WO2001040805A1

WO2001040805A1 - Steroid compounds for steroid receptor binding assays

Info

Publication number: WO2001040805A1
Application number: PCT/EP2000/011803
Authority: WO
Inventors: Wilhelmus G. E. J. Schoonen
Original assignee: Akzo Nobel N.V.
Priority date: 1999-11-30
Filing date: 2000-11-24
Publication date: 2001-06-07
Also published as: EP1238285A1

Abstract

The invention provides a compound having binding affinity for a receptor and comprising a steroid skeleton in its molecular structure, which compound is according to formula (1) wherein: Bu is a sterically bulky structure or Bu is a molecular moiety having high affinity for a sterically bulky molecular structure; A is -NH-, -O-, -C(O)- or -S-; Y is a branched or unbranched, saturated or unsaturated chain of 2 to 18 atoms of carbon, which chain is optionally interrupted by replacements of carbon atoms by oxygen, nitrogen or sulfur atoms and is optionally substituted with keto, hydroxyl, sulfhydryl or halogen groups; X is a carbon atom or an arylene group linked to the steroid skeleton with a carbon or an oxygen atom; Ste is a group with a steroidal skeleton, having binding affinity for a steroid receptor; and dotted lines represent optional double or triple bonds. The invention also provides for a method for determination of binding between a compound having a molecular group L in its molecular structure and a compound having a molecular group R in its molecular structure, in which method L is the group Ste as defined above and R is a steroid receptor.

Description

STEROID COMPOUNDS FOR STEROID RECEPTOR BINDING ASSAYS

The invention relates to a compound having binding affinity for a receptor and comprising a steroid skeleton in its molecular structure, and relates to a binding assay in which such a compound can be used.

Binding assays are based on the principle that the binding of a receptor (R) and a compound having a molecular group which can function as a binding ligand (L) for that receptor form complexes of which the presence can be detected with the help of tracers, also called labels or probes. Usually the tracers are radioactive isotopes incorporated into the molecules of the ligand. For direct ligand-receptor binding assays with ligands of small molecular weight, assays with a radioactively labelled ligand are the only available methods presently. A method without the need of a radioactively labelled ligand would be preferable. A principle for a binding assay without the use of a radioactive label is a fluorescence resonance energy transfer (FRET) assay. For an indirect measurement of ligand-binding with a ligand of small molecular weight and using a FRET assay, see for example WO 99/ 18124. A FRET assay is based on the principle that the binding between the ligand and the receptor brings two different molecular fluorochrome domains in close proximity, such that transfer of excitation energy between the paired fluorochrome domains becomes probable and measurable by light emission. The receptor and a ligand are each made to be associated with one member of the pair of fluorochromes. Usually a fluorochrome absorbs light at a particular wavelength and emits the light in random directions at the same or a longer wavelength. Under special circumstances the emission of energy by light can be replaced by energy transfer to another fluorochrome which emits the energy by light at its own characteristic wavelength. This transfer of excitation energy between fluorochromes is dependent on the close proximity of the two fluorochromes and can be understood as a form of quantum mechanical resonance. When the proximity of the fluorochromes is facilitated by associating each fluorochrome to one molecule of a ligand-receptor pair, the amount and the wavelength of fluorescent light is made to be predictably dependent on the binding of the ligand to the receptor, together forming a complex which also comprises the fluorochromes. The association between a molecular fluorochrome and a ligand or a receptor can be made by a covalent bond between the ligand or receptor and the fluorochrome, creating a new compound with a fluorochrome domain and the ligand or receptor as molecular group. The association can also be effectuated by non-covalent binding, for example by using an intermediary antibody to the receptor. The fluorochrome, in turn, can be bound by a covalent bond to the antibody. In other variants the association is effectuated with the help of an intermediary molecular group such as a covalently linked biotinyl moiety, which binds non- covalently to proteins with the property to bind biotin with high affinity, such as avidin or streptavidin. The fluorochrome is then attached to the avidin or streptavidin. FRET assays are frequently applied since 1998. A difficulty in setting up such a binding assay is to find a method to associate the binding molecules and the fluorochrome domains, while avoiding the complication that the associations interfere with the binding. In the known FRET assays the association succeeds because the molecules for which the binding is determined are large protein-like molecules to which molecular groups are more easily associated without consequences for the binding. For example, the measurement of binding between interleukin 2 with its human receptor is made possible by labelling a monoclonal antibody against the alpha chain of the human interleukin- 2 receptor with the fluorochrome Cy5 and by labelling recombinant human interleukin-2 with a fluorescent europium chelate (Stenroos, Hurskainen, Eriksson, Hemmila, Blomberg and Lindqvist: Homogeneous time resolved IL-2-2R alpha assay using fluorescence energy transfer. Cytokine Nol 10 pp 495-499, 1998). With incoming light absorbed by the europium chelate, the latter can act as donor of energy to the acceptor Cy5 when, in a homogeneous mixture, the labelled interleukin-2, the labelled antibody to the interleukin receptor, and the monoclonal receptor itself are mixed together. The binding between interleukin-2 and the receptor brings Cy5 and the europium chelate into close proximity within a single molecular complex. This can be observed by the measurement of fluorescence light at the emission wavelength of Cy5. With large molecules, such as proteins and peptides, it is relatively easy to construct molecular structures comprising the ligand and a fluorochrome such that the binding properties of the ligand are hardly influenced. However, with molecules with molecular weights smaller than 1000 Dalton, for example with steroid compounds, the linking to other molecules easily interferes with the binding to the receptor (see for example Gao et al.; Chemical Reviews, Nol 99, pp 723-744, 1999). A partial succes is disclosed in EP 0003583, wherein the link of a small fluorescent molecule to a steroid skeleton for detection of the presence of steriod receptors by binding of the fluorescent labelled steroid is described.

This invention provides for a compound and its use thereof for a binding assay, which compound has binding affinity for a receptor and comprises a steroid skeleton in its molecular structure, and which compound is according to the formula

Bu-A-Y≡^ ΞX— Ste formula 1 wherein: Bu is a sterically bulky structure or Bu is a molecular moiety having high affinity for a sterically bulky molecular structure; A is -ΝH-, -O-, -C(O)- or -S-; the latter is preferred;

Y is a branched or unbranched, saturated or unsaturated chain of 2 to 18 atoms of carbon, which chain is optionally interrupted by replacements of carbon atoms by oxygen, nitrogen or sulfur atoms and is optionally substituted with keto, hydroxyl, sulfhydryl or halogen groups; an unbranched, saturated chain of 2 to 18 atoms of carbon, which chain is optionally interrupted by replacements of carbon atoms by oxygen atoms, is preferred; X is a carbon atom or an arylene group linked to the steroid skeleton with a carbon or an oxygen atom; a 1 ,4-phenylene group is preferred; Ste is a group with a steroidal skeleton, having binding affinity for a steroid receptor; and dotted lines represent optional double or triple bonds. Solutions of a compound according to formula 1 are used in binding assays and it is therefore an important aspect of this invention that a solution of a compound as defined by formula 1 is made available by this invention, whereby the solution is made to be biocompatible with natural macromolecules. This compatibility of the solution means, for example, that proteins should not denature in the solution. Solutions in water, possibly containing naturally occurring ions and only minor amounts of organic solvents, will in general be suitable. Such solutions are familiar to the person skilled in the art.

Binding affinity of ligands to receptors is a well known concept in the art and is established with binding assays, of which there are several variants. Usually these are done by assessing competition with a radiolabelled ligand for binding with a receptor, but an assay in which a compound of the present invention is used can also be used to assess binding affinity of ligands for receptors. This will be described in more detail later.

The term 'a sterically bulky molecular structure', as used in this description, means a protein or protein-like structure, having a molecular weight of at least 5000 Daltons. Such a structure can be a fluorochrome, for example allophyco(thio)cyanin, or it can be a protein or protein-like structure to which a fluorochrome is attached. Also, compounds according to formula 1 in which Bu- is a protein which is unrelated to a fluorochrome, but which can still be used to label the ligand, such as luciferase or other enzymes useful to trace a ligand in a binding assay, are also made available by the present invention.

A molecular moiety having high affinity for a sterically bulky molecular structure is for example a biotinyl group. This group has extremely high affinity for avidin or avidin-like proteins, such as streptavidin. A fluorochrome domain can be attached to the avidin-like protein. The fluorochrome domain is, according to such a construct associated with the steroid group and can serve as a label in a binding assay.

The symbol — /\—Y ====χ _m formula 1 represents a spacer. The spacer enables the association of a sterically bulky structure to the steroid ligand without serious interference with the binding of the ligand to the receptor. A spacer is defined here as a molecular chain with two ends with a length of at least 3 atoms, but preferably more than 6 atoms. The length of the spacer is usually not longer than 20 atoms, because a compound wherein the steroid is linked to the spacer is preferably easy to handle by being suitable for solubilisation or purification. The chain length of the spacer is counted by starting from the atom attached to the steroid skeleton and ending with the atom, having been part of the precursor molecule before the bond with the bulky structure or molecular moiety having high affinity for the sterically bulky molecular structure was made and which atom is making the chemical bond to the sterically bulky molecular structure or the molecular moiety which has high affinity for the sterically bulky structure. The spacer is at one of its ends linked to Ste, which is the steroid skeleton of the ligand L. At the other end the spacer enables the association with a sterically bulky structure. This association can be either by a chemical bond to the sterically bulky structure or by a chemical bond to said molecular moiety having high affinity for the sterically bulky molecular structure.

Among various possibilities the spacer can be connected to the steroid skeleton at the atom in position 1 1 without interfering with the binding of the steroid to its receptor. Another suitable position is at the 7 position and for glucocorticoids the 17-position is suitable. Substitutions at the 7 and 11 positions of the steroid skeleton often have similar consequences for affinity to the receptor. Disclosures on steroids with long chains on the skeleton and having affinity for the corresponding receptor are US 4014909, US 3959260 and Jin et al (Steroids; Nol 60 pages 512-518; 1995). The 1 1 position is the most preferred position for connecting the spacer. Steroids with high affinity for the estrogen receptor and having a long chain at the 11 position of the steroid, in particular in the preferrable β-position, are known from the publication WO 93/ 13123 by A.

Claussner, F. Νique, J.-G. Teutsch and P. Nan De Nelde. It is surprising that such a huge structure as a fluorochrome protein or a biotinyl group carrying a protein, can be connected to a steroid without loosing to a major extent the affinity of the steroid for the receptor.

A group -Ste in the formula 1, which group has a steroidal skeleton similar to a steroid with binding affinity for a steroid receptor will usually have the same structure as a known ligand with binding affinity to steroid receptors. Obviously, the structure of the endogenous ligand for a steroid receptor can be chosen for -Ste. Thus estradiol, linked at the 1 1 position to the spacer, is suitable for an assay for estrogenic compounds. The structure in formula 2, wherein Bu, A, Y and X have the same meaning as previously defined, is an example of a suitable compound for an assay for progestogenic compounds. The compounds depicted in formula 3 and 4a or 4b, wherein X, Y, A and Bu have the same meanings as previously defined, are suggested for use in assays with androgen or glucocorticoid receptors, respectively.

formula 2 formula 3 formula 4a

Formula 4b For the preparation of a compound according to formula 1 a compound is first prepared with a molecular structure according to the general formula

Fu-Y≡H_^=X— ste formula 5 wherein:

=-=Y X=^"=^"=Ste i_s according to the previously defined and preferred meanings and Fu is a functional group suitable for coupling reactions such as a sulfhydryl, amino or carboxyl group, each optionally with a protecting group. The Fu group can be used to connect the compound to the bulky structure or to the molecular moiety to which a bulky group can bind non-covalently. Such a functional group is commonly a sulfhydryl, amino or carboxyl group, but other suitable groups are not excluded. Compounds with such groups can be prepared with protecting groups in order to preserve the reactive group during storage or further handling of the compound. Protecting groups are well known in the art. The publication of T.W. Green and P.G.M. Wuts: Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons Inc, New York, 1991 and the regular updates published in Contemporary Organic Synthesis of which a recent issue is by K. Jarowicki and Ph. Kocienski in

Contemporary Organic Synthesis vol 3 pp 397-431, 1996 are included for reference in this text. Most commonly used protecting groups are, optionally halogenated, acyls, such as acetyl for a sulfhydryl group. Protection of an amino group generally takes place by urethane functions such as the acid-labile tert-butyloxycarbonyl group, benzyloxycarbonyl group and substituted analogues, the base-labile 9- fluorenylmethyloxycarbonyl group or the phthaloyl group. Esterification of carboxyl groups, for example with t-butylalcohol, is a commonly available method to protect carboxyl functions. A preferred compound is a compound in which Fu is a protected sulfhydryl, the protection group being an acyl group. For purposes of definition we also refer to this preferred group for Fu as acylthio group. A preferred starting compound for use in this invention towards a fluorochrome-labelled steroid ligand for an assay for estrogen ligand-estrogen receptor binding is 1 lβ, 17β- l l-[4- [5-acetylthiopentyloxy]-phenyl]-estra- l ,3,5(10)-triene-3, 17-diol.

The present invention also provides a method for determination of binding between a compound having a molecular group L in its molecular structure and a compound having a molecular group R in its molecular structure by first bringing the compounds together to form a mixture wherein molecular complexes are formed in which complexes L and R are bound to each other, followed by exposing the mixture to light for addition of excitation energy to the mixture and measuring fluorescence light, said molecular complexes each having a fluorochrome domain of type E and a fluorochrome domain of type D, said domain E emits the fluorescent light as a consequence of donation of the excitation energy by the domain D, whereby the molecular group R is a receptor for the molecular group L, the molecular group L is associated with one of the two types of the said domains and the molecular group R is associated with the domain, which is not associated with the molecular group L, whereby a method for determination of binding according to this aspect of the invention is characterised in that R is a steroid receptor and the molecular group L has a steroid skeleton which is associated with one of the two types of the said fluorochrome domains via a spacer.

It was, unexpectedly, found that a sterically bulky molecular structure comprising a fluorochrome domain can be associated to the steroid without creating steric hindrance for the binding of the steroidal group L to its corresponding receptor by the use of the spacer. The advantage of the method is that the label of the ligand is not radioactive. The meaning of the term 'spacer' has been defined previously.

There are various fluorochromes known for fluorescence detection and several of those are particularly suitable for energy transfer by resonance. Ions of the rare earth elements of the lanthanide group, such as europium or terbium, are suitable fluorochromes useful as energy donors. These are fluorochromes of type D. The ions of these elements can be enclosed in the intramolecular cavity of a macropolycyclic complexing agent for enlargement of the efficiency of light absorption and attachment to other molecules. Europium (III) chelating agents are commercially available for example by EG & G Wallac (Turky, Finland) . Suitable complexing agents for europium¹¹¹ which are made available by this manufacturer are N^:-(p- isothiocyanatobenzyl)diethylene-triamine-N¹,N²,N³,N⁴-tetraacetic acid and AD 0062 LANCE-Eu-W1024.

Rhodamine, Cy5 and allophycocyanin (APC) and their variants are suitable fluorochromes for emission of the light. These are fluorochromes of type E. The emission of the light is at wavelengths outside the absorption spectrum of serum proteins, which prevents excessive quenching of the light signal in an assay. A variant of APC is in general a phycobiliprotein or modifications of such proteins such as cross-linked APC (xl-APC) and allophycothiocyanin. Cross-linked APC is made available by PhycoLink™ (product PJ25C; ProzymeiNc; San Leandro, CA, USA). The cross-link is between the α an β subunits of APC, making the secondary structure of the protein (±100 kDaltons) more stable. A well-known pair and preferred for use in this invention is formed by an europium (III) complex and APC or modified APC. The europium can be excited by a brief pulse of light at wavelength around 340 nm which may come from a laser source. By resonance energy transfer the excitation energy can be donated to APC which emits the energy as light with wavelength of 665 nm.

Distances between 10 and 75 A between europium and allophycocyanin are required to enable resonance energy transfer. The emission energy by europium and the transfer of energy last on average more than 100 μsec. When the brief pulse of incoming light is much shorter than the period for emission, and measurement of fluorescence emission is measured at a point in time chosen to be sufficiently long after light exposure, the background light scatter will be very low. This makes time resolved fluorescence measurements particularly suitable for accurate operation of an assay of the invention. An adapted Victor (EG&G Wallac) or Discovery (Packard Instrument Company, Meriden, CT, USA) can be used for (time resolved) fluorescence measurement. Thus, under brief exposure to light of a wavelength corresponding to the absorption band of europium the emission of fluorescent light at the wavelength which is characteristic for APC is dependent on the proximity of this molecule to europium and therefore the intensity of the emission at 665 nm reflects accurately and sensitively the preponderance of proximity of the europium/ allophycocyanin fluorochromes. From this parameter the degree of binding between the ligand and the receptor can be determined with calibration and calculation methods which are well-known to the skilled person in the field.

The assay of this invention can be used for determination of binding between steroid ligands and the corresponding receptors of the whole family of steroid receptors, more in particular for the classical steroid receptors, i.e. the androgen, estrogen, progestogen, glucocorticoid and mineralocorticoid receptor. A preferred embodiment of the method is the determination of binding between an estrogen and an estrogen receptor.

With the method of the invention it is easy to determine whether any other compound has an influence on the binding between a ligand and a steroid receptor corresponding to the ligand by adding the compound to the mixture of the method of the invention. A compound which has affinity for the steroid receptor or which has an allosteric influence on the receptor in the method can interfere with the binding of the ligand with the steroid receptor. This can be detected with the method of the present invention. In a homogenous time resolved energy transfer fluorescence assay all ingredients can be mixed sequentially and fluorescence measurement can be done after overnight incubation or shorter. The advantage of such an assay over the known radiolabelled ligand-competition assays for steroid receptors is that interaction with the receptor can be identified without a step for separation of the unlabelled receptors and labelled receptors. The method of this invention is non-radioactive, it is as sensitive as the regular radioactive screening assay technique, and it does not need wash or extraction steps.

The steroid receptor used in the assay of this invention is the receptor corresponding to the ligand in the sense that for example an estrogenic ligand corresponds to an estrogen receptor, a glucocorticosteroid corresponds to the glucocorticoid receptor, a progestogenic steroid corresponds to the progesterone receptor etceteras. The steroid receptor is for example, and preferably, one of an estrogen, a progesterone, an androgen, a glucocorticoid or a mineralocorticoid receptor. A more preferred embodiment of the invention is a method for the determination of binding between an estrogenic ligand and the estrogen receptor. The receptors should be used in highly purified form, preferably obtained with specific preparation methods. Such methods are well-known in the art or the receptors can be obtained from commercial sources.

For preparation of the components for the assay the fluorochromes are to be linked directly or indirectly to the ligand and the receptor. In this context the term linked has a meaning equivalent to the terms conjugated and coupled. Where covalent bonds between proteins must be made, such as between a phycobiliprotein and the receptor or an antibody to an avidin, well-known methods can be used. Usually the reagents used are known as linkers or cross-linking reagents. With such linkers among others sulfhydryl, amino or carboxyl groups can be linked to other functional groups. Examples of cross-linking reagents are succinimidyl 4- (N-maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC), N-succinimidyl iodoacetate and N-succinimidyl[4-iodoacetyl]aminobenzoate) (See catalogue of ProzymeiNc; San Leandro, CA, USA). With the reagent SMCC the maleimide group reacts to free sulfhydryl groups. Typically only 1 IgG sulfhydryl reacts with SMCC-protein conjugate due to steric hindrances. The sulfhydryl groups of proteins are exposed by dithiothreitol (HS- CC(OH)C(OH)C-SH; threo- l,4-dimercapto-2,3-butanediol) under nitrogen atmosphere. The coupling of SMCC-xl-APC to globuline is by exclusion of light. For completion, the remaining sulfhydryl groups are blocked by treatment with NEM (N-ethylmaleimide).

Similar methods can be used to make compounds wherein covalent bonds connect a receptor to an intermediary molecular moiety, or wherein a covalent bond connects the spacer to a protein, a fluorochrome or an intermediary molecular moiety. It is also possible to use reagents containing a biotin moiety, such as N-iodoacetyl-N- biotinylhexylenediamine or N-hydroxysuccinimide-long chain-biotin, which can connect a biotinyl moiety to a spacer. Such reagents are for example made available by Pierce™ (Rockford, IL, USA). The reaction conditions are specifically described in the source from which the coupling method, also known as conjugation method, is selected and should not need further description here. For coupling (= conjugating) biotin to the ligand or protein the water soluble sulfo-N-hydroxysuccinimide-long chain-biotin can be used.

Compounds according to formula 5 can be prepared by methods described in WO 93/ 13123 by A. Claussner, F. Nique, J.-G. Teutsch and P. Van De Velde, which is included in this text by reference.

When immunoglobulins are used for associating a fluorochrome with the receptor the immunoglobulins are identified, produced and purified by methods well-known in the art. See for example J.E. Coligan et al. Current protocols in immunology; publisher: John Wiley and Sons Inc, 1996; ISBN: 0 471-52276-7. Purified immunoglobulines are often commercially available. An immunoglobuline will be suitable for the method of this invention when it does not interfere with the binding of the steroid ligand to its corresponding receptor in the assay. It is a matter of standard procedures to test candidate immunoglobulines for such an absence of influence on the binding of ligand to receptor. Testing an antibody to a steroid receptor, having a binding domain to the hormone responsive element of DNA, for suitability in the method of this invention can for example be done in an electrophoretic mobility shift assay. For this technique, see for an example C. Massaad et al Properties of overlapping EREs: synergistic activation of transcription and cooperative binding ofER; Biochemistry volume 37: pp 6023-6032, 1998. In this assay the mobility of the pure antibody-steroid receptor-complex is measured and this mobility is compared with the mobility of the antibody- steroid receptor- complex in the presence of the corresponding steroid ligand and the radiolabelled hormone responsive element for that receptor. If the presence of ligand and hormone responsive element induces a shift in mobility the antibody is suitable for use in the assay of this invention. By biotinylating the antibody it can make the association with an avidin linked fluorochrome and the steroid receptor ligand to the antibody. A suitable antibody for the human α-estrogen receptor is the mouse antibody Mab-a-ERα with molecular weight 66258 Daltons from Stressgen™ (product SRA-1010 and referenced as clone C-542; for further details see Weigel et al Mol. Endocrinol vol 6: pp 1585- 1597, 1992, in which publication the antibody is characterised without referring to the name 'clone C-542').

Legends to the figures Figure 1 :

Dose dependent displacement of the APC-labelled estrogenic compound from the estrogen receptor by the estrogen estradiol- 17β in a 96 well plate according to the procedure as described in example 3. The ingredients were 0.8 pmol APC-labelled estrogen, 0.4 pmol recombinant estrogen receptor α (ERα), 0.4 pmol antibody-ERα-biotin, 0.4 pmol streptavidine europium.

Figure 2: Dose dependent displacement of the APC-labelled estrogenic compound from the estrogen receptor by the estrogen estradiol- 17β under four different incubation procedures in a 96 well plate as described in example 2 and Table 2. The ingredients were again 0.8 pmol APC-labelled estrogen, 0.8 pmol recombinant estrogen receptor α (ERα), 0.4 pmol antibody-ERα- biotin, 0.4 pmol streptavidine europium.

Examples

Example 1 Preparation of estrogen receptor ligand labelled with APC

Allophycocyanin .

a) Synthesis of l lβ, 17β- l l-[4-[5-acetylthiopentyloxy]-phenyl]-estra- l,3,5(10)-triene-3, 17-diol:

A mixture of 362 mg of l lβ-(4-hydroxyphenyl)estra-4,9-diene-3, 17-dione, 240 mg of potassiumcarbonate, 1 ml of 5-bromo- l-chloropentane and 5 ml of acetone was refluxed for 6 hr, while monitoring the reaction by thin layer chromatography (tic). Then water was added followed by acidification of the mixture by 2N HCl. The product was extracted with ethyl acetate. Chromatography of the crude material thus obtained gave 310 mg of 1 lβ- (4-(5-chloropentyloxy)ρhenyl)estra-4,9-diene-3, 17-dione; Mp 220-221 ; NMR 3.56, 3.92 (t,2x2,CH₂O and CH₂C1); 5.80 (s, l ,CH₃) ; 6.80, 7.08 AB, 4,H-aromatic); 0.57 (s, 3, CH₃). A suspension of 300 mg of 1 lβ-(4-(5-chloropentyloxy)phenyl)estra4,9- diene-3, 17-dione in 0.3 ml of acetic anhydride was added 0.15 ml of acetylbromide at 0°C. The product was extracted with ethylacetate. The organic phase was neutralised with NaHCOβ solution , and concentrated. The crude material thus obtained was dissolved in tetrahydrofuran (THF) and treated with a solution of 40 mg of LiOH in 1 ml of water. After stirring for 0.5 h the saponification was complete and the mixture was acidified to pH 3 by addition of aqueous 2N HCL. Followed by extraction of the product with ethyl acetate. The material thus obtained was dissolved in 1 ml of THF and 1 ml of 96% ethanol and treated with 30 mg of NaBH₄ to reduce the carbonyl at C17 into a 17β-hydroxy group. Upon stirring for 1 h the reduction was complete. The mixture was diluted with water and extracted with ethyl acetate. The crude material was chromatographed over silicagel and provided 1 10 mg of l lβ-(4-(5-chloropentyloxy)- phenyl)estra- l,3,5(10)-triene-3, 17β-diol. Mp 165- 166 °C, NMR 0.35 (s,3, CH₃), 3.52, 3.85 (2x t, 4, -CH₂C1 and -CH₂O-), 3.70 (m, 1 , CHOH), 3.95 (m, l ,CH- l lα), 6.4, 6.58, 6.62, 6.82, 6.96 (7 , aromatic H's). To a solution of 100 mg of 1 lβ-(4-(5-chloropentyloxy)-phenyl)estra- l ,3,5(10)-triene-3, 17β-diol in 5 ml of 2-butanone was added 200 mg of sodiumiodide. The mixture was re fluxed for 10 h. The reaction was monitored by tic. The reaction mixture was poured into water and the product extracted with ethyl acetate, to provide 100 mg of essentially pure l lβ-(4-(5-iodopentyloxy)- phenyl)estra- l ,3,5(10)-triene-3, 17β-diol. R_f 0.43

(hept/ ethyl ac. 1 / 1) Rf starting material 0.40. NMR 3.20 (2, t, -CH₂I). A mixture of 100 mg of l lβ-(4-(5-iodopentyloxy)-phenyl)estra-l ,3,5(10)- triene-3, 17β-diol and 40 mg of potassium thioacetate was heated in 4 ml of absolute ethanol for 1 h. The solvent was evaporated and water and ethylacetate were added. The organic phase was dried and concentrated and the residue chromatographed, to give 75 mg of l lβ-(4-(5- acetylthiopentyloxy)- phenyl)estra- l,3,5(10)-triene-3, 17β-diol as an amorphous material. Rf 0.38 (hept/ethyl ac. 1 / 1) , starting material Rf 0.42. NMR 2.90 (t, 2, -CH2S), 2.30 (s, 3 , S(CO)CH ), 0.33 (s, 3, I8-CH3), 3.65 (m, 1 , CHOH), b) Cross-linked APC (Product PJ25K, ProzymeiNc) is linked to succinimidyl 4-{N-maleimidomethyl}-cyclohexane- 1 -carboxylate (SMCC) according to the instructions by ProzymeiNC resulting in a solution containing APC linked to SMCC (APC-SMCC). c) The acetyl group of the acetylthio steroid of example la is removed by adding 78 μl of 0.1 M NaCl in water and 19.5 μl 0.1 M NaOH in water to 30 nmol of said steroid in 61 μl 100% methanol and incubating the mixture during 30 min at room temperature. After the incubation the solution is neutralised with 23 μl 0.1 M hydrochloric acid, followed by the addition of 171 μl methanol, 159 μl of a buffer solution with pH 7.5, consisting of 25 mM Tris-HCl, 0.1 M KC1 and 2 mM EDTA in water, and finally 98 μl of the mixture of APC-SMCC obtained under lb is added. For a second incubation the mixture is left overnight under nitrogen in a refrigerator. Hereafter the steroid-APC compound was desalted on an NAP- 5 column. The steroid-APC was stored at 4°C in a buffer solution with pH 7.5, consisting of 25 mM Tris-HCl, 100 mM KC1 and 0.5 mg/1 pentachlorophenol in water.

Example 2

Coupling of a steroid ligand to biotin with N-iodoacetyl-N- biotinylhexylenediamine (Pierce™; Rockford, IL, USA; product 21334ZZ). The acetylthio steroid of example la is deacetylated by adding 38.4 μl of 0.1 M NaCl in water and 9.6 μl 0.1 M NaOH in water to 30 nmol of said steroid in 30 μl 100% methanol and incubating the mixture during 30 min at room temperature. After the incubation the solution is neutralised with 1 1.4 μl 0.1 M hydrochloric acid, followed by the addition of 45 μl methanol and the conjugation with biotin is obtained by further addition of 16 μl 2 mM Iodoacetyl-LC-biotin (Pierce™; Rockford, IL, USA) in DMF and 78 μl of 50 mM TRIS/HC1 buffer having pH 8.3 and 5 mM EDTA. The mixture is incubated for 90 minutes at room temperature in the dark under nitrogen. Desalting for final purification is done with one sample by ultrafiltration and with another sample by C 18 solid phase extraction. For ultrafiltration a 70% methanol resistant Microsep filter of Filtron with a cut-off of 1 kD is used. Filtration was carried out twice with 1 ml of 40% methanol and the steroid labelled biotin was stored in a mixture of 40% methanol in water containing 25 mM TRIS/HC1 pH 7.5 and 100 mM KC1. For solid phase extraction of a second sample the column was pre-washed with 1 ml of methanol, followed by 2 ml of milli Q water. The biotin labelled steroid was dissolved in 20% methanol and absorbed to the column. The column was washed with 20% methanol /water and washed two times with 1 ml. The steroid biotin derivative was eluted in 1 ml of 100% methanol. The steroid labelled biotin was stored in 40% methanol with 25 mM TRIS/HC1 pH 7.5 and 100 mM KC1.

Example 3

Time resolved fluorescence energy resonance (TR-FRET) assay for binding of estrogen ligand with the α-estrogen receptor (ERα).

The estrogen receptor was a recombinant human estrogen receptor, product P2187 (molecular weight 66 kD) from PanNera Corporation (Madison, Wi, USA)

To the wells of a 96 wells-plate were added:

• 0.4 pmol ERα /well,

• 0.8 pmol steroid-APC of example lc per well and

• a concentration range from 0 to 12.6 pmol/well of 17β-estradiol The plate was incubated overnight at 4°C (Incubation A).

Then, 0.4 pmol 's biotinylated antibody for the ERα receptor of Stressgen™ (product SRA-1010) was added to each well and an incubation followed for 6 h at 4°C (Incubation B). The Stressgen™ antibody was biotinyl labelled with sulfo-N-hydroxysuccinimide-long chain biotin (product 21335 from Pierce™; Rockford, IL, USA) according to laboratory protocol Eu 586.

Finally 0.4 pmol/well of streptavidin europium (product AD 0062 labelling reagent from LANCE EU-W1024) was added and another overnight incubation at room temperature followed (Incubation C). After incubation C the wells were measured for time-resolved fluorescence with an adapted Victor2, type 1420 multilabel (EG&G Wallac) fluorescence counter with 320 nm light for excitation and measurement of 665 nm light for emission and time-resolution set at 50- 150 μseconds. Result:

Emission light counts reflect the binding of APC labelled ligand to the europium labelled receptor via the antibody coupling and diminishment by estradiol reflects selectivity of the binding. Figure 1 shows a good displacement of the APC-ligand by estradiol in a concentration range.

Example 4 Other incubation conditions were tested in this example.

Experiment Incubation A Incubation B Incubation C

1 6h 4°C Overnight 4°C Overnight 4°C

2 Overnight 4°C 6h 4°C Overnight 4°C

3 Overnight 4°C Overnight 4°C Overnight 4°C

4 Overnight RT Overnight RT Overnight RT

RT: Room temperature.

Other conditions were as in example 3

Results

Figure 2 shows the result of this comparison of different experimental conditions.

Conclusion: Experiment 2 gave the most favourable results for measurement of binding.

Claims

1. A compound having binding affinity for a receptor and comprising a steroid skeleton in its molecular structure, characterised in that the compound is according to the formula

wherein:

Bu is a sterically bulky structure or Bu is a molecular moiety having high affinity for a sterically bulky molecular structure; A is -NH-, -O-, -C(O)- or -S-;

Y is a branched or unbranched, saturated or unsaturated chain of 2 to 18 atoms of carbon, which chain is optionally interrupted by replacements of carbon atoms by oxygen, nitrogen or sulfur atoms and is optionally substituted with keto, hydroxyl, sulfhydryl or halogen groups;

X is a carbon atom or an arylene group linked to the steroid skeleton with a carbon or an oxygen atom;

Ste is a group with a steroidal skeleton, having binding affinity for a steroid receptor; and dotted lines represent optional double or triple bonds; or addition salts or solvates thereof.

2. A compound according to claim 1 , characterised in that Bu is cross- linked allophycocyanin, A is -S-, Y is an unbranched, saturated chain of 2 to 18 atoms of carbon, which chain is optionally interrupted by replacements of carbon atoms by oxygen atoms, and X is 1,4- phenylene.

3. A compound according to claim 2, characterised in that Y is pentyleneoxy.

4. The use of the compound according to claim 1 as a binding ligand for a receptor in a binding assay.

5. A method for determination of binding between a compound having a molecular group L in its molecular structure and a compound having a molecular group R in its molecular structure by first bringing the compounds together to form a mixture wherein molecular complexes are formed in which complexes L and R are bound to each other, followed by exposing the mixture to light for addition of excitation energy to the mixture and measuring fluorescence light, said molecular complexes each having a fluorochrome domain of type E and a fluorochrome domain of type D, said domain E emits the fluorescent light as a consequence of donation of the excitation energy by the domain D, whereby the molecular group R is a receptor for the molecular group L, the molecular group L is associated with one of the two types of the said domains and the molecular group R is associated with the domain, which is not associated with the molecular group L, characterised in that the molecular group L has a steroid skeleton which is associated with one of the two types of the said fluorochrome domains via a spacer.

6. The method according to claim 5, characterised in that the compound having a molecular group L is having a structural formula as defined in claim 1.

7. The method according to claim 5, characterised in that the receptor is a receptor selected from the group consisting of an androgen, an estrogen, a progesterone, a glucocorticoid and a mineralocorticoid receptor and the molecular group L is a steroid with affinity for the selected receptor

8. The method according to claim 5, characterised in that the receptor is an estrogen receptor and the molecular group L is a steroid with affinity for the estrogen receptor

. The method according to claim 5, characterised in that the domain E is allophycocyanin or a variant thereof and in that the domain D comprises a europium ion which is bound to avidin or an avidin-like protein.

10. The method according to claim 5, characterised in that the molecular group L is associated with domain E and the molecular group R is associated with domain D.

11. Use of the method according to claim 5 for determination of the influence of a compound on the binding between a ligand and a receptor corresponding to the ligand.

12. A steroid for use in the preparation of the compound according to claim 1 , which steroid has the formula

Fu-Y≡^-X— Ste

wherein: =~=Y=-=-__ :X ^•*■*■=^■= Ste ^₅ j-^g meaning as defined in claim 1 and Fu is a functional group suitable for coupling reactions, optionally with a protecting group.

13. The steroid according to claim 12, characterised in that Fu is an acylthio group.

14. The steroid l lβ, 17β- l l-[4-[5-acetylthiopentyloxy]-phenyl]-estra- l ,3,5(10)-triene-3, 17-diol.