Biologically active peptides: from a laboratory bench curiosity to a functional skin care product.

Small, biologically active peptides (short sequences of amino acids) were first described about 40 years ago: TRH, angiotensin, vasopressin, oxytocin, bradykinin. Since then, many more peptides have been isolated from mammalian tissue and organs, and their activity investigated. Essentially, these molecules play a hormonal (messenger) role: released at one point in the body, they act at specific receptor sites at different locations in the organism. Mostly the peptides are transported from the site of release to the site of biological activity through the blood or lymphatic fluid. The use of these molecules in cosmetics does not appear obvious, as the topical application of these highly soluble, fragile and extremely expensive molecules seems inappropriate, and systemic effects (blood transport) are not desired. This paper shows that the obstacles to using highly specific, powerful peptides as 'actives' in cosmetic products can be overcome. Cosmetically interesting activities such as stimulation of collagen synthesis, chemotaxis, anti-stinging effects and others, can be observed and substantiated with chemically modified peptide sequences. Long chain fatty acid conjugates improve skin penetration, specific activity and economic feasibility of these molecules.

A physical method for preservation of cosmetic products.

To date, the microbiological stability of cosmetics products is assured by means of chemical preservatives, with well-known possible side effects such as irritation or cutaneous allergic events. This paper describes a new concept to protect all types of cosmetic formulae from microbiological contamination: the osmotic effect of a particular form of glycerylpolyacrylate gels. The latter allows the formulator to conceive numerous preservative-free formulae. The basis of its activity lies in the strong osmotic properties of the hydrogel: it absorbs water from its surroundings, thus depriving germs of the vital aqueous environment needed for survival. Challenge tests on various microorganisms, repeat insult challenges and time-stability tests performed on these hydrogels in cosmetic formulas were carried out. At the same time, the hydrogel exhibited high skin moisturizing power. The hydrogel is non-toxic and non-irritant as the Primary Cutaneous Irritation Index observed after 48 h of occlusive patches in 10 adult volunteers is only of 0.10.

The effect of a synthetic ceramide-2 on transepidermal water loss after stripping or sodium lauryl sulfate treatment: an in vivo study.

The integrity of the chemical and physical structure of the horny layer is essential for maintaining the skin in good health. Any disturbance of this integrity may lead to cutaneous reactions of varying degree: dryness, redness, inflammation. The measurement of Transepidermal Water Loss (TEWL) allows one to record this kind of disturbance and to follow the slow return to normal. In this in vivo study two techniques of insulting the epidermis were used: stripping and washing with sodium lauryl sulfate (SLS). A significant increase of TEWL values resulted in both cases. The application of emulsions containing 0.5% and 1% of a synthetic ceramide type-2 (N-stearoyl-DL-erythro-sphinganine) decreased the disturbance measured by TEWL, in a significant fashion in both trials. The placebo emulsions showed no significant effect. The ceramide thus seemed to participate in the restructuring of the horny layer.

Studies on angiotensin II and analogs: impact of substitution in position 8 on conformation and activity.

Affinity, residual agonist activity, and inhibitor properties of a series of angiotensin II analogs modified at the COOH-terminal position (X8-substituted peptides) have been probed for structure/conformation-biological activity relationships. The results emphasize (i) the large impact that subtle conformational variations caused by structural alterations in the position 8 side chain have on biological properties, (ii) the implication of the COOH-terminal carboxyl group in both affinity and intrinsic activity, and (iii) the influence that the bulkiness of the side chain in position 8 of antagonists has on the local conformation at the COOH terminus and thus on the inhibitory properties. In the hormone, the phenylalanine-8 ring is required for its steric influence and aromaticity to ensure a fully active conformation at the COOH terminus. Especially, correct orientation of the position 8 carboxyl group relative to the phenyl group of the phenylalanine residue may be necessary for agonistic activation of the angiotensin receptor complex. Replacement of the aromatic ring on the COOH-terminal residue by a nonaromatic group leads to incorrect orientation of the carboxyl group and causes the appearance of antagonist properties. Although the steric effects of the side chain can be modulated by specific interaction of its chemical groups (if any) with the peptide backbone, we found a good correlation between the size of the side chain-e.g., the steric parameter V gamma (the van der Waals volume consisting of the C alpha, C beta, and C gamma atoms), the conformational properties in the backbone (3J HC alpha-NH), and the binding capacities in all compounds tested.

Conformation and mobility of tyrosine side chain in tetrapeptides. Specific effects of cis- and trans-proline in Tyr-Pro- and Pro-Tyr-segments.

We examined the properties of tyrosine in four free tetrapeptides: Ala-Ala-Tyr-Ala (AATA), Ala-Pro-Tyr-Ala (APTA), Ala-Tyr-Ala-Ala (ATAA) and Ala-Tyr-Pro-Ala (ATPA) by CD, n.m.r. and energy calculations. Experimental data (the aromatic 1Lb signal, rotamer populations around the C alpha-C beta bond (chi 1), rotations around C beta-C gamma(chi 2), chemical shifts of ortho- and meta-protons in the phenolic ring (in aqueous and Me2SO solutions), NH proton temperature coefficients and vicinal coupling constants 3JNH-C alpha H in the backbone (Me2SO solution) were compared with calculated minimum energy conformations. We find qualitative agreement between the results of the different techniques with respect to global tendencies of conformational behaviour: we present experimental evidence showing that the presence of proline in the sequence has a more pronounced effect on the side chain organization of the residues preceding it than on one succeeding it. This steric influence of proline on its immediate neighbor is even stronger in the cis isomer than in the more common trans isomer. The strong preference for Rotamer II (chi 1 = 180 degrees) over Rotamer I (chi 1 = -60 degrees) in ATPA (cis-form) concomitant with a noticeable deviation of chi 2 is a striking example.

Preferred solution conformation of des-Arg9-bradykinin and analysis of structure-conformation-activity relationships in the series [Alan]des-Arg9-bradykinin.

This paper describes the solution conformation of the vasoactive peptide hormone des-Arg9-bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe). 1. By 1H-NMR spectroscopy we studied des-Arg9-bradykinin and its fragments bradykinin-(1-5), bradykinin-(1-6) in aqueous solution as a function of pH (titration) and in dimethylsulfoxide solution at two ionic states (cation and neutral ion species). 2. The preferred solution conformation which is most strongly stabilized in dimethylsulfoxide in the neutral ion species includes a distorted beta-turn II involving the N-terminal sequence of Pro2-Pro3-Gly4-Phe5 and most likely a C7-type bend in the C-terminal part Ser-Pro-Phe. 3. A complete series of analogous ([Alan]des Arg9-bradykinin, with n = 1, 2...8) was then investigated by circular dichroism and 1H-NMR spectroscopy in order to study the conformational role played by each residue and to delineate the local and the long-range effects on conformation brought about by the Xaa leads to Ala substitutions. Chosen spectral parameters (circular dichroic spectra, chemical shift variations and vicinal coupling constants) characteristic of the preferred solution conformation of the native sequence of des-Arg9-bradykinin are followed from analogue to analogue. The important conformational role of the arginine-1 side chain and its positive charge and the spatial proximity of the N-terminal and C-terminal groups, i.e. the folded structure of the peptide can be inferred from these data. 4. A comparison of the biological activities of the analogues with the conformative perturbations caused by the chemical alterations shows des-Arg9-bradykinin conformation and receptor affinity to be equally sensitive to single-residue substitutions. The correct orientation of the arginine-1 side chain, the precise geometry of the turn involving residues 2-5 and of the C-terminal Pro-Phe sequence are of primary importance.

Synthesis of [alpha-methyltyrosine-4]angiotensin II: studies of its conformation, pressor activity, and mode of enzymatic degradation.

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of alpha-methylation of the tyrosine in position 4. [alpha-Methyltyrosine-4]angiotensin II, synthesized by the solid-phase procedure, showed 92.6 +/- 5.3% pressor activity of angiotensin II. Incubation with alpha-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin II was completely degraded to two components. Comparison of the 1H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [alpha-methyltyrosine-4]angiotensin II suggested that alpha methylation of the tyrosine residue in angiotensin II does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by alpha-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.

Amino acid side chain conformation in angiotensin II and analogs: correlated results of circular dichroism and 1H nuclear magnetic resonance.

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding N-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of N-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (i) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (ii) N-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-N-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (i) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (ii) greater impact of N-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.

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.

Conformational features of bradykinin. A circular dichroism study of the aromatic side-chains.

The circular dichroism (CD) of the peptide hormone bradykinin and its analogues, [Phe(H4)5]-bradykinin, [Phe(H4)8]bradykinin, [Phe(H4)5,8]bradykinin, [TyrOMe5]bradykinin, [TyrOMe8]bradykinin and [TyrOMe5.8]bradykinin, is described. The comparison of the CD spectra of these analogues with each other, recorded under a variety of conditions (pH, solvent, temperature), allows the monitoring of the behaviour of the aromatic side-chains (phenylalanine, tyrosine) and an estimation of their respective spectral contributions in both spectral regions (320-250 nm, 250-190 nm) with good precision. Conformational non-equivalence of the residues Phe-5 and Phe-8 together with some overall conformational features of bradykinin are thus established.

Influence of hydrogen bonding on the rotamer distribution of the histidine side chain in peptides: 1H NMR and CD studies.

Both 1H NMR and circular dichroism pH titration studies on histidine, His-Gly, Gly-His and Gly-His-Gly indicate that the side-chain spatial orientation depends strongly on the vicinal charges. The arrangement of the imidazole side-chain (rotamer population) is shown by the histidine beta and beta' and the glycine methylene proton chemical shifts as well as the vicinal 1H-1H coupling constants 3JCalpha-H-beta-H, beta'-H. For His-Gly and Gly-His-Gly a good correlation can be found between the ionization of the glycine COOH group and the increase of rotamer III (g-g) which is also visualized by circular dichroism through an enhancement of the ellipticity at 212 nm. In these two peptides a hydrogen bond between the imidazolium and the carboxylate group is supposed to stabilize rotamer III at pH 4-5.

Circular dichroism studies of angiotensin II and analogues: effects of primary sequence, solvent, and pH on the side-chain conformation.

Conformational aspects of the pressor hormone angiotensin II and 11 of its structural analogues were studied by circular dichroism. Each position of the peptide was singly substituted with an aliphatic residue and alterations of the CD spectra of the resulting analogues in the peptide and aromatic spectral regions (320-250 nm, 250-190 nm) were examined. The spectra of these peptides in 2,2,2-trifluoroethanol solution permit estimation of the relative importance of the various side chains in maintaining the backbone conformation of the hormone. The evolution of the CD spectra in both spectral regions of the peptides in aqueous solution during a titration from pH 1 to pH 12 makes it possible to elucidate further the role of ionizable groups and their interaction with aromatic amino acids such as tyrosine. The results obtained indicate that substitutions in aspartic acid 1, proline 7, and phenylalanine 8 of angiotensin II entail changes in the backbone conformation. On the other hand, the side chains of valine 3, isoleucine 5, and the biologically essential histidine 6 serve mainly to correctly align the phenolic ring of tyrosine in position 4.