Mutagenesis at the human tachykinin NK2 receptor to define the binding site of a novel class of antagonists
Introduction
Neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) is a tachykinin peptide which displays the highest affinity for the tachykinin NK2 receptor, a G-protein coupled with seven helices spanning through the cell membrane, which mediates its biological effects Gerard et al., 1990, Severini et al., 2002.
Antagonists for the peripheral tachykinin NK2 receptors are considered as potential innovating therapies in various diseases, such as neurogenic bladder hyperreflexia and irritable bowel syndrome (Lecci and Maggi, 2003).
We previously constructed a three-dimensional receptor model of the tachykinin NK2 receptor and, by means of site-directed mutagenesis, have identified a subset of amino acids that participate in the binding site of peptide and nonpeptide antagonist ligands Giolitti et al., 2000, Giolitti et al., 2002.
The aim of the present study was the investigation of the binding interaction mode of a new class of tachykinin NK2 receptor antagonists, such as MEN13918 (Nγ{Nα[Nα(benzo[b]thiophen-2-yl)carbonyl]-1-aminocyclohexan-1-carboxy]-d-phenylalanyl}-3-cis-aminocyclohexan-1-carboxylic-acid-N-(1S,2R)-2-aminocyclohexyl)amide trifluoroacetate salt) and MEN14268 (Nα[Nα(benzo[b]thiophen-2-yl)carbonyl)-1-aminocyclopentane-1-carboxyl]-d-phenylalanine-N-[3(morpholin-4-yl)propyl]amide trifluoroacetate salt) (Fig. 1), here termed as linear pseudopeptides. By means of data obtained using site-directed mutagenesis at the human tachykinin NK2 receptor and binding competition experiments, a molecular model of MEN13918 and MEN14268 docked to their recognition site in the human tachykinin NK2 receptor is presented.
Section snippets
Materials
[125I]NKA (specific activity 2000 Ci mmol−1) was provided by Amersham, [3H]SR48968 (specific activity 25.5 Ci mmol−1) (Emonds-Alt et al., 1993) by Perkin Elmer New England Nuclear, and [3H]nepadutant (specific activity 30 Ci mmol−1) (MEN11420; (cyclo-{[Asn(β-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β–5β)}; Renzetti et al., 1998) was synthesized by SibTech (Elmsford, NY). Peptides were obtained from Neosystem. All salts used were purchased from Merck and all other materials from Sigma. NK2 receptor
Antagonist activity of MEN13918 and MEN14268 in various smooth muscle contractility assays for tachykinin receptors
Neither MEN13918 nor MEN14268 produced agonist effects, up to 1 μM concentrations, in any of the smooth muscle preparations tested.
Both compounds potently antagonized NK2 receptor-mediated contractions produced by NKA in the human isolated urinary bladder (pKB 9.1 and 8.3 for MEN13918 and MEN14268, respectively; Table 1). In the guinea pig colon preparation both MEN13918 and MEN14268 antagonized the contractile effects produced by the NK2 receptor agonist [βAla8]-NKA(4–10), without depressing
Discussion
In the field of tachykinin NK2 receptor antagonists we previously reported the pharmacological profile of bycyclic peptide antagonists such as MEN10,627 (cyclo-(Met-Asp-Trp-Phe-Dap-Leu]cyclo(2β-5β)}; Maggi et al., 1994) and nepadutant (Catalioto et al., 1998). These structures were further simplified leading to the monocyclic peptide MEN11558 (cyclo-{-Suc-Trp-Phe-[(R)-NH-CH(CH2-C6H5)-CH2-NH]}; Giannotti et al., 2000), and by combining rational design and site-directed mutagenesis at the human
References (33)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation
FEBS. Lett.
(1997) - et al.
Characterization of the binding sites of [3H]SR 48968, a potent nonpeptide radioligand antagonist of the neurokinin-2 receptor
Biochem. Biophys. Res. Commun.
(1993) - et al.
The human neurokinin A (substance K) receptor
J. Biol. Chem.
(1990) - et al.
Molecular determinants of peptide and nonpeptide NK-2 receptor antagonists binding sites of the human tachykinin NK-2 receptor by site-directed mutagenesis
Neuropharmacology
(2000) - et al.
Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity
Eur. J. Pharmacol.
(1997) - et al.
Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles
Regul. Peptides
(1992) - et al.
Site-directed mutagenesis at the human B2 receptor and molecular modelling to define the pharmacophore of non-peptide bradykinin receptor antagonists
Biochem. Pharmacol.
(2004) - et al.
Effect of nepadutant at tachykinin NK2 receptors in human intestine and urinary bladder
Eur. J. Pharmacol.
(2000) - et al.
Characterization of [3H]MEN 11420, a novel glycosylated peptide antagonist radioligand of the tachykinin NK2 receptor
Biochem. Biophys. Res. Comm.
(1998)
Molecular characterization of rat substance K receptor and its mRNAs
Biochem. Biophys. Res. Commun.
Locating ligand-binding sites in 7TM receptors by protein engineering
Curr. Opin. Biotechnol.
Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands
J. Biol. Chem.
Isolation and pharmacological characterization of a hamster neurokinin A receptor cDNA
Mol. Pharmacol.
A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis
Br. J. Pharmacol.
MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist
Br. J. Pharmacol.
Cited by (16)
Synthesis and structural features of cyclobutane-containing chiral bicyclic ureas
2010, Tetrahedron AsymmetryCharacterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3
2009, Biochemical PharmacologyCitation Excerpt :Despite the potential importance of these residues for binding and the rather large degree of variation between species, we were unable to identify significant differences of the potency of saredutant and ZD6021 at NK2R among the species studied. However, it cannot be excluded that the potency of structurally different compounds could have different potency at NK2R from gerbil and dog [16]. Consistent with previous findings by Ref. [38], both talnetant and osanetant displayed about a 5–10-fold lower potency at the rat NK3R compared to the human NK3R.
Inflammatory pain in the rabbit: A new, efficient method for measuring mechanical hyperalgesia in the hind paw
2008, Journal of Neuroscience Methodsα,α-Cyclic aminoacids as useful scaffolds for the preparation of hNK<inf>2</inf> receptor antagonists
2007, Bioorganic and Medicinal Chemistry LettersMEN15596, a novel nonpeptide tachykinin NK<inf>2</inf> receptor antagonist
2006, European Journal of Pharmacology