Mutagenesis at the human tachykinin NK2 receptor to define the binding site of a novel class of antagonists

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Abstract

The pharmacological profile of novel antagonists endowed with high affinity for the human tachykinin NK2 receptor is presented. 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) were more potent in blocking neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) induced contraction in human, which induced greater contraction in human (pKB 9.1 and 8.3) than rat (pKB 6.8 and <6) urinary bladder smooth muscle preparation in vitro. In agreement with functional data, in membrane preparations of CHO cells stably expressing the human NK2 receptors, both MEN13918 and MEN14268 potently inhibited the binding of agonist ([125I]NKA, Ki 0.2 and 2.8 nM) and antagonist ([3H]nepadutant, Ki 0.1 and 2.2 nM, [3H]SR48968 Ki 0.4 and 6.9 nM) radioligands. Using site-directed mutagenesis and radioligands binding we identified six residues in the transmembrane (TM) helices that are critical determinants for the studied antagonists affinity. To visualize these experimental findings, we constructed a homology model based on the X-ray crystal structure of bovine rhodopsin and suggested a possible binding mode of these newly discovered antagonist ligands to the human tackykinin NK2 receptor. Both MEN13918 and MEN14268 bind amongst TM4 (Cys167Gly), TM5 (Tyr206Ala), TM6 (Tyr266Ala, Phe270Ala), and TM7 (Tyr289Phe, Tyr289Thr). MEN13918 and MEN14268 diverging binding profile at Y289 mutations in TM7 (Tyr289Phe, Tyr289Thr) suggests a relation of their different chemical moieties with this residue. Moreover, the different influence on binding of these two ligands by mutations located deep along the inner side of TM6 (Phe270Ala, Tyr266Ala, Trp263Ala) indicates a nonequivalent positioning, although occupying the same binding crevice. Furthermore, binding data indicate the Ile202Phe mutation, which mimics the wild-type rat NK2 receptor sequence, as a species selectivity determinant. In summary, data with mutant receptors describe, for these new tachykinin NK2 receptor antagonists, a binding site which is partially overlapping either with that of the cyclized peptide antagonist nepadutant (cyclo-{[Asn(β-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β-5β)} or the nonpeptide antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide).

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)

  • Y. Sasai et al.

    Molecular characterization of rat substance K receptor and its mRNAs

    Biochem. Biophys. Res. Commun.

    (1989)
  • T.W. Schwartz

    Locating ligand-binding sites in 7TM receptors by protein engineering

    Curr. Opin. Biotechnol.

    (1994)
  • G. Turcatti et al.

    Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands

    J. Biol. Chem.

    (1997)
  • D. Aharony et al.

    Isolation and pharmacological characterization of a hamster neurokinin A receptor cDNA

    Mol. Pharmacol.

    (1994)
  • F. Bellucci et al.

    A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis

    Br. J. Pharmacol.

    (2003)
  • R.-M. Catalioto et al.

    MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist

    Br. J. Pharmacol.

    (1998)
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