NK1 receptor antagonist | EPFL Graph Search

DISPLAYTITLE:NK1 receptor antagonist Neurokinin 1 (NK1) antagonists (-pitants) are a novel class of medications that possesses unique antidepressant, anxiolytic, and antiemetic properties. NK-1 antagonists boost the efficacy of 5-HT3 antagonists to prevent nausea and vomiting. The discovery of neurokinin 1 (NK1) receptor antagonists was a turning point in the prevention of nausea and vomiting associated with cancer chemotherapy. An example of a drug in this class is aprepitant. Chemotherapy-induced emesis appears to consist of acute and delayed phases. So far, the acute phase emesis responds to 5-HT3 antagonists while the delayed phase remains difficult to control. The discovery and development of NK1 receptor antagonists have elicited antiemetic effect in both acute and especially in delayed phases of emesis. Casopitant, netupitant and rolapitant are some newer additions in this group. Rolapitant has a significantly longer half-life of 160 hours and was appro

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

Gerardo Turcatti

Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5- dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the N(alpha) position were synthesized and characterized. Peptides modified at the alpha-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18,000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl CP96,345 was much higher than for any of the dansyl labeled peptides indicating that the micro- environment of the binding site is more hydrophobic for the nonpeptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.

1997

Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes

Jonathan Knowles, Gerardo Turcatti, Horst Vogel

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that read-through at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodol. Fluorescence-labeled NK2 mutants contg. an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionally active. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topol. for G protein-coupled receptors and detd. a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helixes required for receptor function. [on SciFinder (R)]

1997

Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites

Jonathan Knowles, Gerardo Turcatti, Horst Vogel

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants contg. an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calcd. by the theory of Forster, permit to fix the ligand in space and define the structure of the receptor in a mol. model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of exptl. distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the anal. of spatial relationships in integral membrane proteins such as receptors or channels. [on SciFinder (R)]

1996

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

Gerardo Turcatti

1997