octa, nona and a larger peptide of 13 residues, although N-acetyl tripeptide showed only weak activity and N-acetyl penta and hexa peptides showed almost no activity. Since the small subunit is identical in calpains I and II, the results indicate that both calpains could be precursor proteins of chemotactic factors for neutrophils.     

12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.     

[12-Homoarginine]glucagon: synthesis and observations on conformation,biological activity,and copper-mediated peptide cleavage

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content, the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.     

An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides

We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two- to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.     

Selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity

The role of adhesive selectin molecules in the process of atherogenesis is an open question. These molecules are known as markers of atherosclerosis activity, however, only some biological mechanisms are known up to now. In this study we examined the levels of soluble forms of E-, P-selectin and monocyte chemotactic protein (MCP-1) in the process of extracorporeal cholesterol elimination by LDL-apheresis. We measured the levels of sE-, sP-selectin and MCP-1 in the plasma before and after LDL-apheresis and in the washout solution from immunoabsorption columns Lipopak. Eighty measurements were performed repeatedly in 6 patients with severe familial hypercholesterolemia (FH) on long-term LDL-apheresis treatment. Before the procedure P-selectin levels were 204+/-179 ng/ml, E-selectin 32.1+/-33.7 ng/ml, MCP-1 323.8+/-121 pg/l, whereas after the procedure we found P-selectin levels 131.6+/-34 ng/ml, E-selectin 33.1+/-51 ng/ml, and MCP-1 200.4+/-15 pg/l. Levels of P-selectin were increased in the blood of patients with FH in spite of long-term intensive extracorporeal LDL-elimination, documenting thus the activity of atherosclerosis. The levels of P-selectin and MCP-1 decreased significantly after the hypolidemic procedure and could be used as another marker showing the effectivity of the extracorporeal LDL-cholesterol elimination (immediately after the procedure), and, after further verification, may serve as a marker for controlling the therapy efficacy.