WO2007057428A1

WO2007057428A1 - Chromophore containing ligands for characterisation of receptors for binding and activation

Info

Publication number: WO2007057428A1
Application number: PCT/EP2006/068553
Authority: WO
Inventors: Jörg GRANDL; Rudolf Hovius; Horst Vogel; Daniel Bertrand; Elias Sakr; Florence Kotzyba-Hibert; Maurice Goeldner
Original assignee: Epfl - Ecole Polytechnique Federale De Lausanne
Priority date: 2005-11-17
Filing date: 2006-11-16
Publication date: 2007-05-24

Abstract

The present invention is directed to conjugates of chromophores with agonistic ligands directed to ligand-gated ion channels (LGICs) of the Cys loop-family. The conjugated ligands bind specifically and with high affinity to their cognate receptors, allowing to measure directly the binding of these ligands to the receptors, to quantify the affinity of unlabelled compounds for the receptor, to detect receptor activation, and to inspect and image presence of and behaviour of said receptors in living or fixed cells or tissues, either in situ or ex vivo, or on (semi-) purified receptor preparations.

Description

Chromophore containing ligands for characterisation of receptors for binding and activation

Field of the invention

The present invention describes conjugates of chromophores with receptor ligands for receptors of small neurotransmitters, in particular for ligand-gated ion channels, and their use to study these receptors and their activation.

Background of the invention

To study receptors and their activation mainly ligands labelled with either radioactive isotopes or groups emitting photons are presently used. A serious drawback of the former is the very nature of the label; it is radioactive. Moreover compared to the second method it is far less sensitive, and not suited for on-line in vivo/in situ data acquisition. Emitting ligands have been widely used in scientific research and are exploited for practical applications (see e.g. N. Baindur and D.J. Triggle, Drug Dev. Res. 33, 373 (1994)).

Ligand-gated ion channels (LGICs) of the Cys-loop family count more that 40 gene sequences in man, coding for both inhibitory and excitatory receptors (M. O. Ortells and G. G. Lunt, Trends Neurosci. 18(3), 121 (1995); H. Lester et al., Trends Neurosci. 27, 329 (2004)). LGICs are receptors for small neurotransmitters, and upon binding an activating ligand, i.e. an agonist, an internal transmembrane channel is opened allowing ions to flow according to their electrochemical gradient. Acetylcholine receptors are involved in the fast excitatory synaptic signal transduction amongst neurons and between neurons and e.g. muscle cells, and are involved in many disorders (see e.g. S. Weiland et al., Behav. Brain Res. 1 13, 43 (2000); A.G. Engel et al., Nature Reviews Neuroscience 4, 339 (2003); A.A. Jensen et al., J. Med. Chem. 48(15), 4705 (2005)). Despite their importance still many questions about the function of such receptors remain unanswered.

Some fluorescent nicotinic acetylcholine receptor (nAChR) ligands are known. Such compounds are either derivatives of acetylcholine or of toxins.

Derivatives of acetylcholine: The choline part of acetylcholine has been conjugated with several fluorophores. Dansyl, pyrene and NBD (7-nitrobenz-2-oxa-1 ,3-diazol-4-yl) were attached through an ω-amino-fatty acid to the hydroxyl group of choline (J. B. Cohen and J.-P. Changeux, Biochemistry 12, 4855 (1973); FJ. Barrantes et. al., Proc. Natl. Acad. Sci. USA 72, 3097 (1975); R. Jϋrrs et al., Proc. Natl. Acad. Sci. USA 76, 1064 (1979)). Pyrene-C4-choline was shown to be an antagonist as tested on frog (FJ. Barrantes et al., supra). Agonists were obtained for dansyl and NBD when the fatty acid was from 4 to 8 carbon atoms long (G. Waksman et al., MoI. Pharm. 18, 20 (1980); H.W. Meyers et al., Eur. J. Biochem. 137, 399 (1983)). The majority of these activity studies have been performed on nAChR obtained from electric fish or frog. The dansyl compounds described by Waksman et al. {supra) featured agonistic effects at low concentrations with apparent EC₅₀ values ranging from 10^~7 to 10^~3 M, whereas at high concentrations they showed pronouncedly reduced efficacy, potentially due to antagonistic effects of a non-competitive block. Neither tests on mammalian muscle nor on neuronal nAChRs are reported by these authors. Jϋrss et al. (supra) determined the activity of a fluorescently labelled acetylcholine (NBD linked to choline through a 6 carbon atom linker) on a mammalian nAchR; it was found to stimulate contraction of rat diaphragm. However, the same publication reports a strong interaction of this compound with acetylcholine esterase. Therefore this latter compound cannot be considered to be a specific nAChR ligand.

Derivatives of other nAchR ligands: A 4-carboxynorcotinine has been labeled and used for an immuno-based assay for the evaluation of ligand binding (US 5,527,686).

Derivatives of toxins: Peptides from the venom of e.g. snail (α-conotoxins) and snake (α- bungarotoxin, α-Naja toxin) are potent inhibitors of the nAChR. These molecules are easily modified and have been produced with a variety of fluorophores (C. Schreiter et al., ChemBioChem 6, 2187 (2005); K.R. Rogers et al., Biosensors & Bioelectronics 6, 507 (1991 ); M.M.S. Lo et al., FEBS Lett. 1 1 1 , 407 (1980)); several fluorescent analogues of α- bungarotoxin are commercially available. However, as the toxins, these emitting conjugates are all receptor antagonists.

It is of great interest, on the one hand, to investigate in detail ligand-gated ion channels and to develop new medicines based on results obtained, and, on the other hand, to develop new tools to facilitate or enable these studies. Summary of the invention

The present invention is directed to conjugates of chromophores with agonistic ligands directed to ligand-gated ion channels (LGICs) of the Cys loop-family. The conjugated ligands bind specifically and with high affinity to their cognate receptors, allowing to measure directly the binding of these ligands to the receptors, to quantify the affinity of unlabelled compounds for the receptor, to detect receptor activation, and to inspect and image presence of and behaviour of said receptors in living or fixed cells or tissues, either in situ or ex vivo, or on (semi-) purified receptor preparations. The invention is furthermore directed to a method of characterisation and quantification of receptors, and also to uses of the compounds of the invention.

Brief description of the figures

Figure 1. Radioligand competition assay of EPB-chlorocoumarin (10).

Inhibition of [³H]-cytisine and [¹²⁵l]-α-bungarotoxin binding to rat brain (Fig. 1 A) and Torpedo (Fig. 1 B) membranes, respectively, by increasing concentrations of EPB- chlorocoumarin, compound (10). These data illustrate a strong interaction of the EPB- chlorocoumarin probe on α4β2 neuronal nAChRs and the specific binding to muscle-type- like nAChRs, yielding IC₅₀ values of 63.4 nM and 16 μM, respectively. The observed values are to be compared with the highly specific interactions of the parent EPB molecule: 0.18 nM and 61 nM, respectively. X-axis: molar concentration, y-axis: cpm.

Figure 2. Activation of various subtypes of nAChRs by fluorescent epibatidines. The ability of the fluorescent EPBs to activate the nAChR was investigated by whole-cell electrophysiology where the human neuronal α3β4 and α4β2 nAChRs were expressed in Xenopus oocytes (Fig. 2A), and the fetal human muscle nAChR was expressed in HEK cells (Fig. 2B and 2C). (2A) Human neuronal α3β4 and α4β2 nAChR subtypes expressed in oocytes were challenged with 1 μM EPB-chlorocoumarin (EPB-CI-Cou) or 1 μM EPB-Cy5; for comparison the currents evoked by 100 μM ACh are shown. (2B) Recording of electric current during patch clamp experiments on HEK293-cells expressing human muscle nAChR. Activation of channels can be seen during application of an EC50 concentration of fluorescent ligand (20 nM EPB-Cy5). The duration of ligand addition is indicated by the diagram on top. X-axis: time in seconds, y-axis: current in pA. (2C) Fetal human muscle nAChR with the mutation 5S268K expressed in HEK293 cells were challenged with the indicated concentrations of EPB-coumarin (O), EPB- chlorocoumarin (D), EBP-Atto610 (O), EPB-Cy3 (A) and EPB-Cy5 (■). Responses are normalized to currents evoked by 10O μM ACh and fitted by Hill-equations (dotted lines) from which EC₅₀ values are evaluated to be 26±5 nM, 29±4 nM, 127±30 nM, 1 15±24 nM and 30±10 nM, respectively, which compares favorably with the EC₅₀ of epibatidine (53±4 nM). The Cy5 and both coumarin EPB compounds are full agonists, whereas the Cy3 and Atto610 labelled epibatidine are partial agonists activating the nAChR to an extent of 50%. X-axis: concentration (MoI), y-axis: normalized current.

Figure 3. Fluorescence intensity on the membrane of a HEK293 cell during addition of EPB-Cy5.

(3A) Fluorescence intensity on the membrane of a HEK293 cell, expressing the adult wild- type nAChR, as a function of time during addition of fluorescent ligand (50 nM epibatidine- Cy5) and rinsing with buffer. X-axis: image number, y-axis: fluorescence intensity in arbitrary units.

(3B) Overlay of fluorescence intensity plots acquired during the dissociation phase and their fits for different acquisition rates. X-axis: image number, y-axis: fluorescence intensity in arbitrary units. (3C) Values of k_depι obtained from mono-exponential fits to intensity profiles as a function of the acquisition rate. k_depι is dominated by k_off, which does not depend on the acquisition rate. X-axis: acquisition rate in Hz, y-axis: depletion per second.

Figure 4. Fluorescence imaging of single molecules EPB-Cy5 labelled nAChRs in live cells.

(4A) Transmission micrograph of a HEK293 cell expressing wild-type adult muscle nAChR.

(4B) Fluorescence imaging of the cell shown in (4A) after addition of 5 nM EPB-Cy5. The

AChRs, in the plasma membrane of the cells, that bind the fluorescent agonist appear as single fluorescent spots as the one shown in the white box.

(4C) Trajectory of the single fluorescent receptor boxed in (4B) during its diffusion on the cell membrane during the acquisition of a series of fluorescence images. The chess board like background represents the pixels of the CCD-camera; the grey values scale with the fluorescence intensity of the image taken in which the receptor's position is indicated by the cross; the line shows the trajectory of this molecule over 52 frames (-4.3 s). Scale bar: 10 μm in (4A) and (4B), 450 nm in (4C). Detailed description of the invention

The present invention is directed to conjugates of chromophores with agonistic ligands directed to ligand-gated ion channels (LGICs) of the Cys loop-family, as illustrated, for example, by conjugates of chromophores to agonists of ionotropic acetylcholine receptors. The chromophore may be an emitting or absorbing chromophore, such as a fluorophore or a fluorophore quencher, respectively.

The ligand-gated ion channels of the Cys loop family carry the so-called Cys-loop signature C-X-[LIVMFQ]-X-[LIVMF]-XX-[FY]-P-X-D-XXX-C. Particular ligand-gated ion channels considered are ionotropic acetylcholine receptors, such as nicotinic acetylcholine receptors (nAChRs), for example mammalian and insect receptors. Ligands to neuronal and muscle-type nAchRs are preferred, in particular to human muscle and neuronal nAchRs.

In particular the present invention is directed to emitting chromophores conjugated to ligands for LGICs other than acetylcholine or acetylcholine derivatives and other than toxins binding to LGICs. The advantages of the described emitting compounds and their applications resides in the following points: (i) the compounds are emitting, thus allowing sensitive on-line and in vivo data acquisition; (ii) the compounds comprise a ligand giving rise to a selective receptor recognition allowing specific receptor binding assays and the study of receptor properties; (iii) the compounds are agonists thus allowing one to study receptor activation and effects thereof; (iv) the compounds allow combined measurement of receptor binding and activation.

Furthermore the present invention is directed to a method of characterisation and quantification of receptors, wherein the conjugates of chromophores with agonistic ligands as described herein are contacted with the corresponding receptors, and the amount of chromophore bound to the receptor or the activation of the receptor is measured. At the same time this method allows determination of ligand binding and of activation of these receptors. The method can be performed on (semi-)purified receptors, membrane preparations from cells (heterologously or naturally) expressing the receptor, on cells expressing the receptor both in suspension and surface-attached, on tissues comprising such cells or preparations thereof, and in situ using any modality for the detection of an optically signal emitted or modified by the chromophore in the compounds of the invention. Furthermore the present invention is directed to the use of the compounds of the invention, i.e. the use of conjugates of chromophores with agonistic ligands directed to ligand-gated ion channels (LGICs) of the Cys loop-family in a method of characterisation and quantification of cognate receptors, in particular for characterisation of the receptor proteins and/or their interaction with the agonistic ligands. This use also includes the parallel use of unconjugated ligands in competition experiments to study the effect of such unlabelled ligands on a receptor, to study receptor activation, to characterise the receptors, the compounds and other unlabelled ligands for competition or modulation.

The general structure of the labelled ligands of the invention corresponds to

wherein A is the ligand for receptor recognition, preferably a receptor ligand of high affinity (K ≥ 10⁶ M^"1) and high selectivity (> 10x), B is a bond or a spacer, and C is the chromophore. Chromophore C may be an absorbing or emitting group, but is preferably an emitting group. Absorbing and emitting groups considered are described hereinbelow, and are in particular fluorophores. Moiety C is connected to moiety A through covalent bonds either directly or via a spacer moiety B, which can be linear but also branched, and which may comprise a further functionality, for example an antibody epitope, a detectable tag such as biotin, or a chemically reactive group.

The chromophore containing ligands of the present invention are directed towards ligand- gated ion channels, preferably against channels of the Cys loop-family. Within this group, the ionotropic acetylcholine receptors are considered with priority, and in particular mammalian ionotropic acetylcholine receptors.

Preferred ligands A display a high affinity (K ≥ 10⁶ M^'1) and specificity (> 1 Ox) for their cognate receptor, therefore allowing one to discretely probe this receptor. Ligands considered are those, which either before conjugation with a chromophoric reporter group were agonists and remained agonists after conjugation, or through the conjugation have acquired the ability to activate the channel. Some of the most preferred substances are pyridine-containing compounds, e.g. those of Scheme 1.

Scheme 1 shows the structures of typical recognition ligands A of nicotinic acetylcholine receptors. These molecules are pyridine-containing compounds, in particular pyridine- containing derivatives substituted at position 5 by monocyclic or bicyclic aliphatic amines and at position 3, indicated by X, by a bond or linker B connected to a chromophore as nicotinic probes, e.g. compounds of formula (1 ) wherein R is an alkyl group and the circle represents a optionally substituted mono- or bicyclic ring structure with 5 to 10 ring members. Particular examples of such molecules are epibatidine (EPB) (2, X=H) and derivatives thereof, nicotine (3, X=H) and derivatives thereof, and aza-bicyclo[2.2.2]octane derivatives (4, X=H), but the invention encompasses all types of mammalian nicotinic receptor ligands. A further ligand A of the invention is represented by imidacloprid (5, X=H), which targets invertebrate nicotinic receptors.

Derivatives are compounds wherein the mono- or bicyclic amine residue of formula (1 ) to (5) is further substituted at a carbon atom, for example by alkyl, in particular lower alkyl with 1 to 6 carbon atoms, e.g. methyl or ethyl, hydroxy, alkoxy, in particular d-C₆-alkoxy, e.g. methoxy, halogen, e.g. fluoro or chloro, oxo, acyloxy, in particular CrC₆-alkyl- carbonyloxy, CrCe-alkylaminocarbonyloxy and optionally substituted phenylamino- carbonyloxy, e.g. acetoxy or phenyl-, fluorophenyl-, chlorophenyl-, bromophenyl- or methoxyphenyl-aminocarbonyloxy, acylamino, in particular Ci-C₆-acylamino and optionally substituted phenylaminocarbonylamino, e.g. acetamino or phenylamino- carbonylamino, and the like, and/or wherein the pyridine nucleus of formula (1 ) to (5) is further substituted by halogen, e.g. fluoro or chloro. Particular derivatives are those wherein R in formula (1 ) is alkyl, in particular lower alkyl with 1 to 6 carbon atoms, e.g. methyl or ethyl, and those wherein R in formula (4) is hydrogen or has any of the mentioned meanings for substituents, in particular optionally substituted phenylaminocarbonyloxy or optionally substituted phenylaminocarbonylamino.

Scheme 1

Scheme 1 (continued)

The chromophores C used as reporter groups to be attached to the ligands A have to meet certain criteria, e.g. absorption coefficient > 25'00O M^~1cm^~\ quantum yield in water > 10% and absorption maximum > 400 nm. They should ideally neither hinder the interaction of the modified ligand to the receptor nor affect its selectivity. Moreover, the chromophores must confer an optically detectable signal to the ligand in order to allow the quantification of the receptors and bound ligands.

Chromophores C can be absorbing or emitting. In the case of absorbing chromophores, interaction of the conjugated ligands with the receptors can be monitored through quenching of a fluorescence signal originating from a fluorescently labelled receptor protein. Examples of such receptor proteins might be fluorescent protein labelled receptors, receptor proteins labelled through (bio)chemical or enzymatic modification or through reversible interactions. Examples of suitable absorbing groups useful as chromophores C are the QSY-dyes (Invitrogen), the Blackhole quenchers (Biosearch Technologies), Cy5Q or Cy7Q (GE Healthcare), FluoQuench, Dabcyl, and others.

The (preferred) light emitting chromophores C of the conjugated ligands, which are subject of the present invention, are selected from emitting molecules such as organic dyes (e.g. carbopyronin, coumarin, (indo-)cyanines, 4,4,-difluoro-4-bora-3a,4a-diaza-5- indacenes, rhodamine, oxazines, or derivatives thereof); fluorescent proteins (e.g. phycoerythrine and the green fluorescent proteins, or homologues thereof); chelates, cryptates or complexes of metals such as europium, rubidium or osmium, e.g. europium or rubidium complexes; semiconductor nanoparticles (e.g. CdSe quantum dots) or similar chromophores and modifications thereof. Also included are non-emitting moieties like fluorogenic derivatisation reagents, which once conjugated to the ligand become fluorescent, such as compounds known under the name of Atto-Tag (Invitrogen).

It is important that the fluorescent emitting moieties within the compounds of this invention have excellent properties, i.e. feature a quantum yield of more than 10% in water and a wavelength of maximal excitation of ≥ 400 nm, as to be compatible with optimal detection of single receptor molecules or low abundant receptors in a complicated matrix like membrane preparations, cells or tissues.

A spacer B consists of optionally substituted straight or branched chain alkylene group with 1 to 30 carbon atoms, wherein optionally

(a) one or more carbon atoms are replaced by oxygen, in particular wherein every third carbon atom is replaced by oxygen, e.g. a poylethyleneoxy group with 1 to 10 ethyleneoxy units; (b) one or more, e.g. two, three or four, carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function -NH-CO-;

(c) one or more, e.g. two, three or four, carbon atoms are replaced by oxygen, and the adjacent carbon atoms are substituted by oxo, representing an ester function -O-CO-; or a combination of alkylene and/or one, two or three modified alkylene groups as defined under (a) to (c) hereinbefore, optionally containing substituents.

Substituents considered are, for example, CrC₆-alkyl, e.g. methyl, d-C₆-alkoxy, e.g. methoxy, CrC₆-acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro, and also antibody epitopes (peptide fragments consisting of 8 to 30 naturally occurring α-amino acids), biotin or (photo)reactive groups.

Further substituents considered are e.g. those obtained when an α-amino acid, in particular a naturally occurring α-amino acid, is incorporated in the spacer B wherein carbon atoms are replaced by amide functions -NH-CO- as defined under (b). In such a spacer B, part of the carbon chain of the alkylene group is replaced by a group -(NH-CHR-CO)_n- wherein n is between 1 and 10 and R represents a varying residue of an α-amino acid.

A spacer B is preferably a straight chain alkylene group with 1 to 25 carbon atoms or a straight chain polyethylene glycol group with 1 to 8 ethyleneoxy units, optionally attached to the chromophore C by an amide function -NH-CO-.

The most preferred spacer B is formed of a straight chain alkylene group with 1 to 10 carbon atoms containing two or three amide bonds, one connecting the spacer B to the chromophore C. This spacer offers the possibility to use e.g. commercially available fluorescent probes C containing activated carboxylic acids. The amide bond formation allows to modulate the chain length by varying the number of carbon atoms in the straight chain alkylene group as well as to incorporate additional functionalized groups at branching points (antibody epitopes, biotin or (photo)reactive groups). Alternatively, the polymethylene chain can be replaced by a polyethylenoxy spacer to be connected to the chromophore C through an amide bond offering again the possibility to modulate the chain length and to incorporate additional functionalized groups.

The conjugates of the invention are manufactured by methods known in the art, although hitherto not applied to the particular compounds envisaged. General methodology is shown in Scheme 2, and a particular example is described in Example 1 (Scheme 3).

Care must be taken that labelling does not abolish receptor recognition by the ligand, e.g. through steric hindrance or electrostatic effects. To circumvent adverse effects of this sort the chromophoric label is, for example, attached to the ligand through a spacer (Moiety B) of variable length and chemical structure.

It can be of importance in certain applications of the preferred embodiment that the emitting properties of the label change upon the interaction of the conjugated ligand with its cognate receptor. In other applications it is preferred that these fluorescence properties do not change.

Scheme 2 shows examples where either amide-type or ether-type of spacers are used. The spacer is introduced by substituting an amino or a hydroxymethyl group on the ligand A pyridine ring at position C-3 using standard procedures. The fluorophore ("fluo") C is coupled to the spacer through an amide bond by coupling the fluorescent probe- containing carboxylic acid with a primary amine on the spacer B using usual activating

methods, for example EDC/NHS. Alternatively, commercially available fluorescent probes linked through a spacer to an active ester group (i.e. NHS-esters) are coupled to an aminomethyl or an α-aminoacetamido group on C-3 of the pyridine ring.

Significant biological activity of the conjugates (labelled ligands) is desired in the embodiments of the invention. It is meant that their efficacy in binding to the cognate receptors is with an EC₅₀ ≤ 10^~6 M and/or that their efficacy is not diminished by more than 1000-fold compared to the unlabelled ligand. It is preferred that the conjugates (labelled ligands) of this invention activate the channel (as agonists or partial agonists) of the receptors.

The signal emitted by the chromophore moiety of the compounds of interest can be detected by a variety of methods or combination of methods. Non-limiting examples of detection modalities and experimental constellations are described.

Receptor-ligand interactions can be investigated to determine the presence of the receptor of interest, to quantify the amount of receptor present, and its affinity for the labelled compound, or of unlabelled compounds in a competition experiment. This can be done by e.g. methods for detection or imaging of the intensity or anisotropy of this emission either by steady-state or time-resolved methods. Moreover fluctuation of emission signals can be detected and evaluated using (cross) correlation or intensity distribution analysis methods. In the case that (i) several ligand molecules bind to a receptor or receptor oligomer, or (ii) an emitting ligand binds to a receptor protein labelled with an emitting moiety, methods like fluorescence or bioluminescence energy transfer can be applied to detect and quantify the interaction between receptor and ligand. For that purpose the receptor can be rendered emitting by any means known to those skilled in the art, e.g. by fusion with proteins (e.g. fluorescent proteins, carrier proteins and enzymes like transferases), chemical labelling with reactive emitters, or with small biochemical recognition elements like antibody epitopes or oligo-histidine tags.

Receptor activation can be detected and quantified by means of (i) electrophysiology on whole cell or patches from a cell using the pipette patch-clamp, planar patch clamp on a chip or automated (microfluidic) systems; (ii) ion indicators that report on the flux of certain ion species through the activated receptor; (iii) indicators that report on changes in membrane potential due to activation of the receptor's ion channel; (iv) monitoring changes in receptor diffusion and distribution within the plasma membrane; or (vi) monitoring receptor modification by enzymes (e.g. kinases) and/or internalisation. These different means can be applied to cells, featuring either native or induced expression of the receptor of interest, or membrane fragments thereof.

Simultaneous detection of ligand binding and receptor activation can be accomplished by any combination of the methods mentioned above. Receptor behaviour such as its location within a cell or tissue and co-localisation or interaction with other molecules can be studied as to determine the quantity, distribution and diffusion of these receptors using any kind of imaging set-up, in particular fluorescence detection.

Instruments that can be used to detect the emission can be any of e.g. a spectrometer, microscope, multi-well reader or imager, micro-fluidics device, cell sorter or cytometer. The acquisition mode in which the parameters are detected can be at specific time/end point values or time-dependent evolutions of the signal, and can be measured once or repeatedly under identical or dissimilar conditions. The repeated acquisition of data can be as detailed in C. Schreiter et al., ChemBioChem 6, 2187 (2005).

The receptor of interest can be present in cells or tissues which either feature native or heterologous expression of this protein. Cells can be cultured in suspension or attached to surfaces. Heterologous expression includes both transient and stable expression methods. Moreover the receptor can be formulated as homogenates or membrane preparations of the above cells or tissue. Furthermore, the receptor proteins may be presented in a solubilized and/or purified or reconstituted form. The above receptor containing preparations can also be immobilized by specific or unspecific chemisorption/physisorption on to a surface, or be trapped in solution by any electromagnetic means.

Examples

Example 1 - Synthesis of a chromophore labelled liqand, compound (10)

The synthesis of EPB-chlorocoumarin, compound (10), wherein the fluorescent coumarin is connected to C-3 of pyridine by a linker containing three amide bonds is described below and illustrated in Scheme 3. Scheme 3

1) N-Hydroxysuccinimide DCC/DMF

(±)-7-tert-Butoxycarbonyl-2-exo-(2'-chloro-3'-(2-aminoacetyl)amino-5'-pyridinyl)-7- azabicyclor2.2.11heptane (7):

3-(α-Chloracetamido)-substituted epibatidine (6) is synthesized according to a described procedure. Sodium iodide (1 10 mg, 0.48 mmol) is added to a solution of (6) (200 mg, 0.48 mmol) in THF (20 ml_) and stirred under reflux for 2 h. After cooling of the reaction mixture in a dry ice acetone bath, liquid ammonia (20 drops) is added in one portion to the mixture at -78⁰C, and the mixture is left overnight at room temperature (RT). After evaporation in vacuo, the residue is purified by chromatography using ethyl acetate/methanol (95/5) to obtain 180 mg (98%) of the title compound. ¹H NMR (200 MHz, CDCI₃): δ (ppm) = 10.08 (s large, 1 H, N-H), 8.68 (d, 2.2 Hz, 1 H), 8.10 (d, 2.2 Hz, 1 H); 4.37 (s large, 1 H); 4.21 (s, 1 H); 3.53 (s, 2H); 2.88 (m, 1 H), 2.04 (dd, 1 H); 1 .97 (m, 1 H); 1.86 (m, 1 H); 1.67 (m, 1 H); 1 .43 (s, 9H). N-(5'-Carboxypentyl)-6-chloro-7-hvdroxy-coumarin-3-carboxamide (9): 6-Chloro-7-hydroxy-coumarin-3-carboxylic acid (8) is synthesized according to described procedures (Y. Zhao et al., J. Amer. Chem. Soc.,126, (2004)). To a solution of (8) (200 mg, 0.833 mmol) in DMF (3 ml_), N-hydroxysuccinimide (95 mg, 0.833 mmol) is added. After dissolving, the reaction mixture is cooled at O'C for 1 h and DCC (88 mg, 0.916 mmol) is added. The reaction mixture is stirred for 30 min at O'O followed by an additional 2 h at RT. To this mixture is added a solution of 6-aminocaproate ethyl ester hydrochloride (200 mg, 1 .25 mmol) and triethylamine (350 μl_, 2.5 mmol) in DMF (1 ml_). The resulting mixture is stirred at RT overnight before being extracted with ethyl acetate. The residue is purified by chromatography using ethyl acetate as eluent to obtain 220 mg of the ethyl ester of compound (9), yield 70%. ¹H NMR (CDCI₃) : δ (ppm) 1 .26 (t, J = 7.08 Hz, 3H); 1 .57-1.70 (m, 6H); 2.32 (t, J = 7.34 Hz, 2H); 3.5 (m, 2H); 4.13 (q, J= 7.1 ; 14.2 Hz, 2H); 7.07 (s, 1 H); 7.68 (s, 1 H); 8.78 (s, 1 H). To a solution of this ethyl ester (220 mg, 0.5789 mmol) in dioxane (15 ml_) is added 2N hydrochloric acid (20 ml_) for 1 h followed by an addition of 5 drops of 12 N hydrochloric acid. The reaction mixture is stirred overnight at room temperature. The solvent is concentrated under reduced pressure. The remaining solution is lyophilized to yield the title compound (9) (155 mg, 70% yield from ethyl ester).

Synthesis of EPB-chlorocoumarin (10):

To a solution of (9) (10 mg, 0.0283 mmol) in DMF (3 ml_), N-hydroxysuccinimide (3.5 mg, 0.0283 mmol) is added. After dissolution the reaction mixture is cooled at O'O for 1 h. DCC (6.7 mg, 0.031 1 mmol) is added. The reaction mixture is stirred for 30 min at O'O and an additional 2 h at RT. To this mixture is added a solution of (2) (20 mg, 0.0526 mmol) and diisopropylamine (90 μl_, 0.526 mmol) in DMF (2 ml_). The resulting mixture is stirred at RT overnight. The mixture is purified by HPLC. The elution is performed at a flow rate of 4 ml_/min with a linear gradient of acetonitrile in an aqueous solution of TFA (0.1%) from 0 to 100% (v/v) over 30 min. The remaining solution is freeze dried to give the BOC- protected derivative of compound (10). To this BOC derivative dissolved in 2 ml_ anhydrous methylene chloride, 2 ml_ of trifluoroacetic acid is added dropwise. The mixture is stirred at room temperature for 2 h. The solvent is evaporated under reduced pressure and the compound is finally triturated in an anhydrous HCI/ether solution to obtain the hydrochloride salt of compound (10). The mixture is purified by HPLC. The elution is performed at a flow rate of 4 ml_/min with a linear gradient of acetonitrile in an aqueous solution of TFA (0.1%) from 0 to 100% (v/v) over 30 min. The yield is 35% (6 mg). ¹H NMR (CD₃OD): δ (ppm) 8.68 (s, 1 H); 8.31 (s, 1 H); 8.12 (s, 1 H); 7.82 (s, 1 H); 6.85 (s, 1 H); 4.53 (br s, 1 H); 4.33 (s, 1 H); 4.07 (s, 2H); 3.42 (m, 2H); 2.45 (dd, 1 H); 2.34 (t, 2H); 1 .90- 2.12 (m, 6H); 1.61 - 1 .78 (m, 4H); 1.45-1 .53 (m, 2H).

The compound is further characterised by high resolution MS; m/z: 616.3049/618.3046. In a buffer of pH 7.2 the maximum of absorbance is 408 nm with an extinction coefficient of 44000 M^"1cm^"1. Upon excitation at 408 nm, emission is characterised by a maximum at 450 nm and a quantum yield of 0.77.

Example 2 - Synthesis of additional chromophore labelled EPBs

A set of fluorescent EPBs was prepared using methods as in Example 1. Compound 1 1 , EPB-coumarin, was made as compound 10 except using the N-hydroxysuccinimide (NHS) ester of 7-hydroxy-coumarin-3-carboxylic acid. Compounds 12 to 14 were prepared using commercially available NHS-activated fluorophores yielding EPB-Cy5 (12), EPB-Cy3 (13) and EPB-Atto610 (14), see Scheme 4 for the structures of the NHS-activated fluorophores and the chromophore labelled EPBs.

Scheme 4

Example 3 - Binding of a chromophore labelled liqand to nAChR subtypes

Binding studies for the three synthesized probes on membranes from rat brain containing mainly the α4β2 subtype and Torpedo membranes containing muscle-type nAChR.

[³H](-)-Cytisine binding assay with rat brain membranes: Binding conditions use a modified procedure described by L. A. Pabreza et al., MoI. Pharmacol. 39, 9 (1991 ). Samples that contain 200 to 400 μg of protein, 6 nM [³H](-)cytisine (30.4 Ci/mmol) and various concentrations of ligands (0.1 nM to 1 μM) are incubated in a final volume of 200 μl for 75 min at 4⁰C in triplicate. The suspensions are filtered through GF-C (Whatman) and counted. Non-specific binding is determined in the presence of 10 μM (-)-nicotine. The result is shown in Fig. 1A.

[¹²⁵I]-O-BgTx binding assays with Torpedo membranes: [¹²⁵I]-O-BgTx binding is determined in membranes prepared from Torpedo marmorata (T. Saitoh and J. -P. Changeux, Eur. J. Biochem. 105, 51 (1980), T. Grutter et al., Biochemistry 38, 7476 (1999)). The result is shown in Fig. 1 B.

Example 4 - Activation of nAChR by chromophore labelled ligands

The demonstration that said components act as agonists is delivered with electrophysiology using nAChR subtypes expressed in Xenopus oocytes or HEK 293 cells.

Fig. 2A shows the activation of neuronal α4β2 and α3β4 nAChR expressed in oocytes by EPB-chlorocoumarin (10) and EPB-Cy5 (12), demonstrating that these compounds are agonists for these neuronal nAChR subtypes. Fig. 2B shows the activation of human muscle nAChR expressed in HEK 293 cells by EPB-Cy5 (12).

Fig. 2C shows the concentration dependency of human muscle nAChR by chromophore labelled EPB. All ligands are able to elicit channel currents and are thus agonists for this nAChR subtype. EPB-chlorocoumanin, EPB-coumarine and EPB-Cy5 are full agonists whereas EPB-Cy3 and EPB-Atto610 are partial agonists.

Example 5 - In cellulo labelling

Binding and dissociation of epibatidine-Cy5 is measured as an accumulation and depletion of fluorescence on the membrane of a living HEK293 cell, expressing adult wild- type nAChR (Fig. 3). HEK cells growing on glass coverslips placed on a sample holder of a microscope are illuminated and fluorescence is detected. Using a solution changer either buffer or buffer containing Cy5-modified epibatidine is flown over the cells. Thus, the binding and dissociation of the labelled ligand can be quantified (Fig. 3A). Fluorescence intensity on the membrane of a HEK293 cell, expressing the adult wild-type nAChR, is shown as a function of time during addition of fluorescent ligand (50 nM epibatidine-Cy5) and rinsing with buffer. Kinetic constants can be determined by single exponential fits. The accumulation kinetics is fitted by a single exponential curve assuming its rate constant k_acc being a linear combination of the binding constant k_on, the dissociation constant k_off and the rate of photobleaching k_bι_each : k_acc = k_on + k_off + k_bι_each- Identically, the depletion rate constant k_depι is given: k_depι = k_Oft + k_bι_eac_h- By repetition of the experiment on the same cell and variation of the acquisition rates it is shown that for the settings used (laser intensity, exposure time) k_depι is dominated by k_off, since it is approximately independent of the acquisition rate.

Overlay of fluorescence intensity plots acquired during the dissociation phase and their fits for different acquisition rates is shown in Fig. 3B. Curves do not overlap because they are plotted in the frame space and not in time space. Values of k_depι obtained from monoexponential fits to intensity profiles as a function of the acquisition rate. k_depι is dominated by k_off, which does not depend on the acquisition rate (Fig. 3C). The average rate constants are k_on = 5.6 ± 0.5 nM-s^"1 and k_off = 0.34 ± 0.01 s^"1. The resulting binding constant K_d = 16±2 nM is in good agreement with the obtained EC₅₀ value of 30±10 nM.

Example 6 - Observation in cellulo of individual nAChR labelled with EPB-Cv5

The demonstration that the said components can be used for highly sensitive investigations of nAChRs in live cells is given in Fig. 4.

Human muscle nAChR are expressed in HEK 239 cells (Fig. 4A). Upon incubation of such cells with low concentrations of EPB-Cy5, single EPB-Cy5 molecules bound to cell surface exposed nAChR are observed as small fluorescent spots (Fig. 4B). These spots are considered to be single molecules as they feature single step photo-bleaching or disappearance, and a diffraction-limited size.

The diffusion of single receptor molecules within the plasma membrane of the living cell was monitored by acquiring a sequence of images and subsequent determination of the position of the fluorescent spots. Fig. 4C shows the trajectory of one such nAChR molecule.

The said components thus allow observation of nAChR with ultimate sensitivity, i.e. detection of a single molecule.

Claims

1 . A conjugate of an agonist ligand directed to ligand-gated ion channels (LGICs) of the Cys loop-family with a chromophore.

2. The conjugate as claimed in claim 1 of the general structure

wherein A is an agonist ligand directed to ligand-gated ion channels (LGICs) of the Cys loop-family of high affinity and high selectivity, B is a bond or a spacer, and C is a chromophore.

3. The conjugate as claimed in claim 2 wherein the agonist ligand A has an affinity K ≥ 10⁶ M^"1 and a selectivity > 1 Ox to ligand-gated ion channels (LGICs) of the Cys loop- family.

4. The conjugate of anyone of claims 1 to 3 wherein the ligand is directed to a ionotropic acetylcholine receptor.

5. The conjugate of anyone of claims 1 to 3 wherein the ligand is directed to a mammalian ionotropic acetylcholine receptor.

6. The conjugate of claim 4 or 5 wherein the ligand is a specific nicotinic acetylcholine receptor (nAChR) agonist.

7. The conjugate of anyone of claims 1 to 6 wherein the ligand is a compound of formula (1 )

wherein the circle represents a mono- or bicyclic aliphatic amine, n is 0 or 1 and R is hydrogen or alkyl and wherein X represents a bond to the chromophore C or spacer B; or a derivative thereof.

8. The conjugate of claim 7 of formula (1 ), wherein R is alkyl.

9. The conjugate of claim 7 or 8 of formula (1 ), wherein the mono- or bicyclic aliphatic amine has between 5 and 10 ring members; or a derivative thereof.

10. The conjugate of claim 9, wherein the derivative is a compound further substituted in the mono- or bicyclic amine at a carbon atom by alkyl, hydroxy, alkoxy, halogen, oxo, acyloxy or acylamino, and/or further substituted in the pyridine nucleus by halogen.

1 1 . The conjugate of claim 7 or 10, wherein the ligand is epibatidine of formula (2)

(2) wherein X represents a bond to the chromophore C or spacer B; or a derivative thereof.

13. The conjugate of claim 7, 8 or 10, wherein the ligand is the compound of formula (3)

wherein X represents a bond to the chromophore C or spacer B; or a derivative thereof.

14. The conjugate of claim 7, 8 or 10, wherein the ligand is the compound of formula (4)

(4) wherein X represents a bond to the chromophore C or spacer B; or a derivative thereof.

15. The conjugate of anyone of claims 1 to 6 wherein the ligand is the compound of formula

16. The conjugate of claim 15, wherein the derivative is a compound further substituted in the five-membered heterocyclic ring at a carbon atom by alkyl, hydroxy, alkoxy, halogen, oxo, acyloxy or acylamino, and/or further substituted in the pyridine nucleus by halogen.

17. The conjugate of anyone of claims 2 to 16, wherein spacer B consists of an optionally substituted straight or branched chain alkylene group with 1 to 30 carbon atoms, wherein optionally (a) one or more carbon atoms are replaced by oxygen;

(b) one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function -NH-CO-;

(c) one or more carbon atoms are replaced by oxygen, and the adjacent carbon atoms are substituted by oxo, representing an ester function -0-C0-; or a combination of alkylene and/or one, two or three modified alkylene groups as defined under (a) to (c) hereinbefore, optionally containing substituents.

18. The conjugate of claim 17 wherein spacer B consists of a straight chain alkylene group with 1 to 10 carbon atoms, wherein one, two or three carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function -NH-CO-.

19. The conjugate of anyone of claims 2 to 18 wherein the chromophore is a fluorophore.

20. The conjugate of claim 19 wherein the fluorophore is a QSY-dye, a Blackhole quencher, Cy5Q, Cy7Q, a FluoQuench, or Dabcyl.

21 . The conjugate of claim 19 wherein the fluorophore is a carbopyronin, coumarin, (indo-)cyanine, 4,4,-difluoro-4-bora-3a,4a-diaza-5-indacene, rhodamine, oxazine, or phycoerythrine; a green fluorescent protein or a homologue thereof; a chelate, cryptate or complex of the metals europium, rubidium or osmium; or a CdSe quantum dot.

22. The conjugate of claim 19 wherein the fluorophore is coumarin, chloro-coumarin, Cy5, Cy3 or Atto610.

23. A method of characterisation and quantification of receptors, wherein a conjugate as claimed in anyone of claims 1 to 22 is contacted with the corresponding receptor, and the amount of chromophore bound to the receptor or the activation of the receptor is measured.

24. The method of claim 23 wherein the receptor is present in a cultured cell in suspension or attached to a surface.

25. Use of a conjugate as claimed in anyone of claims 1 to 22 for the characterisation of the receptor proteins and/or their interaction with the agonistic ligand.

26. Use according to claim 25 wherein the interaction with the agonistic ligand is measured in a competition experiment with an unlabelled compound.