WO2009049791A2 - Radiolabeled nk3 receptor antagonist - Google Patents

Abstract

The present invention relates to a radiolabeled Neurokinin 3 receptor antagonist of the general formula (I) wherein R1 is a radiolabeled lower alkyl and a method for identifying a compound that can bind to a NK3R comprising a) contacting a compound of interest with a sample comprising the NK3R in the presence of the radiolabeled NK3R antagonist of any one of claims 1 to 3; and b) monitoring whether the compound of interest influences the binding of said radiolabeled NK3R antagonist to said NK3R. Furthermore, the present invention relates also to a mutant NK3R and uses thereof.

Description

RADIOLABELED NK3 RECEPTOR ANTAGONIST

The tachykinin family comprises the neuropeptides; substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). SP, NKA and NKB act as neurotransmitters and neuromodulators through three types of membrane receptors termed neurokinin 1 (NK₁), neurokinin 2 (NK₂) and Neurokinin 3 (NK₃) receptors, respectively. NK receptors are belonging to the class A family of G-protein coupled receptors (GPCRs) that couple via G_q/π to the activation of phospholipase C leading to phosphoinositide (PI) hydrolysis and elevation of intracellular Ca²⁺ levels. The tachykinins rank order of potency at the NK receptors: SP>NKA>NKB for the NK₁R, NKA>NKB>SP for the NK₂R and NKB>NKA>SP for the NK₃R. Among NK receptors, NK₃ receptors are of particular interest due to their brain distribution and their possible role in the pathophysiology of psychiatric disorders including schizophrenia {Spooren W., et al, Nat. Rev. DrugDis., 4, 967-975, 2005). Senktide, which is a synthetic peptide, has been found to be a highly selective and potent agonist at the NK3 receptor. Based on in situ hybridization histochemistry and NKB/senktide binding, the expression OfNK₃ receptors was detected in brain regions including cortex (frontal, parietal and cingulate cortex), various nuclei of the amygdala, the hippocampus, and in midbrain structures (the substantia nigra, ventral tegmental area, and raphe nuclei). SR- 142801 (osanetant) and SB 223412 (talnetant), which are from two distinct chemical classes, have been shown to be potent antagonists at the NK3 receptor as determined by means of Ca²⁺ mobilization studies with IC₅₀ values of ~6 and -16 nM, respectively. In a double blind placebo controlled clinical trial, osanetant was shown to be active in schizophrenia patients with improved efficacy and side effect profiles (Meltzer H. Y., et al., Am J Psych 161, 975-984, 2004).

Therefore, potent and selective NK3 receptor antagonists have recently attracted special attention as an alternative therapeutic for the treatment of psychiatric disorders such as schizophrenia over the current mainstay treatments of anti-psychotic drugs. The present invention provides a radiolabeled NK₃ antagonist of formula I

formula I

wherein Rl is a radiolabeled lower alkyl. Preferably, Rl is tritium-labeled methyl (-CT₃) or a methyl-group comprising a radioisotope of carbon. A preferred radioisotope of carbon is

[¹¹C] Or [¹⁴C]. The term "T" as used herein refers to a Tritium atom.

In the present description the term "alkyl", alone or in combination with other groups, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to twenty carbon atoms, preferably one to sixteen carbon atoms, more preferably one to ten carbon atoms. The term "lower alkyl" or "Cl-C7-alkyl", alone or in combination, signifies a straight- chain or branched-chain alkyl group with 1 to 7 carbon atoms, preferably a straight or branched- chain alkyl group with 1 to 6 carbon atoms and particularly preferred a straight or branched- chain alkyl group with 1 to 4 carbon atoms. Examples of straight-chain and branched C1-C7 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.-butyl, the isomeric pentyls, the isomeric hexyls and the isomeric heptyls, preferably methyl and ethyl and most preferred methyl.

The term "Me-talnetant" refer to the unlabeled compound of the formula I, wherein Rl is CH₃. The term "R-Me-talnetant" as used herein refers to the radiolabeled NK₃ receptor antagonist of the present invention.

The term "SB 223412" refers to the unlabeled compound of the formula I, wherein Rl is H. The term "R-SB223412" as used herein refers to the tritium or carbon radiolabeled NK₃ receptor antagonist of the present invention. The structure of the unlabeled compound SB 223412 has been disclosed in the European Patent No. EP 95920894.

hi addition, the present invention provides a process of synthesizing a radiolabeled NK₃ receptor antagonist hereinbefore described which comprises reacting a compound having the formula (II)

formula (II)

with a radiolabeled lower alkyl, for example [³H]CH₃I, in presence of a base such as for example caesium carbonate in a suitable solvent like e.g. toluene.

The present invention also relates to a radiolabeled NK₃receptor antagonist obtainable by the process hereinbefore described.

The present invention provides the use of a radiolabeled NK₃ receptor antagonist hereinbefore described for identifying a compound that may bind a NK₃ receptor. Moreover, the present invention provides a method for identifying a compound that can bind to a NK₃R comprising a) contacting a compound of interest with a sample comprising the Neurokinin 3 receptor in the presence of the radiolabeled NK₃R antagonist hereinbefore described, and b) monitoring whether the compound of interest influences the binding of said radiolabeled NK₃R antagonist to said NK₃R.

Preferably, the compound of interest is a NK₃R antagonist.

The sample comprising NK₃R may be a tissue sample, primary cell, cultured cells which either naturally express a NK₃R, or which either transiently or stably transfected with a NK₃R. Method of transfecting cells are well known in the art (Sambrook et al, Molecular Cloning: a Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, new York, USA). The sample also includes a membrane preparation of the cells indicated above. The tissue sample and -A- primary cells may be obtained from a human or non-human animal. The non-human animal may be a wildtype or a NK₃R transgenic animal. Methods of producing transgenic animals are well known in the art (see e.g. Suitable methods are i.e. in Hogan B., Beddington R., Costantini F. & Lacy E. Manipulating the mouse embryo. A laboratory manual. 2nd Edition (1994). Cold Spring Harbor Laboratory Press).

Monitoring whether the compound of interest influences the binding of said radiolabeled NK₃R antagonist to said NK₃R involves determining the amount of bound radiolabeled NK₃R antagonist hereinbefore described.

Preferably, the amount of bound radiolabeled NK₃ antagonist hereinbefore described is compared with a control. Said the control may be the amount of bound radiolabeled NK₃R antagonist hereinbefore described to the NK₃R in the sample without the compound of interest or another ligand; or in the presence of reference compound.

The separation of the receptor and with bound antagonist from free ligand is usually achieved by filtration and centrifugation.

To determine whether a compound of interest influences the binding of said radiolabeled

NK₃R antagonist to said NK₃R, the skilled person may use the term Ka (dissociation constant) to represent the affinity of a compound to the receptor. The K_d is based on the molar concentration of a drug occupying 50% of the receptor population. So the lower the IQ, the higher is the "affinity" or stickiness of the ligand for its receptor.

The specificity of the binding of the radiolabeled may be determined by methods well known to the skilled in the art. In principle, unlabeled (cold) ligand is added in access to the receptor and the radiolabeled ligand. The unlabeled ligand competes with the radiolabeled ligand for the binding sites. In these conditions the labeled ligand is completely displaced from recognition sites by the cold ligand. However, the non-specific binding of the radiolabeled ligand is not altered by the large amount of unlabeled ligand because the unspecifϊc binding is not saturable.

Furthermore, the present invention relates to the use of a radiolabeled NK₃ receptor antagonist hereinbefore described for identifying the activity of a compound as a NK₃R.

Further to this, the present invention pertains to a method of identifying a cellular receptor expressed in a host as a Neurokinin 3 receptor comprising contacting the cellular receptor with the radiolabeled NK₃R antagonist hereinbefore described and determining whether binding of said radiolabeled NK₃R antagonist has occurred. The host may be a tissue, a primary cell or a cultured cell suspected of expressing a Neurokinin 3 Receptor.

Methods for determining the binding of the radiolabeled NK₃R antagonist hereinbefore described are well known to the skilled person in the art. Suitable methods are for example described in (T. P. Kenakin, A pharmacology primer: Theory, Applications, and Methods, 2^nd edition (2006) Academic press).

Preferably, said compound is used as radiolabel in binding assays, in particular for identifying NK₃ receptor antagonists. Binding assays are well known to the skilled person in the art and include but are not limited to competitive binding assays and saturation binding assays.

The NK3 receptor used in the hereinbefore described methods can be a wildtype receptor (SEQ. ID NO: 1), or the receptor may comprise mutations.

Preferably, the mutant NK₃ receptor comprises one of the following mutations:

^■ an alanine instead of an asparagine at position 142 of SEQ ID NO:1 (N142A),

" a methionine instead of a valine at position 169 of SEQ. ID NO: 1 (V169M),

^■ a phenylalanine instead of a tyrosine at position 315 of SEQ. ED NO: 1 (Y315F),

^■ a methionine instead of a phenylalanine at position 342 of SEQ. ED NO: 1 (F342M),

^■ an isoleucine instead of a serine at position 341 of SEQ. ED NO: 1 and a methionine instead of a phenylalanine at position 342 of SEQ. ED NO: 1 (S341I/F342M),

■ an alanine instead of a valine at position 95 of SEQ. ED NO: 1 (V95 A), or

^■ an isoleucine instead of a valine at position 95 of SEQ. ED NO: 1 and a serine instead of an alanine at position 99 of SEQ. ED NO: 1 (V95I/A99S), , or

^■ an alanine instead of a methionine at position 346 of SEQ. ED NO:1 (M346A)

Osanetant and Talnetant, two structurally different NK3R antagonists, have a different mode of action (see Beaujouan et al., Eur J Pharmacol. (1997) 319,307-316; Tain et al, MoI Pharmacol, (2007) 71(3)902-911). For the development of drugs it is interesting to determine whether a potential drug candidate (NK₃R antagonist) has the mode of action and binding pocket, respectively, like osanetant ("osanetant-like") or like talentant ("talnetant-like"). The NK₃ receptors proteins comprising V95A, N142A, Y315F and/or M346A mutation may be used to differentiate between the binding pockets of "osanetant-like" and "Me-talnetant-like" compounds. In addition, the NK₃ receptors proteins comprising V95A, Nl 42 A, Y315F and/or M346A mutation may also be used for the alignment of ligand-based modelling of compounds and /or refinement of such models, especially in terms of orientation.

The NK₃ receptor proteins comprising F342M, S341I/F342M, Y315F and/or V95I/A99S mutations may be used for testing the selectivity of a NK₃R ligand towards Neurokinin- 1 receptor (NK|R). Therefore, the current application also pertains a method for determining the selectivity of a NK₃R ligand towards Neurokinin- 1 receptor comprising a) contacting a NK3 ligand with a sample comprising the Neurokinin 3 receptor comprising F342M, S341I/F342M, Y315F and/or V95I/A99S in the presence of the radiolabeled NK₃R antagonist hereinbefore described, b) monitoring whether the compound of interest influences the binding of said radiolabeled NK₃R antagonist to said NK₃R. A NK₃R ligand having a cross reactivity with a NKiR reduces the amount of bound radiolabeled NK₃R antagonist hereinbefore described. The term "reduces the amount" means that the amount is significant smaller than the amount measured in a control. The control is the crossactivity determination method described above wherein the NK₃R ligand has no crossactivity with NKiR. Significantly smaller means that the difference of the measured amount to the control amount is statistically relevant (p < 0.05, preferably, p < 0.01).

NK₃R proteins comprising a V169M mutation are useful for testing the selectivity of a NK₃R ligand towards Neurokinin-2 receptors (NK₂R).

Therefore, the current application also pertains a method for determining the selectivity of a NK₃R ligand towards Neurokinin-2 receptor comprising a) contacting a NK₃ ligand with a sample comprising the Neurokinin 3 receptor with a point mutation V169M in the presence of the radiolabeled NK₃R antagonist hereinbefore described, b) monitoring whether the compound of interest influences the binding of said radiolabeled NK₃R antagonist to said NK₃R.

A NK₃R ligand having a high cross reactivity with a NK₂R reduces the amount of bound radiolabeled NK₃R antagonist hereinbefore described. The term "reduces the amount" means that the amount is significant smaller than the amount measured in a control. The control is the crossactivity determination method described above wherein the NK₃R ligand has no crossactivity with NK₂R. Significantly smaller means that the difference of the measured amount to the control amount is statistically relevant (p < 0.05, preferably, p < 0.01). Furthermore, the present invention provides mutant NK₃R comprising

^■ an alanine or a leucine at position 95 of SEQ. ID NO:1 instead of a valine (V95A, V95L)

^■ an isoleucine instead of a valine at position 95 of SEQ. ID NO: 1 and a serine instead of an alanine at position 99 of SEQ. ID NO: 1 (V95I/A99S)

^■ an alanine instead of methionine at position 134 of SEQ. ID NO: 1 (M134A)

^■ an alanine instead of asparagine at position 138 of SEQ. ID NO: 1 (N138A)

^■ an alanine instead of threonine at position 139 of SEQ. ID NO: 1 (Tl 39A)

^■ an leucine instead of a valine at position 95 of in SEQ. ID NO: 1 and an threonine instead of an alanine at position 139 of in SEQ. ID NO: 1 (V95L/T139A)

^■ an alanine instead of an asparagine at position 142 of SEQ ID NO:1 (N142A),

^■ a methionine instead of a valine at position 169 of SEQ. ID NO: 1 (V169M),

^■ an alanine instead of a leucine at position 232 of SEQ. ID NO: 1 (L232A)

^■ a phenylalanine instead of a tyrosine at position 315 of SEQ. ID NO: 1 (Y315F),

" an alanine instead of a serine at position 341 of SEQ. ID NO: 1 (S341A);

^■ an alanine or methionine instead of a phenylalanine at position 342 of SEQ. FD NO: 1

(F342A/M),

^■ an isoleucine instead of a serine at position 341 and an methionine instead of a phenylalanine at position 342 of SEQ. ID NO: 1 (S341I/F342M)

" an alanine instead of a methionine at position 346 of SEQ. ID NO: 1 (M346A), or

^■ an alanine instead of a serine at position 348 of SEQ. ID NO: 1 (S348A).

The present invention also pertains to the radiolabeled ligand, compounds, methods, process and uses substantially as hereinbefore described, especially with reference to the following examples. Figures

Figure 1 shows the chemical structure of A) [³H]Me-talnetant (T=Tritium), B) SB 223412 (tanetant), C) SB 222200 and D) SR142801 (osanetant).

Figure 2 shows a graphical representation of the saturation bindings of A) [³H]osanetant and B) [³H]Me-talnetant to membranes from HEK293-EBNA cell transfected transiently with hNK₃ receptor.

On the y-axis is the amount of bound radiolabel in pmol per mg protein and on the x-axis is the amount of free radiolabel. Each data point is mean ± SEM (bars) of 3 individual experiments performed in triplicate. The data were analyzed by nonlinear regression analysis using GraphPad Prism 4.0 software and a single-site binding model.

Figure 3 shows an Alignment of the amino acids forming the binding site OfNK₃Rs. The first row gives the Ballesteros-Weinstein numbering scheme (Ballesteros and Weinstein, (1995) Integrated methods for construction three-dimensional models and computational probing of structure- function relations in G protein-coupled receptors. Methods Neurosci. 25:366-428). The numbers above the NK3R HUMAN receptor gives the sequence number of the positions of the mutations carried out in this study. The amino acid sequences of the human NK₃ (accession number: P29371), rat NK₃ (accession number: P16177), mouse NK₃ (accession number: P47937), gerbil NK₃ (accession number: AM157740), human NKi (accession number: P25103) and human NK₂ (accession number: P21452) receptors were retrieved form the Swiss-Prot database (Apweiler et al., (2004) UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 32:D115-119).

Figure 4 shows a graphical representation of competition binding experiments with wildtype and mutant NK₃R. Radioisotope labeled ligand is [³H]Me-talnetant ([³H]Me-tal). The binding analysis were done with membranes isolated from HEK293-EBNA cells transiently expressing WT and mutated NK₃ receptors. The [³H]Me-talnetant at a concentration equal to its

K_d value was used in these competition binding experiments. Each data point is mean ± SEM

(bars) of 3 individual experiments performed in duplicate.

A) unlabeled ligand: [MePhe⁷]NKB; Receptor: wildtype (WT) or mutant NK₃R comprising

V95A, V95L, V95I/A99S or N138A. B) Unlabeled ligand: osanetant, wildtype or mutant NK₃R comprising V95A, V95L, V95I/A99S or Nl 38 A.

C) unlabeled ligand: [MePhe⁷]NKB; Receptor: wildtype (WT) or mutant NK₃R comprising T139A, V95L/T139A, N142A or L232A

D) unlabeled ligand: osanetant; Receptor: wildtype or mutant NK₃R comprising T139A, V95L/T139A, N142A or L232A E) unlabeled ligand:[MePhe⁷]NKB; Receptor: wildtype or mutant NK₃R comprising Y315A, S341A, M346A or S348A

F) unlabeled ligand: osanetant; Receptor: wildtype or mutant NK₃R comprising Y315A, S341 A, M346A or S348A.

Figure 5 A, 5C. 5E and 5F show a Dose-response curve (DRCs) for [³H]IP formation stimulated by [MePhe⁷]NKB in the absence and presence of various concentrations of Me-talnetant (A) and osanetant (C, E, F) in CHO cells expressing transiently the hNK₃R WT, T139A and M346A. Figures 5B and 5D show a graphical representation of Schild plot analyses for antagonism of [MePhe⁷]NKB-induced accumulation of [³H]IP by Me-talnetant and osanetant. Schild plots for antagonism by Me-talnetant (B) and osanetant (D).

hNK3-WT: human wildtype NK3 receptor, Me-tal: Me-talnetant; os: Osanetant; hNK3R- T139A: human NK3 receptor comprising T139A mutation, hNK3R-M346A: : human NK3 receptor comprising M346A mutation

The EC₅₀ and EC₅₀' values, which derived from NKB DRCs in the absence and presence of increasing fixed concentrations of Me-talnetant or osanetant (panels A and C), were used to calculate the dose ratios (DR = EC_5O'/EC_5O) and plotted according to Schild regression in panels B and D. Each curve represents the mean of 8 dose-response measurements from a minimum of two independent transfections.

Figure 6 shows a graphical representation of a time course for the association (A) and dissociation (B) of [³H] Me-talnetant ([³H]Me-tal) binding to hNK₃R WT membrane and [³H]osanetant ([³H]os) binding to membranes comprising the hNK₃R wildtype or mutant hNK3R with T139A and M346A.

Each data point is mean ± SEM (bars) of three individual experiments performed in quadruplet.

Figure 7 Proposed docking modes of Me-talnetant ES and osanetant B . Shown are only the residues that were mutated in this study. Indicated are those mutations (italic letters) that either led to a complete loss of affinity for both osanetant and Me-talnetant ( ) or that influenced mainly only one of the two antagonists ( : influence on osanetant (partial loss of osanetant affinity, no influence on Me-talnetant , : influence on Me-talnetant (partial loss of

Me-talnetant affinity, no or smaller influence on osanetant). Examples

Compounds

SRl 42801 (osanetant) : (S)-(N)-( 1 -(3-( 1 -benzoyl-3 -(3 ,4-dichlorophenyl)piρeridin-3 - yl)propyl)-4-phenylpiperidin-4-yl)-N-methyl acetamide) (Emonds-Alt X., et al. SR142801, the first potent non-peptide antagonist of the tachykinin NK3 receptor. Life Sci. 56, PL 27-32 (1995).);

[³H]SR142801 ([³H]Osanetant), Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, was purchased from Amersham, GE Healthcare UK limited (Buckinghamshire, UK).

Talnetant (SB223412, (5)-(-)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4- carboxamide) (Sarau H.M. et al. Nonpeptide tachykinin receptor antagonists: I. Pharmacological and pharmacokinetic characterization of SB223412, a novel, potent and selective neurokinin-3 receptor antagonist. J. Pharm. Exp. Ther. 281, 1303-1311 (1997))

SB222200: [(S)-(-)-N-(α-ethylbenzyl)-3-methyl-2-phenlquinoline-4-carboxamide] (Giardina, Giuseppe A. M. et al. 2-Phenyl-4-quinolinecarboxamides: A Novel Class of Potent and Selective Non-Pep tide Competitive Antagonists for the Human Neurokinin-3 Receptor. Journal of Medicinal Chemistry (1996), 39(12), 2281-2284.)

Me-talnetant ((5)-(-)-N-(α-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide); (Giardina, Giuseppe A. M. et al.; Discovery of a Novel Class of Selective Non-Peptide Antagonists for the Human Neurokinin-3 Receptor. 2. Identification of (S)-N-(I -Phenylpropyl)- 3-hydroxy-2- phenylquinoline-4-carboxamide (SB 223412). Journal of Medicinal Chemistry (1999), 42(6), 1053-1065 )

[³H] -Me-talnetant (tritiumlabeled Methoxy-Talnetant): specific activity: 84.6 Ci/mmol)

[MePhe⁷]Neurokinin B (Asp-Met-His-Asp-Phe-Phe-NMe-Phe-Gly-Leu-Met-NH₂, Catalog No. SC981) was purchased from NeoMPS SA (Strasbourg, France). Example 1: Generation of tritiumlabeled Me-talnetant

C25H22N2O2 C26H24N2O2 382.47mg/mmol 402.47mg/mmol

Procedure:

A flask was treated with 0.5 ml of dichlorodimethylsilane, heated with a flame dryer and washed twice with 1 ml of THF prior to the reaction. Then 4.6 mg of RO3301510-000 (0.012 mmol), 1.0 ml of acetone, and 14.8 mg of cesium carbonate (0.046 mmol) were added. The reaction mixture was evacuated under liquid nitrogen, then a solution of 0.64 μlof [³H]-MeI (875 mCi, 0.010 mmol) in toluene was condensed to the reaction flask via vacuum transfer technique.

The reaction mixture was stirred at 50 ⁰C for 2.75 hours and at room temperature for 13 h. The reaction flask was evacuated under liquid nitrogen and then the solvent was removed by lyophilization. The residue was treated with 1 ml of methanol and evacuated again. The resulting residue was dissolved in 1 ml of methanol. After filtration of the methanolic solution through a nylon filter and evaporation of the solvent, 90 mCi of crude product was obtained. Purification:

HPLC: column: XTerra RP-8, 5 urn, 7.8x150 mm, mobile phase: water/MeCN/TFA 45:55:0.1 flow: 4.0 ml/min temperature: 23 °C sample: 300 mCi/ml in MeOH injection: 60 μl, 5 runs detection: UV, 254 ran

Retention time: t_r: 3.6 min

After HPLC purification 60 mCi of SB22412-001 was obtained in a radiochemical purity of >98 % (overall yield of 6.9 %).

Analysis:

HPLC: column: Symmetry C8, 250x4.6 mm, 5 μm mobile phase: [A]: 10mM KH₂PO₄-buffer pH=6.5; [B]: acetonitrile, [C]: 5%[B] in water; gradient: 00.0 min : 20% [A], 40% [B], 40% [C] 30.0 min : 20% [A], 80% [B], 00% [C] flow: 1.0 ml/min temperature: 40 °C detector: DAD, 254 nm

retention time: t_r (product): 19.41 min t_r (starting material): 13.75 min

Specific activity (by MS analysis): 84.7 Ci/mmol Example 2:

2.1 experimental procedures

2.1.1 Construction of point-mutated hNK₃ receptors. cDNA encoding the human NK₃ receptor (SW: P29371) was subcloned into pCI-Neo expression vectors (Promega Corporation, Madison, WI). All point-mutants were constructed using the QuikChange™ site-directed mutagenesis kit (Cat.#200518, Stratagene, La Jolla, CA) according to the manufacturer's instructions and using pCI-Neo-hNK₃R as a DNA template. Complementary oligonucleotide primers (sense and antisense) containing the single site or double sites of mutations were synthesized by Microsynth AG (Balgach, Switzerland). The following PCR conditions were used for repeated extensions of the plasmid template: 95°C for 1 min and 20 cycles of 95°C for 30 s, 55°C for 1 min and 68°C for 8 min using 50 ng plasmid DNA, 100 ng each of primers and 2.5 units Pfu Turbo DNA polymerase (Stratagene). The entire coding regions of all positive point-mutants were sequenced from both strands using an automated cycle sequencer (Applied

2.1.2 Cell culture, large-scale transient transfection, and membrane preparation.

Human embryonic kidney (HEK)293-EBNA cells (Invitrogen, Carlsbad, CA) were adapted to grow in suspension in spinner flask at 95-105 rpm. For transfection experiments, HL media (DHI special, Invitrogen) (Schumppand Schlaeger, J Cell Sci 97, 639-647, 1990; Schlaeger, J Immunol Methods 194, 191-199, 1996) w/o heparin and for the gene delivery, the transfection reagent X-tremeGENEQ₂ (Cat. No. 03045595001, Roche Applied Science, RAS, Rotkreuz, Switzerland) which consists of substances A and B, were used.Cells were cultured to a density of 6-10 X 10⁵ cells/ml, centrifuged for 3 min at 600 rpm and resuspended HL media ( w/o heparin). The cell density was adjusted to 5 x 10⁵ cells/ml, and the culture was incubated for at least 3 h before transfection. The transfection complexes were generated in 1/10 of the culture volume in HL media w/o heparin at room temperature. For 1 ml culture, first 0.4 μg DNA was added to 0.1 ml medium, mixed, after 2 min followed by 0.15 μg X-tremeGENEQ A (Roche Applied Science, RAS, Rotkreuz, Switzerland), mixed and followed after further 2 min by 0.5 μg X-tremeGENEQ B (Roche Applied Science, RAS, Rotkreuz, Switzerland). The mixture was incubated for 15 min at room temperature to allow DNA complex formation before it was added to the cells. 48 h post-transfection, the cells were harvested and washed three times with cold PBS and frozen at -80°C. The pellet was suspended in ice-cold 50 mM Tris pH 7.4 buffer containing 10 mM EDTA (10 X volume) and homogenized with a polytron (Kinematica AG, Basel, Switzerland) for 30 s at 16 000 rpm. After centrifugation at 48 000 x g for 30 min at 4°C, the pellet was suspended again in ice-cold 10 mM Tris pH 7.4 buffer containing 0.1 mM EDTA (10 x volume), homogenized and spun again as above. The pellet was resuspended in ice-cold 10 mM Tris pH 7.4 buffer containing 0.1 mM EDTA and 10% sucrose (5 x volume). After homogenization for 15 s at 16,000 rpm, the protein content was measured using the BCA method (Pierce, Socochim, Lausanne, Switzerland) with bovine serum albumin as the standard. The membrane homogenate was frozen at -80°C before use.

2.1.3 [³H]Me-talnetant and [³H]osanetant bindings.

After thawing, the membrane homogenates were centrifuged at 48,000 x g for 10 min at 4°C, the pellets were resuspended in the binding buffer (50 mM Tris-HCl, 4 mM MnCl₂, 1 μM phosphoramidon, 0.1% BSA at pH 7.4) to a final assay concentration of 5 μg protein/well. Saturation isotherms were determined by addition of various concentrations of [³H]Me-talnetant (0.005 to 10 nM) or [³H]osanetant (.009 to 3 nM) to these membranes (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI + 0.3% BSA, Packard BioScience, Meriden, CT) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer.

Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zurich, Switzerland) and shaking for 1 h. Saturation experiments were analyzed by Prism 4.0 (GraphPad software, San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B = (B_max * [F])/(K_D + [F]), where B is the amount of ligand bound at equilibrium, B_n^_x is the maximum number of binding sites, [F] is the concentration of free ligand and K_d is the ligand dissociation constant. For inhibition experiments, membranes were incubated with [³H]Me-talnetant or [³H]osanetant at a concentration equal to K_<j value of radioligand and ten concentrations of the inhibitory compound (0.0003-10 μM). IC₅₀ values were derived from the inhibition curve and the affinity constant (K₁) values were calculated using the Cheng-Prussoff equation K₁ = IC₅o/(l+[L]/K_d) where [L] is the concentration of radioligand and K_d is its dissociation constant at the receptor, derived from the saturation isotherm. To measure association kinetics, membranes were incubated at RT (22 ⁰C) in the presence of radioligand (~ InM [³H]Me- talnetant or ~ 0.25 nM [³H]osanetant) for 0, 1, 3, 5, 7, 10, 15, 20, 30, 60, 90 or 120 min, then terminated by rapid filtration. Dissociation kinetics were measured by adding at different times before filtration, 10 μM SB222200 to membranes preincubated at RT for 30 min in the presence of- InM [³H]Me-talnetant or for 60 min in the presence of- 0.25 nM [³H]osanetant. Binding kinetics parameters, K_ob and K^ values (observed on and off rates) were derived from association-dissociation curves using the one phase exponential association and decay equations (Prism 4.0, GraphPad software), respectively. K_0n, half-life and K_d were calculated using the K_on=(K_0b-K_ofτ)/[ligand], ti_/2=ln2/K and K_d=K_0HZK_0n equations, respectively. Statistical significance was determined using the Two-tailed t-test (Prism 4.0, GraphPad software).

2.1.4 [³H]Inositol phosphates (IP) accumulation assay.

[³H]Inositol phosphates accumulation was measured as described previously (Malherbe P, Kratochwil N, Muhlemann A, Zenner MT, Fischer C, Stahl M, Gerber PR, Jaeschke G and Porter RH (2006) Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP. J Neurochem. 98:601-615) with the following adaptations. The Chinese hamster ovary (CHO) was maintained in DMEM:F12/ISCOVE supplemented with 5% dialyzed fetal calf serum, 100 μg/mL Penicillin/Streptomycin. The CHO cell was transfected with the wildtype (WT) or mutant hNK₃ receptor cDNAs in pCl-Neo using Lipofectamine Plus™ reagent (Invitrogen) according to the manufacturer's instruction. 24 h post-transfection, cells were washed twice in labeling medium: DMEM w/o inositol (ICN 1642954), 10% FCS, 1% Pen/Strep, 2mM Glutamate. Cells were seeded at 8x10⁴ cells/well in poly-D-lysine-treated 96- well plates in the labeling medium supplemented with 5 μCi/mL of myo-[l,2-³H]-inositol (Amersham Biosciences TRK911, specific activity: 16.0 Ci/mmol). On the day of assay (48 h post-transfection), cells were washed three times with the buffer (I X HBSS, 20 mM HEPES, pH 7.4) prior to the addition of agonists or antagonists in assay buffer (1 X HBSS, 20 mM HEPES, pH 7.4 containing 8 mM LiCl, final concentration, to prevent phosphotidyl-inositide break- down). When present, antagonists were incubated for 5 min at RT prior to stimulation with agonist [MePhe⁷]NKB, concentrations ranging from 10 μM- 0.1 nM. After 45 min incubation at 37°C with [MePhe⁷]NKB, the assay was terminated by the aspiration of the assay buffer and the addition of 100 μL 20 mM formic acid to the cells. After shaken for 30 min at RT, a 20μL aliquot was mixed with 80μl of yttrium silicate beads (Amersham Biosciences RPNQ0013, 12.5 mg/ml) that bind to the inositol phosphates (but not inositol) and shaken for 30 min at RT. Assay plates were centrifuged for 2 min at 2500 rpm prior to counting on a Packard Top-count microplate scintillation counter with quenching correction (Canberra Packard SA). The relative efficacy (E_m3x) values of [MePhe⁷]NKB was calculated as fitted maximum of the dose-response curve of each mutated receptors expressed as a percentage of fitted maximum of the wild-type dose-response curve from cells transfected and assayed on the same day.

2.1.6 Residue numbering scheme.

The position of each amino acid residue in the seven transmembrane domain (7TMD) was identified both by its sequence number and by its generic numbering system proposed by Ballesteros and Weinstein (Ballesteros and Weinstein, (1995) Integrated methods for construction three-dimensional models and computational probing of structure- function relations in G protein-coupled receptors. Methods Neurosci. 25:366-428) which is shown as superscript. In this numbering system, amino acid residues in the 7TMD are given two numbers: the first refers to the TMD number; the second indicates its position relative to a highly conserved residue of family 1 GPCRs in that TM which is arbitrarily to 50. The amino acids in the extracellular loop EC2 are labeled 45 to indicate their location between the helix 4 and 5. The highly conserved cysteine thought to be disulfide bonded, was given the index number 45.50 and the residues within the EC2 loop are then indexed relative to the "50" position.

2.1.7 Alignment and Model building.

The amino acid sequences of the human NK₃ (accession number: P29371), rat NK₃ (accession number: P16177), mouse NK₃ (accession number: P47937), human NKi accession number: P25103) and human NK₂ (accession number: P21452) receptors were retrieved form the Swiss-Prot database (Apweiler et al., (2004) UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 32:D115-119). The sequence of the gerbil NK₃ receptor was solved internally. These amino acid sequences were aligned to the sequence of bovine rhodopsin (accession number: P02699) using the ClustalW multiple alignment program (Thompson et al., (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680). A slow pairwise alignment using the BLOSUM matrix series (Henikoff and Henikoff, (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA. 89:10915-10919) and a gap opening penalty of 15.0 were chosen for aligning the amino acid sequences. Other parameters were those given as default. The sequences were aligned in two steps: (i) from the N-terminus to the first 5 residues of the third intracellular loop 13, (ii) from the last 5 residues of the 13 loop to the C-terminus. The 13 loop was excluded from the alignment since it shows too high variability in amino acid composition and length. The alignments were then verified to ensure that conserved residues of the transmembrane regions were aligned and manually adjusted in the second extracellular loop (E2) in order to align the conserved cysteine which takes part in the disulfide bridges occurring between the third transmembrane segment (TM3) and the second extracellular loop (E2).

Using this alignment and the X-ray structure of bovine rhodopsin (Palczewski et al., (2000) Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 289:739-345) as template, the software package MOE (MOE v.2005.05, Chemical Computing Group, Montreal, Quebec, Canada) was used to generate a three-dimensional model of the human NK₃ receptor. 10 intermediate models were generated and the best one selected as final MOE model. No minimization was used in order to keep the backbone coordinates as in the X-ray structure. After the heavy atoms were modeled, all hydrogen atoms were added in appropriate locations with the preparatory program PROTONATE of AMBER6 (AMBER 6.0. University of California, San Francisco). Osanetant was then manually docked into the transmembrane cavity of the human NK₃ model. The docking mode was based on the following hypotheses: 1) The ligand should make a direct interaction with M134 (2.53) since this residue has been shown to be responsible for species selectivity of SR48968 (Wu et al., (1994) Identification of methionine 134 and alanine 146 in the second transmembrane segment of the human tachykinin NK3 receptor as reduces involved in species-selective binding to SR 48968. Biochem Biophys Res Commun. 198:961-966). 2) Phenyl-piperidine substructures are privileged fragments for the subpocket formed by the transmembrane domains 3, 5 and 6. The resulting protein-ligand complex was then minimized using AMBER6. The minimization was carried out by 5000 steps of steepest descent followed by conjugate gradient minimization until the rms (root mean square) gradient of the potential energy was less than 0.05 kcal/mol A. A twin cut-off (10.0, 15.0 A) was used to calculate non-bonded electrostatic interactions at every minimization step and the non-bonded pair-list was update every 25 steps. A distance-dependent (ε=4r) dielectric function was used. Removing the ligand from the complex yielded the final coordinates of the human NK₃R model. Me-talnetant was then manually docked into the receptor. The proposed docking mode is based on the SAR of Talnetant according to which at position 3 similar side chains as used in the osanetant series can be added. The 3-methoxy group of Me-talnetant has thus to point into the direction of the subpocket TM 3, 5 and 6.

14 single point and 3 double mutations of residues surrounding Me-talnetant and osanetant were chosen for binding and displacement studies to get information about the different residues involved in Me-talnetant and osanetant binding and their selectivity toward ItNK₁ and hNK₂ receptors.

2.2 Results

2.2.1 [³H] Me-talnetant and [³H]osanetant binding and displacement studies.

Talnetant and osanetant have been extensively characterized by in vitro pharmacology; both antagonists potently inhibited [¹²⁵I]iodohistidyl-[MePhe⁷]NKB binding at hNK₃R expressed in CHO cells with k, values of 1.0 and 0.2 nM, respectively (Emonds-Alt et al., (1995) SR 142801, the first potent non-peptide antagonist of the tachykinin NK3 receptor. Life Sci. 56:PL27-32; Giardina et al., (1999) Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 2. Identification of (S)-N-(I -phenylpropyl)-3- hydroxy-2-phenylquinoline-4-carboxamide (SB 223412). J Med Chem. 42: 1053-1065; Langlois et al., (2001) Detailed distribution of Neurokinin 3 receptors in the rat, guinea pig and gerbil brain: a comparative autoradiographic study. Neuropharmacology. 40:242-253; Sarau et al., (1997) Nonpeptide tachykinin receptor antagonists: I. Pharmacological and pharmacokinetic characterization of SB 223412, a novel, potent and selective neurokinin-3 receptor antagonist. J Pharmacol Exp Ther. 281:1303-1311). The structures of talnetant and its CNS penetrant analogue, SB222200 (Sarau et al., (2000) Nonpeptide tachykinin receptor antagonists. II. Pharmacological and pharmacokinetic profile of SB-222200, a central nervous system penetrant, potent and selective NK-3 receptor antagonist. J Pharmacol Exp Ther. 295:373-381) belong to a chemical class different from osanetant (Fig. 1). Although the radioligand [³H]SB222200 is available from Amersham, GE Healthcare, our in-house binding studies showed that [³H]SB222200 is not a suitable radioligand for in vitro binding due to low specific binding (< 20%) in hNK₃R transfected cell membranes, in comparison with [³H]SR142801 ([³H]osanetant) which gave an excellent signal to noise ratio, almost 97.5% specific binding in hNK₃R cell membranes.

To characterize the in vitro binding of [³H]Me-talnetant (Fig.lA)and [³H]osanetant, saturation binding analyses were performed at binding equilibrium (75 min incubation at room temperature), as outlined in "Experimental Procedures" on membranes isolated from the HEK293-EBNA transiently transfected with the hNK₃R. The saturation isotherm was monophasic ([³H]Me-talnetant concentrations 0.005-10 nM and [³H]osanetant 0.009-3 nM) and best fitted to a one-site model for both radioligands (Fig. 2). As seen in Fig. 2, both [³H]Me- talnetant and [³H]osanetant bind to a single saturable site on recombinantly expressed human NK₃ receptor (B_max of 34.3 and 26.0 pmol/mg protein, respectively) with high affinity (K_d of 0.8 and 0.2 nM, respectively). At the K_d values, the nonspecific binding for [³H]Me-talnetant and [³H]osanetant was approximately 6.9% and 2.5% of total bound radioactivity for both radioligands, respectively. Osanetant and SB222200 were able to displace the [³H]Me-talnetant binding from hNK₃R membrane with K₁ values of 1.0 ± 0.3 nM and 5.4 ± 0.5 nM; Hill values of 1.1 ± 0.2 and 0.9 ± 0.0, respectively and vice versa, Me-talnetant and SB222200 fully displace [³H]osanetant from hNK₃R membranes with K₁ values of 5.7 ± 0.3 and 8.7 ± 0.6 nM; Hill values 1.2 ± 0.1 , 1.1 ± 0.1 , respectively. Therefore, Me-talnetant shares a common binding pocket in the transmembrane region of the receptor, at least overlapping with that of osanetant.

2.2.2 Alignment of 7TM domains of the NK receptors towards rhodopsin and generation of human NK₃ point-mutated receptors.

To elucidate the binding modes of Me-talnetant and osanetant, an alignment of the seven transmembrane helices of the whole NK family towards the transmembrane helices of bovine rhodopsin (pdb ref code If88) was made. The inverse agonist of rhodopsin, cis-retinal, was employed as a template for the locations of Me-talnetant and osanetant. Amino acids, which were found 6.0 A away from retinal in the X-ray crystal structure of rhodopsin (Palczewski et al., (2000) Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 289:739-345), were considered as likely candidates to affect bindings of Me-talnetant and osanetant. Since it was previously reported that the residue M134^{2 53} of hNK₃R is responsible for species selectivity of SR48968 (Wu et al., (1994) Biochem Biophys Res Commun. 198:961-966), this information also guided us initially in docking of osanetant. The alignment of these amino acids of the NK family toward rhodopsin is shown in Fig. 3. From this preselection, amino acids in the TM 1, 2, 3, 6, 7 and EC2 regions were chosen for mutational studies (Fig. 3, bold). As outlined in "Experimental Procedures", 14 point-mutations and 3 double mutations in hNK₃R were introduced in the 7TMD region by site-directed mutagenesis.

2.2.3 Comparison of binding properties of [³H]Me-talnetant and [³H]osanetant to wild-type and mutated hNK₃ receptors.

Saturation binding analyses of [³H]Me-talnetant and [³H]osanetant were performed on membranes isolated from the HEK293-EBNA transfected with the wild-type (WT) and mutated receptors. The dissociation constants (K_d) and the maximum binding sites (B_m3x) derived from the saturation isotherms are given in Table 1. The mutations V95L, T139A, V95L / T 139 A, L232A, S341A and S348A did not significantly affect the binding affinity of both [³H]Me- talnetant and [³H]osanetant compared to the WT hNK₃ receptor (Table 1). Four mutations, M134A, V169M, F342M and S341I / F342M abolished both [³H]Me-talnetant and [³H]osanetant bindings to undetectable levels (Table 1). While the mutation N 142 A completely abolished [³H]osanetant binding, the same mutation led to an increase in binding affinity of [³H]Me- talnetant by 8-fold that was statistically significant (P = 0.0047, Two-tailed t-test). The mutation F342A that abolished [³H]Me-talnetant binding affinity led to statistically significant 17-fold decrease in binding affinity of [³H]osanetant (P = 0.009, Two-tailed t-test). The mutation Y315F, which had no effect on [³H]Me-talnetant binding affinity, led to 9-fold decrease in binding affinity of [³H]osanetant with high statistical significance (P = 0.0007, Two-tailed t-test). The binding affinities of [³H]Me-talnetant and [³H]osanetant were decreased by 4.0- and 2.3-fold by mutation V95A, respectively, and were statistically significant (P = 0.0002 and = 0.02, Two- tailed t-test). The double mutation V95I / A99S resulted in decrease in binding affinities of

[³H]Me-talnetant and [³H]osanetant by 3.0- and 3.5-fold, which were statistically significant (P = 0.003 and = 0.009, Two-tailed t-test), respectively. The mutation N138A led to decreases in binding affinities of [³H]Me-talnetant and [³H]osanetant by 3.5- and 3.8-fold that were statistically significant (P = 0.004 and = 0.01, Two-tailed t-test). The mutation M346A, which had no effect on [³H]osanetant binding affinity, led to statistically significant 2.5-fold decrease in binding affinity of [³H]Me-talnetant (P = 0.004, Two-tailed t-test).

2.2.4 Effect of mutations on the displacement of [³H]Me-talnetant by [MePhe⁷]NKB, osanetant or SB222200.

The mutations, which had no effect on or partially affected the [³H]Me-talnetant binding affinity, were chosen further for the competition binding studies with [MePhe⁷]NKB, osanetant and SB222200. Table 2 summarizes the affinity constant (K₁) and Hill slope (n_H) values for the [³H]Me-talnetant displacement by [MePhe⁷]NKB, osanetant or SB222200 on HEK293-EBNA cell membranes expressing 10 point-mutated and 2 double-mutated hNK₃ receptors. In the competitive inhibition of [³H]Me-talnetant binding by [MePhe⁷]NKB, the mutants Nl 38 A, L232A, Y315A and M346A showed 59.4-, 10.0-, 6.2- and 14.1-fold, respectively, lower affinity for [MePhe⁷]NKB than the WT (Fig.4A, C, E & Table 2). The mutation N 142 A resulted in complete loss of [MePhe⁷]NKB binding affinity (Fig. 4C & table 2). The mutations V95 A, V95L, V95I / A99S, T139A, V95L / T139A, S341 A and S348A had no effect on the competition binding by [MePhe⁷]NKB (Fig.4A, C, E & Table 2).

As seen in Fig.4B, D, F & Table 2, the mutations V95A, N138A, N142A, Y315F, that caused decreases in binding affinity of [³H]osanetant (Table 1), resulted similarly to increased affinity constant of osanetant for displacing of [³H]Me-talnetant by 13.6-, 11.2-, 124.3- and 12.2- fold, respectively. The double-mutant V95I / A99S, which decreased the [³H]osanetant binding affinity by 3.5-fold (Table 1), led to small increase in K₁ value of osanetant for displacing of [³H]Me-talnetant (Table 2). SB222200 (a close analogue of Me-talnetant) in competition binding assay behaved similarly to Me-talnetant (Table 2). The mutations V95A and N138A caused increases in the K₁ values of SB222200 for displacing of [³H]Me-talnetant by 5.0 and 3.9-fold, respectively. Interestingly, the N 142 A mutant, which had led to increased binding affinity of [³H]Me-talnetant (Table 1), displayed similarly a 500-fold higher affinity for SB222200 in displacing of [³H]Me-talnetant than the WT (Table 2).

2.2.5 Effect of mutations on the [MePhe⁷]NKB-evoked accumulation of [³H]IP.

To obtain more information about the NK₃R agonist binding pocket, the effects of mutations on NKB-induced formation of [³H]IP were investigated in CHO cell expressing transiently the WT and mutated hNK₃ receptors. [MePhe⁷]NKB (0.1 nM-10 μM) elicited a concentration-dependent increase in the accumulation of [³H]IP in the cells expressing WT and mutated hNK3 receptors. The EC₅₀, nn and relative E_m2x values, calculated from concentration- response curves of [MePhe⁷]NKB in the cells expressing WT and mutated receptors, are given in Table 3. The [MePhe⁷]NKB showed a lower functional potency (by 918.9-, 14.4- and 117.1- fold) and efficacy (relative E_n13x of 45%, 66% and 80%) at the mutants N138A, N142A and F342A, respectively, in comparison to the WT. The mutation M348A caused a 21.6-fold decrease in potency of [MePhe⁷]NKB without any effect on its efficacy. The mutants L232A, Y315F, F342M and S341I / F342M exhibited moderate increases in the [MePhe⁷]NKB EC₅₀ values (5.3-, 5.2- 9.0- and 9.0-fold, respectively) compared to WT. Although the same mutations have been observed to affect both the functional potency (Table 3) and affinity constant (Table 2) of [MePhe⁷]NKB, there were differences in the extent of the effect in two assays; e.g., the mutation N142A that abolished the affinity of [MePhe⁷]NKB in competition binding (K₁ > 10,000 nM), decreased [MePhe⁷]NKB potency by only 14.4-fold in functional assay and vice versa, the mutation Nl 38 A, which had a dramatic effect on NKB functional potency (EC₅₀ values 1020 versus 1.1 nM for WT), increased NKB K, value by 59-fold in competition binding.

2.2.6 Effect of mutations on the Schild analyses of Me-talnetant and osanetant as measured by [MePh e⁷]NKB-induced [³H]IP accumulation assay.

To characterize the effects of mutations on antagonism potency and the inhibition mode of Me-talnetant and osanetant, the dose-response curves (DRCs) for [³H]IP formation stimulated by [MePhe⁷]NKB have been measured in the presence of 0, 10 nM, 30 nM and 100 nM Me- talnetant or 0, 30 nM, 100 nM, 300 nM osanetant in CHO cell expressing transiently the WT and mutated hNK₃ receptors. As seen in Fig. 5A & C, both Me-talnetant and osanetant behave as a competitive antagonist at WT IiNK₃R, shifting the NKB DRC to the right without changing its maximal response. Me-talnetant displayed an apparent antagonist potency pA₂ = 8.1 (or K_t,^a = 7.2 nM) and a Schild slope of 0.8 (Fig. 5B), which is in good agreement with its affinity constant and is consistent with a competitive mode of action. However, osanetant had an apparent antagonist potency pA₂ = 7.5 (or K_b ^a = 33.9 nM), which is almost two log values lower than its binding constant (pK, = 9.6), and a Schild slope of 1.8, which has deviated from simple competitive antagonism with unit slope (Fig. 5D).

As observed above (Table 3), NKB has kept its potency on most of the mutated receptors except for the mutant N138A where it was inactive. Therefore, the antagonism potency of Me- talnetant and osanetant were determined at the mutated receptors using Schild analyses. The apparent antagonist potency (pA₂) and Schild slope values of Me-talnetant and osanetant at the mutated hNK3 receptors are given in the Table 4. hi general, there was a good agreement between the effects of mutations on binding affinity and functional potency results for both antagonists (Tables 1 &4). In cells expressing four mutants, M134A, V169M, F342M and S341I / F342M, which did not bind [³H] Me-talnetant and [³H]osanetant, both antagonists were inactive and not able to produce right shift of NKB DRC. In good agreement with the binding experiments, the mutant N142A, which caused an increase in binding affinity of Me-talnetant, also resulted in an increase of Me-talnetant antagonism potency, whereas the same mutation abolished the osanetant affinity and potency. The mutation F342A, which led to the loss of Me- talnetant affinity, similarly resulted in a loss of antagonism potency. In the case of osanetant, the mutant F342A decreased the antagonist potency, a result consistent with the 17-fold decrease of osanetant binding affinity. Among the mutated receptors, the mutants T139A and M346A exhibited the NKB DRC in presence of increasing fixed concentrations of osanetant that shifted to the right with a concomitant decrease in NKB maximal response, a fact inconsistent with a simple competitive mode of antagonism (Fig. 5E, F & Table 4). 2.2.7 Binding kinetics of [³H]Me-talnetant and [³H]osanetant to JiNK₃R WT and mutants T139A and M346A.

Binding of [³H]Me-talnetant and [³H]osanetant to the WT receptor was rapid with half- maximal binding occurring at 1.4 min and 4 min, and reaching equilibrium within 15 min and 30 min, respectively. The data from both antagonists were fit to a one-phase exponential model with the association rate constants of 0.34 ± 0.06 and 0.33 ± 0.06 nlVf'min^'1, respectively (Fig. 6A & Table 5). The association bindings of [³H]osanetant to the mutants T139A and M346A were similar to the WT with half-maximal binding, X_\a values of 4.6 min and 5 min, respectively (Fig. 6A & Table 5). The dissociation rates for [³H]Me-talnetant and [³H]osanetant binding to the WT receptor was determined by the addition of an excess amount of SB222200 after equilibrium (30 min and 1 h, respectively) was reached. The reversal of binding for both antagonists was complete with ti_/2 values of 4.6 min and 10 min, respectively (Fig. 6B & Table 5). The rates of [³H]osanetant dissociation from the mutants T139A and M346A were decreased as compared to the WT, with half-reversal binding occurring, ti_/2 values of 21 min and 18 min, respectively (Fig. 6B & Table 5). The calculations of the apparent K_d values derived from the kinetic experiments are given in Table 5. The apparent K_d value of [³H]Me-talnetant (0.44 ± 0.04 nM) was lower than that of equilibrium K_d value (0.8 ± 0.1 nM). [³H]osanetant had an apparent Kd value of 0.22 ± 0.06 nM at WT receptor, which is in good agreement with the equilibrium K_d value (0.2 ± 0.0). However, the apparent K_d values of [³H]osanetant at the mutants T139A and M346A (0.12 ± 0.01, 0.11 ± 0.01 nM) were lower that those of equilibrium K_d values (0.3 ± 0.1, 0.2 ± 0.0 nM, respectively).

2.3 Discussion

Various preclinical studies have demonstrated the involvement of NK₃ receptor-mediated activation in the release of dopamine, especially in ventral and dorsal striatal regions. Furthermore, recent phase II clinical results of osanetant and talnetant have indicated that blocking the NK₃ receptor could be beneficial for the treatment of schizophrenia and possibly other psychoses (Dawson et al., (2007) In Vitro and hi Vivo Characterization of the Non-peptide NK(3) Receptor Antagonist SB-223412 (Talnetant): Potential Therapeutic Utility in the Treatment of Schizophrenia. Neuropsychopharmacology 29, PMID: 17728699; Meltzer et al., (2004) Placebo-controlled evaluation of four novel compounds for the treatment of schizophrenia and schizoaffective disorder. Am J Psychiatry. 161:975-984; Meltzer et al., (2006) NK3 receptor antagonists for the treatment of schizophrenia. Drug Discovery Today: Therapeutic Strategies. doi:10.1016/j.ddstr.2006.11.013; Spooren et al., (2005) Opinion: NK3 receptor antagonists: the next generation of antipsychotics? Nat Rev Drug Discov. 4:967-975). In the current study, the likely binding pockets of Me-talnetant and osanetant, the selective and potent NK₃R antagonists from two distinct chemical classes, were determined using site-directed mutagenesis and rhodopsin-based modeling of hNK₃R 7TMD. Among fourteen point-mutations and three double-mutations that are located in TMl, -2, -3, -6, -7 and EC2 of hNK₃R, we observed that the M134^{2 53}A, V169^{3 36}M, F342^{7 39}M and S341⁷³⁸I / F342^{7 39}M mutations resulted in the complete loss of both [³H]Me-talnetant and [³H]osanetant binding affinities and also abolished their functional potencies in a NKB- evoked accumulation of [³H]IP assay, while the mutations V95^{1 42}A, N142^{2 61}A, Y315⁶⁵¹F, F342^{7 39}A and M346⁷⁴³A behaved differently between two antagonists' interacting modes. These mutated hNK₃Rs have also allowed us to probe the NKB-binding pocket using [MePhe⁷]NKB-evoked accumulation of [³H]IP and competition binding with [³H]Me-talnetant. The residues N138^{2 57}, N142^{2 61}, Y315⁶⁵¹, F342^{7 39} and M346⁷⁴³ were found to be crucial for the NKB-binding site. To visualize the mutation data, we have constructed a 3D model of the 7TMD of the hNK₃ receptor using the atomic coordinates of bovine rhodopsin (pdb code If88). Fig. 7 shows the amino acids in the TM region mutated in this study, and it suggests the possible binding modes for Me-talnetant and osanetant (Highlighted in color are those mutations that either led to a complete loss of affinity for both osanetant and Me-talnetant (red) or that influenced mainly only one of the two antagonists (magenta: influence on osanetant, cyan: influence on Me-talnetant)).

In the TMl region, the V95^{1 42}A mutation had a significant effect onto the binding of Me- talnetant, indicating that Me-talnetant forms a direct contact with this residue. However, this mutation had a lesser effect on osanetant binding affinity. This is in agreement with the proposed docking mode according to which the benzylamine substructure of Me-talnetant reaches deeply into the pocket formed by the TMDl, -2 and -7, while osanetant does not fill this region of space (Fig. 7). The aromatic ring of this Me-talnetant chain is thus located closely to V95^{1 42}. The position 95 is not conserved in the IiNK₁ and hNK₂ receptors (Fig. 3). The incorporation of a leucine (V95^{1 42}L) as in hNK₂R did not affect either of the ligands. While V95^{1 42} obviously forms a hydrophobic interaction with Me-talnetant that contributes to the binding affinity, there is still enough space to incorporate the larger leucine residue. Also the double mutant V95^{1 42}UTl 39^{2 58}A incorporating the residues of the hNK₂ receptor did not influence the binding affinities. This double mutant had been selected since both residues are located closely together in 3D forming a subpocket. The double mutant V95^{1 42}I / A99^{1 46}S incorporating the residues of the ILNK₁ receptor leads for both ligands to an almost equal loss of affinity. These two residues are thus involved in the ILNK₁ selectivity of Me-talnetant and osanetant.

In the TM2 region, the mutant M134^{2 53}A did not bind either Me-talnetant or osanetant. For osanetant, this result was expected since M134^{2 53} had previously been shown to be an important factor for rat and mouse selectivity (in form of the rat double mutant V121^{2 53}M and G133^{2 65}A) (Wu et al., (1994) Biochem Biophys Res Commun. 198:961-966). According to our proposed docking modes, both ligands indeed form direct interactions with M134^{2 53}, osanetant via its benzoyl chain, Me-talnetant via the ethyl side-chain. N138^{2 57} is located one turn above M134^{2 53}. Its mutation to alanine leads for both ligands to a 3.5x loss of affinity. Surprising is the result of the mutation Nl 42^{2 61}A. While binding of osanetant is completely lost, Me-talnetant binding is significantly increased. Because Nl 42^{2 61} is located closely to several other polar residues, it is most likely part of a larger H-bonding network and the observed effect could thus be direct or indirect.

In the TM3, TM6 and TM7 regions, the mutation V169^{3 36}M led for both ligands to a complete loss of affinity. This mutation incorporates the residue OfNK₂ into the NK₃ receptor. Obviously, this residue is responsible for NK₂ selectivity of both Me-talnetant and osanetant. It is interesting to note that in the 3D model, the residue Vl 69^{3 36} is located in proximity to

W263⁶⁴⁸ in the TM6 helix, thus it is likely to be part of the intramolecular TM network involved in receptor activation as shown previously for rhodopsin (W265⁶⁴⁸) and other group A GPCRs (Ballesteros et al., (2001) Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. MoI Pharmacol. 60:1-19; Sheikh et al., (1996) Rhodopsin activation blocked by metal-ion- binding sites linking transmembrane helices C and F. Nature 383:347-350). The mutation of Y315^{6 51} to its corresponding residue in the NK₁ receptor (F) affected only osanetant binding, which was partially lost. This shows that Y315^{6 51} is one of the factors OfNK₁ selectivity of osanetant, which binds to !1NK₁R with K, of 744 nM, whereas K₁ of Talnetant at IiNK₁R is > 10,000 nM. This result is in agreement with our proposed docking pose that allows the phenolic OH of Y315^{6 51} to form a hydrogen bond with osanetant, while Me-talnetant can interact only with the aromatic ring but not the OH group (Fig. 7). Mutation of F342^{7 39} to methionine, the corresponding residue in NK₁, led to a complete loss affinity for both ligands that make hydrophobic interactions with this residue, Me-talnetant via its 2-phenyl ring, osanetant via its di-chloro substituted phenyl ring. A different behavior of osanetant and Me-talnetant is observed with the M346⁷⁴³A mutant that does not affect osanetant binding but leads to a significant loss of Me-talnetant affinity. According to our docking mode, Me-talnetant can indeed form a hydrophobic interaction with M346 while osanetant is located too far from this residue.

The functional potencies of Me-talnetant and osanetant on mutated receptors as determined by Schild plot analyses are mostly in agreement with the binding affinities. Although the Schild slope of osanetant at WT and some mutated receptors exhibited deviation from simple competitive antagonism with unit slope, in general, osanetant behaved competitively at the mutated receptors except for the mutants T139A and M346A, which displayed abnormal Schild plots. Our binding kinetics showed that osanetant had a slower dissociation rate on these mutants than that of WT; this might explain the abnormal Schild analyses observed in the [MePhe⁷]NKB- evoked accumulation of [³H]IP assay. However, [³H]Me-talnetant and [³H]osanetant binding kinetic studies at WT receptor are in good agreement with previously reported data for osanetant and SB222200 (Tian et al., (2007) Neurokinin-3 receptor-specific antagonists talnetant and osanetant show distinct mode of action in cellular Ca2+ mobilization but display similar binding kinetics and identical mechanism of binding in ligand cross-competition. MoI Pharmacol. 71:902-911).

These observations show that the binding pocket of osanetant and Me-talnetant are overlapping, but not identical. While Me-talnetant binding is more influenced by residues on TMl and -7, osanetant binding is affected by the mutation Y315⁶⁵¹F. This is in agreement with our proposed docking modes where Me-talnetant reaches deeply into the pocket formed by TMl, -2 and -7, while osanetant fills the pocket TM3, -5 and -6 with its phenyl-piperidine fragment (Fig. 7).

When the critical residues involved in the NKB-, Me-talnetant- and osanetant-binding site of hNK₃R were compared with those of reported ligand recognition sites of other neuropeptide GPCRs (Table 6), a striking conservation in the TM helix position of many critical residues among NK₁, NK₂, NK₃, V]₃ and Vi_b receptors was observed. As seen in Table 6, there is a high homology between NK3 and Vl receptors; where the ligands all probe similar helix positions in the 7TM. The hNK₃R binding pocket has a higher similarity to hNK₂R (5 a. a. differences in TM region and EC2 loop) than that of hNKjR (8 a. a. differences in TM region and EC2 loop) (Fig. 3). Hence, there is a high homology between the conserved residues involved in NKA and NKB binding sites (Table 6). Interestingly, the TM helix position 2.61 (located at the rim of 7TM cavity) has similar contact sites with the SP, NKA, NKB and AVP (nonapeptide, arginine vasopressin) peptide ligands. Of note are also the TM helix positions 3.36, 6.51, 7.43 (all located deep in TM cavity) and 7.39 (at the top of TM cavity): the amino acids occupying these helix positions are the most frequently involved in interaction with the diverse ligands of the class A and C family of GPCRs (Ballesteros et al., (2001) Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. MoI Pharmacol. 60:1-19; Malherbe et al., (2003) Mutational analysis and molecular modeling of the allosteric binding site of a novel, selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor. J Biol Chem. 278: 8340- 8347; Malherbe et al., (2006) Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP. J Neurochem. 98:601-615; Petrel et al. (2004) Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor. J Biol Chem. 279:18990-18997). Table 1. [³H]Me-talnetant and [³H]osanetant binding properties at human wild-type and mutated NK₃ receptors. Saturation binding isotherms of [³H]Me-talnetant and [³H]osanetant were performed on membrane preparations from HEK293-EBNA cells transiently transfected with the WT and mutated hNK₃ receptors as described under "Experimental Procedures". The K_d and B_max values are mean ± SEM, calculated from three independent experiments (each performed in triplicate). The mutations that affected the [³H]Me-talnetant and [³H]osanetant binding affinities are shown in boldface type. Statistical significance was determined using the Two-tailed t-test: *P<0.05, **P<0.01, ***P<0.001.

to

Os

Table 2. Effects of the mutations on [³H]Me-talnetant displacement by [MePhe7]NKB, osanetant and SB222200 in the HEK293-EBNA cell membrane transiently expressing WT and mutated hNK₃ receptors. Kj and Hill slope (Π_H) values for [³H]Me-talnetant binding inhibition by [MePhe⁷]NKB, osanetant or SB222200 were calculated as described under "Experimental Procedures". Values are mean ± SEM of the Kj calculated from 3 independent experiments, each performed in duplicate. Affected mutations are shown in boldface type.

[MePhe⁷]NKB osaneta nt SB222200 hNK₃

K, K, (m utant) / n_H K, K₁ (m utant) / K₁ K, (m utant) / n_H receptor nM K, (WT) nM K, (WT) nM K, (WT)

WT 14.7 ± 2.8 0.6 ± 0.0 1.0 ± 0.3 1.1 ± 0.2 5.4 ± 0.5 0.9 ± 0.0

V95A 19.4 ± 4.1 1.3 0.9 ± 0.1 14.0 1 3.9 13.6 0.6 ± 0.1 27.1 1 5.4 5.0 0.7 ± 0.1

V95L 7.9 ± 0.9 0.5 0.7 ± 0.1 1 .8 ± 0.4 1.8 1 .0 ± 0.1 4.6 ± 0.2 0.9 0.9 ± 0.0

V95I / A99S 12.9 ± 0.6 0.9 0.8 ± 0.1 1.7 ± 0.4 1 .7 0.8 ± 0.1 12.5 ± 5.5 2.3 0.8 ± 0.2

N138A 875.0 ± 103 .0 59.4 0.9 ± 0.1 11.5 1 0.7 11.2 0.9 ± 0.3 21.0 1 4.9 3.9 0.8 ± 0.0

T139A 24.7 ± 8.8 1.7 0.9 ± 0.1 0.9 ± 0.7 0.9 0.6 ± 0.2 15.7 ± 2.1 2.9 0.9 ± 0.0

V95L / T139A 7.8 ± 2.9 0.5 0.6 ± 0.1 1 .7 ± 0.1 1 .6 1 .0 ± 0.0 8.9 ± 0.8 1.7 1.0 ± 0.0

N142A >10,000 128.0 1 4.9 124.3 1.1 ± 0.1 0.01 1 0.001 0.002 0.5 ± 0.0

L232A 147.0 ± 7. 3 10.0 0.8 ± 0.1 1.5 ± 0.6 1.5 0.4 ± 0.0 15.0 ± 3.8 2.8 0.8 ± 0.0

Y315F 90.8 1 18.£ 6.2 1 .1 ± 0.1 12.6 11.8 12.2 1 .1 ± 0.3 8.0 ± 0.9 1 .5 0.9 ± 0.1

S341A 17.1 ± 3.7 1 .2 0.7 ± 0.1 1.2 ± 0.3 1.2 1 .0 ± 0.2 5.8 ± 0.5 1 .1 0.9 ± 0.0

M346A 207.0 ± 31. 8 14.1 1.0 ± 0.1 0.9 ± 0.4 0.9 0.7 ± 0.0 10.6 ± 0.6 2.0 0.7 ± 0.0

S 348 A 27.6 ± 4.4 1.9 0.8 ± 0.1 2.4 ± 1.0 2.3 0.5 ± 0.1 9.2 ± 0.2 1 .7 0.9 ± 0.1

Table 3. Effect of mutations on the [MePhe⁷]NKB-evoked accumulation of [³H]IP. EC50, Hill slope (n_H), and relative efficacy (E_max) values for the NKB-induced formation of [³H]IP in CHO cell expressing transiently the WT and mutated hNK₃ receptors. The data is mean ± SEM of 8 dose-response measurements (each performed in duplicate) from 4 independent transfections.

to

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Table 4. Schild constants for antagonism of [MePhe⁷]NKB-induced accumulation of [³H]IP by Me-talnetant and osanetant in CHO cells expressing transiently the WT and mutated hNK3 receptors. The apparent antagonist potency (pA₂) and Schild slope values of Me-talnetant and osanetant at the WT and mutated hNK₃ receptors were determined from NKB DRCs in the absence and presence of various concentrations of antagonist and Schild plot analyses shown in Fig 5.

Table 5. Kinetic binding parameters for association and dissociation of ³H]Me-talnetant to ITNK₃R WT membrane and [³H]osanetant to the hNK₃R WT, T139A and M346A membranes. The K_ob (observed on rate), K₀^ (observed off rate), K_0n, W₂ (half-maximal binding) and Kj (apparent dissociation constant) values are mean ± SEM, calculated from three independent experiments (each performed in quadruplet) as described under "Experimental Procedures".

U) O

Table 6. Comparison of binding pockets of neurokinin, Bradykinin and vasopressin. The key amino acids located at helix positions in the Ballesteros numbering ((Ballesteros and Weinstein, (1995) Methods Neurosci. 25:366-428) that have been implicated in the peptide agonist and selective nonpeptide antagonist binding pockets were compared among hNKi, hNK₂, hNK₃, BKB₂, hV]_a and hVi_b receptors. Reported data from: a, ( Huang RR, et al (1994) Interaction of substance P with the second and seventh transmembrane domains of the neurokinin-1 receptor. Biochemistry. 33:3007-3013); b, ( Huang RR, et al (1995). Biochemistry. 34:10048-10055); c, (Labrou et al., (2001) J Biol Chem. 276:37944- 37949); d, (Renzetti et al, J Pharmacol Exp Ther. 290:487-495); e, (Meini et al., (2005) Eur J Pharmacol. 516:104-111); f, (Meini et al., (2004) Site-directed mutagenesis at the human B2 receptor and molecular modelling to define the pharmacophore of non-peptide bradykinin receptor antagonists. Biochem Pharmacol. 67:601-609); g, (Mouillac et al., (1995) The binding site of neuropeptide vasopressin Via receptor. Evidence for a major localization within transmembrane regions. J Biol Chem. 270:25771-25777); h, (Tahtaoui et al., (2003) Identification of

10 the binding sites of the SR49059 nonpeptide antagonist into the Via vasopressin receptor using sulfydryl-reactive ligands and cysteine mutants as chemical sensors. J Biol Chem. 278:40010-40019); i, (Derick et al., (2004). MoI Endocrinol. 18:2777-2789). j, A334 is the crucial residue for U) selectivity SR49059 for hVlaR, (Derick et al., (2004) MoI Endocrinol. 18:2777-2789). The NK3R data are from current study.

Claims

Claims

1. A radiolabeled Neurokinin 3 receptor antagonist of the general formula (I) formula (I)

wherein Rl is a radiolabeled lower alkyl.

2. The compound of claim 1 wherein Rl is a tritium-labeled methyl (-CT₃) or a methyl comprising a radioisotope of carbon.

3. The compound of claim 2 wherein the radioisotope of carbon is [¹¹C] or [¹⁴C].

4. A method for identifying a compound that can bind to a NK3R comprising: a) contacting a compound of interest with a sample comprising the NK3R in the presence of the radiolabeled NK3R antagonist of any one of claims 1 to 3; and b) monitoring whether the compound of interest influences the binding of said radiolabeled NK3R antagonist to said NK3R.

5. The method according to claim 4 wherein the sample comprising NK3R is a tissue sample, primary cell or cultured cells, which either naturally express a NK3R, or which are either transiently or stably transfected with a NK3R.

6. The method according to claim 4 wherein the sample comprising NK3R is a membrane suspension obtained from cells or tissue which either naturally express a NK3R, or which are either transiently or stably transfected with a NK3R.

7. The method according to any one of the claims 4 to 5 wherein the NK3R comprises point mutations.

8. The method according to any one of claims 4 to 5 wherein the NK3R has an alanine at position 142 of SEQ. ID NO: 1 instead of an asparagine, or a phenylalanine at position 315 of SEQ. ID NO: 1 instead of a tyrosine, or a methionine at position 342 of SEQ. ID NO: 1 instead of a phenylalanine, or an isoleucine at position 95 of SEQ. ID NO: 1 instead of a valine and a serine at position 99 of SEQ. ID NO: 1 instead of an Alanine, or a methionine at position 169 of SEQ. ID NO: 1 instead of a valine, or an isoleucine at position 341 of SEQ. ID NO:1 instead of a serine and a methionine at position 342 of SEQ. ID NO: 1 instead of a phenylalanine, or an alanine at position 95 of SEQ. ID NO: 1 instead of a valine, or an alanine at position 346 of SEQ. ID NO: 1 instead of a methionine.

9. A NK3 receptor with an alanine at position 142 of SEQ. ID NO: 1 instead of an asparagine, or a phenylalanine at position 315 of SEQ. ID NO: 1 instead of a tyrosine, or an methionine at position 342 of SEQ. ID NO: 1 instead of a phenylalanine, or an isoleucine at position 95 of SEQ. ID NO: 1 instead of a valine and a serine at position 99 of SEQ. ID NO: 1 instead of an Alanine, or a methionine at position 169 of SEQ. ID NO: 1 instead of a valine, or an isoleucine at position 341 of SEQ. ID NO:1 instead of a serine and a methionine at position 342 of SEQ. ID NO: 1 instead of a phenylalanine, or an alanine at position 95 of SEQ. ID NO:1 instead of a valine, or an alanine at position 346 of SEQ. ID NO:1 instead of a methionine .

10. A method for determining the selectivity of a NK3R ligand towards Neurokinin- 1 receptor comprising a) contacting a NK3 ligand with a sample comprising the Neurokinin 3 receptor comprising F342M, S341I/F342M, Y315F and/or V95I/A99S in the presence of the radiolabeled NK3R antagonist hereinbefore described, b) monitoring whether the compound of interest influences the binding of said radiolabeled NK3R antagonist to said NK3R.

11. A method for determining the selectivity of a NK3R ligand towards Neurokinin-2 receptor comprising a) contacting a NK3 ligand with a sample comprising the Neurokinin 3 receptor with a point mutation V169M in the presence of the radiolabeled NK3R antagonist hereinbefore described, b) monitoring whether the compound of interest influences the binding of said radiolabeled

NK3R antagonist to said NK3R.

12. Compounds, receptors, methods and uses substantially as herein before described especially with reference to the foregoing examples.

***