Definition
N-ureido derivatized chemotactic peptides are potent agonists and antagonists of the neutrophil fMLF (N-formyl-Met-Leu-Phe) receptor.
Discovery
In 1996, J.D.Higgins et al., described that the stimulation of the leukocyte N-formylpeptide receptor (FPR) induces chemotaxis, cell adhesion, free radical release, and degranulation, responses associated with infection and inflammation. Under conditions where continuous activation of the receptor prevails, neutrophil-dependent tissue damage ensues. They synthesized and evaluated a series of amino-terminal carbamate analogues of the peptide Met-Leu-Phe (MLF) in order to determine the structural requirements for imparting agonist or antagonist activity at the human neutrophil FPR. Antagonists of the FPR have potential for use as diagnostic and therapeutic agents. 1, 2.
Structural Characteristics
A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with 99mTc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with 99mTc produced a ternary ligand complex [99mTc (fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [99mTc (fMLFK-HYNIC)(tricine)2] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [99mTc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. Unbranched carbamates (methoxycarbonyl, ethoxycarbonyl, and n-butyloxycarbonyl) resulted in agonist activity, whereas branched carbamates (iso-butyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl) were antagonists. 1,2,3. The biological action of several X-Phe-Leu-Phe-Leu-Z (X=3',5'-dimethylphenyl-ureido; Z=Phe, Lys, Glu, Tyr) analogues was analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-Leu-Phe-Leu-Phe analogues obtained as C-terminal olo or amido derivatives and T-Phe-Leu-Phe-Leu-Phe analogues (T=thiazolyl-ureido) were also analysed. The activities of pentapeptide derivatives were compared with those of X-Phe- -Leu-Phe- -Leu-Phe chosen as reference antagonist. Results demonstrate that X-Phe-Leu-Phe-Leu-Phe-olo, X-Phe-Leu-Phe-Leu-Glu and X-Phe-Leu-Phe-Leu-Tyr are more active antagonists than X-Phe-Leu-Phes-Leu-Phe 4.
Mode of Action
Peptides were evaluated in three in vitro assays: receptor binding, superoxide anion release, and cell adhesion. The peptide antagonists were more potent inhibitors of superoxide anion release than cell adhesion by 4-7-fold. When iso-butyloxycarbonyl-MLF (i-Boc-MLF) was further modified at the carboxy terminus with Lys, antagonist potency was retained but without functional selectivity. Further C-terminal modification with the radionuclide linker diethylenetriaminepentaacetic acid did not alter the potency of i-Boc-MLFK. These results indicate that the switch from agonist to antagonist activity can be achieved by modifying the overall size and shape of the amino-terminal group; that modifications at both the amino and carboxy termini can alter the functional selectivity of the peptide; and that modifications can be tolerated at the carboxy terminus to allow for development of an antagonist for diagnostic applications 1,2,3.
Functions
RP463: A stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes 1.
Antagonist for diagnostic, switch from agonist to antagonist activity can be achieved by modifying the overall size and shape of the amino-terminal group; that modifications at both the amino and carboxy termini can alter the functional selectivity of the peptide; and that modifications can be tolerated at the carboxy terminus to allow for development of an antagonist for diagnostic applications 2,3.
Receptor antagonist properties, the presence of Lys (X-Phe-Leu-Phe-Leu-Lys) seems, instead, to inhibit the formyl-peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor antagonist properties of T-Phe-Leu-Phe-Leu-Phe-OH. The introduction of the C-terminal methyl ester (T-Phe-Leu-Phe-Leu-Phe-OMe) or amido group (X-Phe-Leu-Phe-Leu-Phe-NH2) appears detrimental for the affinity and formyl-peptide receptor antagonist properties of the Phe-Leu-Phe-Leu-Phe derivatives. These peptides inhibit superoxide anion production and lysozyme release more efficaciously than neutrophil chemotaxis 5.
N-Formylmethionylleucyl phenylalanine tripeptides containing either L-difluoro- or L-trifluoromethionine as replacements for methionine were synthesized by solution methods. The fluorinated peptides were found to have potent chemoattractant activities on human neutrophils. Ab initio calculations were utilized to further understand the changes in electronic properties of the methionine side chain upon fluorination.
Ligands, based on the sequence of the prototypical chemotactic tripeptide Met-Leu-Phe-OH (fMLF) and by taking into account the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCO-Met-Leu-gPhe-COOMe and Boc-Met-Leu-gPhe-COOMe were synthesized. These two new ligands are characterized by the presence of a gem-diamino residue (gPhe) replacing the C-terminal Phe and a carbamate functionality positioned at both the ends of the molecule. The agonist and antagonist activity of the two new compounds has been determined on human neutrophils 6.
References
1. Higgins JD 3rd, Bridger GJ, Derian CK, Beblavy MJ, Hernandez PE, Gaul FE, Abrams MJ, Pike MC, Solomon HF (1996). N-Terminus urea-substituted chemotactic peptides: new potent agonists and antagonists toward the neutrophil fmlf receptor. J. Med. Chem., 39:1013-1015.
2. Derian CK, Solomon HF, Higgins JD 3rd, Beblavy MJ, Santulli RJ, Bridger GJ, Pike MC, Kroon DJ, Fischman AJ (1996). Selective inhibition of N-Formyl peptide-induced neutrophil activation by carbamate-modified peptide analogues. Biochemistry, 35(4):1265-1269.
3. Edwards DS, Liu S, Ziegler MC, Harris AR, Crocker AC, Heminway SJ, Barrett JA, Bridger GJ, Abrams MJ, Higgins JD 3rd (1999). RP463: A stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging. Bioconjugate Chem., 10(5):884–891.
4. Dalpiaz A, Ferretti ME, Vertuani G, Traniello S, Scatturin A, Spisani S (2002). C- and N-terminal residue effect on peptide derivatives' antagonism toward the formyl-peptide receptor. European Journal of Pharmacology, 436(3):187-196.
5. Houston ME, Harvath L, Honek JF, (1997). Syntheses of and chemotactic responses elicited by fMet-Leu-Phe analogs containing difluoro- and trifluoromethionine. Bioorganic & Medicinal Chemistry Letters, 7(23):3007-3012.
6. Zecchini GP, Paradisi MP, Torrini I, Nalli M, Lucente G, Spisani S (2000). Biscarbamate analogues of the chemotactic tripeptide fMLF---OMe. Farmaco., 55(4):308-313.