Elsevier

Bioorganic Chemistry

Volume 34, Issue 5, October 2006, Pages 298-318
Bioorganic Chemistry

Review
Structure–activity relationship of for-l-Met l-Leu-l-Phe-OMe analogues in human neutrophils

https://doi.org/10.1016/j.bioorg.2006.07.001Get rights and content

Abstract

Neutrophils constitute the first line of defence against bacterial invasion. They migrate to infected tissues along a concentration gradient of chemoattractant molecules, the most important of which is for-Met-Leu-Phe-OH (fMLP). Different responses arise from formylpeptides binding to different isoforms of the specific receptor. The aim of the studies reported herein was to clarify (i) the role of fMLP-OMe amide bonds in receptor–ligand cross-linking, (ii) the nature of the group occupying the N- and C-terminal positions, (iii) the features peculiar to the Met, Leu, and Phe receptor pockets, and (iv) the features which determine the specific neutrophil response.

Graphical abstract

Neutrophils migrate to infected tissues along a concentration gradient of chemoattractant molecules, e.g. for-Met-Leu-Phe-OMe (fMLP-OMe). The aim of the studies reported herein was twofold: to clarify the mechanisms whereby the ligand hooks its specific receptor and to verify the biological consequences arising from every possible variations on the fMLP-OMe prototype.

Introduction

In spite of the numerous studies into the biological responses of human neutrophils arising from their interaction with the chemotactic formyl-l-Met-l-Leu-l-Phe-OMe (fMLP-OMe) [1], [2], [3], [4], [5], [6], [7], [8], [9], nobody has yet clarified which chemical mechanisms are involved in the binding of this ligand to its receptor. The receptor has been studied in order to understand its position in the membrane and the steric characteristics which allow the best biological responses [10], [11], [12], [13], [14], but not to establish how the ligand hooks into its receptor pocket.

Moreover, the consequences arising from introducing unconventional residues in place of Met, Leu, and Phe amino acids have still to be verified. Previous studies have indicated that the Met residue may be fundamental for good biological activity, due to its side-chain electron-rich sulphur atom which allows the peptide to interact with a restricted positive area on the receptor. The Leu residue has always been substituted with bulky hydrophobic amino acids, since the receptor pocket, in which the second residue is located, was thought to be a hydrophobic area [15], [16], [17]. Studies have also determined that the C-terminal amino acid should be aromatic, Phe being the preferred choice [18], [19], [2].

The aim of the research reported here was twofold: to clarify the mechanisms whereby the formyl-l-Met-l-Leu-l-Phe-OMe ligand hooks the specific receptor on human neutrophils and to verify the biological consequences of introducing unusual amino acids in place of the Met, Leu, and Phe residues. We demonstrated that: (i) the amidic bonds are involved, although with varying importance, in the binding of formylpeptides to the receptor; (ii) the ability to activate human neutrophils response is not strictly due to the presence of the formyl group; (iii) the Met pocket is narrow in dimension, and carries a positive charge located at a well defined distance from the peptide backbone; it is oriented in a specific position, surrounding most of the internally located side-chain; (iv) the Met residue is essential for chemotaxis, but specific substitutions can introduce selectivity towards killing mechanisms, which can be strongly stimulated; (v) hydrophobic residues at position 2 are decisive solely for chemotaxis, which is depressed by introducing hydrophilic amino acids, while both superoxide anion production and lysosomal lytic enzymes are strongly and selectively stimulated; (vi) hydrophobicity is not a mandatory feature for the third residue, and (vii) the requirement of a C-terminal carboxylic group, either free or esterified, to trigger a biological response in human neutrophils.

The studies herein reported allow us to modulate the neutrophil biological responses in two ways:

  • (1)

    variation of the amidic bonds between amino acids allows us to modulate the intensity of the biological response from zero to maximum;

  • (2)

    variation of the character (more or less hydrophilic or lipophilic) of the residues allows us, in some way, to select either chemotaxis or killing mechanisms, i.e. superoxide anion production or lytic enzyme release.

Section snippets

Role of the amide bond in ligand–receptor cross-linking

Several studies have attempted to correlate structure–activity data in order to establish the structural features necessary for biological activity. Optimal agonist potency occurs when (a) the N-terminal group is formylated, (b) the first amino acid is Met, (c) the second amino acid has a bulky hydrophobic side-chain, and (d) the third amino acid is Phe [1], [2], [3], [19]. A critical interaction of the Phe Cdouble bondO group with the receptor, possibly via hydrogen bonding, has also been hypothesized [3]

Substitution of the formyl group

The formyl group on the N-terminal methionine is small enough to reach the bottom of the binding cavity through a narrow passage; it participates as hydrogen-donor in linking to the receptor and it seems that its role in this interaction cannot be ruled out.

As a consequence, all studies performed are directed, on the one hand, to finding groups small enough to penetrate the specific receptor pocket and, on the other, to finding substitutions which can allow a hydrogen bond in the receptor

Variations in the Met residue

With the aim of clarifying the structure–activity relationship peculiar to the Met receptor pocket, a large number of formylpeptides modified at position 1 have been synthesized and biologically tested. These analogues can be grouped into two categories (Fig. 6):

  • (i)

    analogues with a residue carrying a sulphur atom on the side-chain, such as S-methylcysteine Cys(Me) and ethionine Eth [2], 4-amino-tetrahydrothiopiran-4-carboxylic acid (Thp) [41], methionine sulphoxide Met(O) and methionine sulphone

Variation at carboxylic group

A few alterations of the C-terminal function have been made to understand whether and how to change the carboxylic group in the search for an increase in the activity and/or biological selectivity. It has been shown that the ability to activate human neutrophils (using as biological reference the release of β-glucuronidase) is single bondCONH2<single bondCOOMe<single bondCOOH<<single bondCOOBzl [74].

Phenylalaninol analogues have been introduced at position 3, thereby varying both the third residue and the carboxylic group [75]. The

Cyclic analogues

Some studies on the formyl tripeptide structure–activity relationship have been directed toward cyclic analogues, aimed at introducing conformational restrictions into the backbone. Cyclopeptides offer several advantages, among which are reduced conformational heterogeneity, enhanced stability toward enzymatic degradation and the possibility of exerting a certain control on the relative spatial orientation of the side-chains. This latter property is relevant for the design of bioactive ligands

Conclusions

The important points evidenced by this review are:

  • 1.

    Amide bonds are able to modulate and/or select biological responses.

  • 2.

    The formyl group is not mandatory for triggering the physiological functions of human neutrophils.

  • 3.

    Both hydrophilic and hydrophobic residues can fit the specific receptor pockets. In particular, hydrophilic substitutions at position 2 are optimal for triggering killing mechanisms.

  • 4.

    Cyclic analogues, such as the cyclic dimeric tripeptide, can exhibit good activity and a promising

Acknowledgments

This work was supported by MURST (Research Funds ex 60%) and Fondazione Cassa di Risparmio di Ferrara, Italy. We thank Anna Forster for the English revision of the text.

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