METHOD FOR THE IDENTIFICATION AND CHARACTERIZATION OF NUCLEAR RECEPTOR LIGANDS

Abstract: The present invention is directed to a methodology to study the effect of ligands that can modulate functions of nuclear receptors, and specifically, their interaction with specific DNA or other transcription factors. These receptors play a major role in the cascade of inter and intracellular physiological and biochemical events. They generally have a modular construction which enables them to recognize and bind to certain DNA sequences, or binding elements, such as hormone response elements. Various ligands, such as hormones, affect this interaction. In accordance with the methodology presented herein, measurements of such interactions are taken in real time and kinetic pattern. This allows the user to identify and to characterize ligands that are able to affect a receptor's interaction with its binding element. Hence, the present invention provides a useful tool for the elucidation, and development of ligands that affect transcription of target genes, either directly or indirectly.

Description: METHOD FOR THE IDENTIFICATION AND CHARACTERIZATION OF NUCLEAR RECEPTOR LIGANDS Field of the Invention The present invention is directed to assay methodology to determine a kinetic pattern of the interaction between a nuclear receptor and the element it binds to, in the presence of a ligand that may have an affect on such interaction. Using the techniques herein described, it is now possible to assay for substances that upregulate, downregulate, in some other way modulate this interaction. Background of the Invention Steroid hormones are widely distributed small, lipophilic molecules that participate in mtracellular communication, and control a wide spectrum of developmental and physiological processes. Their effects are mediated by specific mtracellular receptors, a family of proteins that are characterized by a high affinity for the corresponding hormones and an ability to discriminate between structurally closely. related ligands. These iigand-activated receptors can modulate transcription of target genes by virtue of their binding to a specific sequence on DNA in target promoters known as hormone response elements. Though distinct proteins, these receptors are members of a large superfamily of hormone receptors and share many common structural and functional features ( 1 -4). Binding of 1 7β-estradιol (E 2 ) to estrogen receptor (ER) is followed by a conformational change, leading to dissociation of the receptor from the complex with the heat shock proteins hsp90 and p59 (5, 6), dimeπzation (7, 8) and activation of DNA binding (9) . The activated receptor can interact with basal transcription factors. These interactions are thought to stabilize the preinitiation complex at the promoter, allowing RNA polymerase to initiate transcription ( 10) . Recently, a number of transcπptional intermediary factors (TIFS) have been identified that can modify estrogen responsiveness, and several of these proteins interact with the ligand binding domain of the ER in gand-dependent manner ( 1 1 - 1 3). The ligand is generally accepted as playing a key role in the initiation of this cascade of events. However, it remains controversial as to whether estrogen affects the receptor's ability to bind specific DNA (ERE) . Initially, in vitro analysis performed with hER (HEO) expressed in HeLa cells, Xenopus oocytes, yeast, or produced by in vitro transcription/translation indicated that binding of ER to an estrogen response element (ERE) was hormone-dependent, and that E 2 induced the formation of receptor dimers (8). It was subsequently found that this protein (HEO) has an artifactual mutation (Gly 400→Val )whιch decreases hormone binding at 25°C, but not at 4°C ( 14). Subsequently it was reported that wild type hER (HEGO) binds DNA in absence of ligand ( 1 5, 16). Hormone independent formation of ER-ERE complex was also reported with crude extracts ( 1 7) and purified ER from calf uterus ( 1 8), rat uterine extracts ( 1 9), mouse uterine extracts (20) , transfected COS-1 cells (21 ,22) and SF9 cells infected with recombinant baculovirus (21 ) In contrast, gand-induced DNA-binding was reported with HEGO, produced by in vitro transcription/translation and in SF9 cells (23) In vivo ligand dependent and ligand-independent ERE-association was reported for hER and Xenopus ER using promoter interference assay (24,25) Conversely, it was also shown that hormone may be required to promote DNA-binding at low, but not at high concentrations of ER (26) Genomic footpπnting indicated that occupation of the ERE present in the apoVLDLII promoter region is hormone dependent (27) which suggests that the hormone is effecting ER interaction with ERE. The present inventors have discovered that ligand binding affects the kinetics of receptor interaction with its binding element, and particularly, nuclear receptor interaction with DNA It was previously reported that binding of hormone accelerates the kinetics of glucocorticoid and progesterone receptor binding to DNA (33), affects dimeπzation status and the kinetics of DNA-binding of vitamin D 3 receptor (28). Thus, the discrepancies reported previously with the ligand effect on ER-ERE interactions are due to the fact that the methodologies used could not detect the dynamics of the protein-DNA complex formation. There has long been a need to study such ligand affected interactions in a manner that will allow the dynamics of the interaction to be ascertained, and not merely the binding affinities. The present invention provides such a novel methodology, which among other attributes, allows one to examine receptor/response element interactions, and particularly, hormone receptor/hormone response element interactions, and the effect various substances have on these interactions. The use of surface plasmon resonance (SPR) methodology to accomplish this task is a particularly useful embodiment. Using this approach, the presently claimed methodology demonstrates that ligand binding dramatically affects the kinetics of receptor interaction with specific binding elements, such as DNA Further, the interaction of agonists or antagonists with such a receptor has effects on the kinetics of such receptor/response element, which can be measured by rates of such interactions. Summary of the Invention The present invention is directed to a methodology for determining a kinetic pattern of the interaction of a binding element with its nuclear receptor binding partner, wherein the binding complex formed by the interaction of the binding element with its nuclear receptor binding partner is further contacted with a substance whose effect on such complex formation, and the rate of dissociation of the binding, it is desired to determine. In preferred embodiments, a surface plasmon resonance detector device is utilized to assay this interaction The nuclear receptor binding partner may be derived from tissues selected from any of a variety of biological sources, including bone, cardiovascular, thyroid, brain, mammary gland, and reproductive tissues. The nuclear receptor binding partner may also be synthetically constructed utilizing recombinant techniques or techniques of o gonucleotide synthesis generally known in the art. The construction of an appropriate nuclear receptor may be carried out in a manner consistent with the needs of the user of the assay system, and the receptor may in fact comprise a synthesized portion of its naturally-occurring counterpart, or a hybrid or other chimeπc construct containing portions of two or more such nuclear receptors. Co-activators or co-suppressors may also be provided in the assay system. Nuclear receptors play an important role, and are thought to interact with, components of DNA transcπptional machinery with sequence specific transcription factors. Various ligands are believed to affect these interactions. In the present methodology, measurements of such interactions, which are taken in real time and kinetic pattern, allow the identification and characterization of ligands that are able to affect the receptor's interaction with its binding element. Brief Description of the Figures Figure 1 . Gel-filtration of ER-ERE complexes. 35 μg of purified hER in 50 μl of 50 mM Tπs-HCI, 1 50 mM NaCI, 10 mM MgCI 2 , pH 7.5. were incubated with 20 ng of [ 32 P]-labeled ohgonucleotide in the presence of [ 3 H]-Estradιol at 10 6 M for 30 mm at room temperature before this mixture was applied on Superdex 200 column. Radioactivity in fractions from the column was detected by liquid scintillation spectrometry. Figure 2. Analysis of the specificity of ER-ERE interaction using SPR First 2400 RU of Streptavidin were immobilized in all four flow cells, then surfaces with 1094 RU of DR, 943 RU of Vιt.A2, and 1063 RU of C3 (see Materials and Methods) were obtained by injection of 50 μl of biotinylated ohgonucleotide solution at 33 ng/μl over the surface with immobilized Streptavidm. 30 μl hER at 1 50 nM bound with E 2 was injected over the surface with different ohgonucleotides immobilized. Figure 3. Gradient DNA immobilization. A - Gradient of immobilized streptavidm in different flow cells was obtained by varying the Streptavidm injection time. The flow rate was 5 μl/min. The flow path was first set to allow serial injections through FC 1 to 4. After activation with EDC/NHS ( 1 ), the Streptavidm was injected at 10 μg/ml (2) and at first directed only to FC 1 . During injection the flow path was redirected to a serial injection, first in FCs 1 and 2 and later in FCs 1 to 3 and FCs 1 to 4. Thus, the total reaction time was 6, 4, 2 and 1 mm in flow cells 1 , 2, 3 and 4, respectively. The immobilization procedure was completed with blocking of excess (unreacted) ester groups on the surface with ethanolamine (3) and rinsing with 10 μl of 0.1 % SDS, to remove Streptavidm that was not bound covalently. As a result, a surface with 291 8 RU, 1 922 RU, 1 363 RU and 908 RU of Streptavidm in FCs 1 , 2, 3 and 4 respectively was obtained. B - Injection of 50 μl of biotinylated Vιt.A2 at 5 ng/μl over the flow cells 1 -4 with different amounts of immobilized Streptavidm resulted in a surface with 1 81 7 RU, 1 290 RU, 91 7 RU and 605 RU of immobilized Vit. A2 in FCs 1 to 4, respectively Figure 4. Titration of the immobilized DNA with hER at different concentrations Serial injections of hER premcubated overnight with 10 6 m of E 2 at protein concentrations: 35, 87.5, 1 75 and 270 nM were run over the A 605 RU, B - 91 7 RU, C - 1 290 RU and D - 1 81 7 RU of Vit. A2 -immobilized on a surface of sensor chip. Figure 5. Estrogen receptor ligands modulate it's interaction with DNA. A - Overlaid sensograms of hER injections over the surface with 943 RU of immobilized Vit. A2 Unhganded hER and hER premcubated overnight with 1 0 6 M of E 2 , 4(OH)tamoxιfen, raloxifene, and ICI- 1 82,780 at protein concentration -89.5 nM were used for this experiment. B - Chemical modifications of the ligand molecules affect receptor- DNA interactions. hER at 89.5 nM was premcubated overnight with 10 6 M of the 4(OH)tamoxιfen, 3(OH)tamoxιfen, tamoxifen, zuclomiphen and toremifene and injected (50 μl) over the surface with 943 RU of immobilized Vit. A2. Detailed Description of the Invention The present invention provides an improved methodology for the study of the effect of small molecules that can modulate functions of nuclear receptors, e.g., interaction with specific DNA and/or other transcription factors. The term "DNA" as used herein is meant to include all forms of nucleotide sequences, including RNA and also nucleotide sequences that may contain modified nucleotide bases, and the like. The term is sometimes used interchangeably with the term "nucleotide sequences" throughout the present specification. Receptors generally play a major role in the cascade of inter and mtracellular physiological and biochemical events. For example, nuclear receptors play an important role in transcriptional control. Steroid hormone receptors exert their influence in embryonic development and adult homeostasis as hormone activated transcriptional regulators. They have a somewhat modular construction, as manifested by a (i) DNA binding domain, (n) nuclear localization signals, a (in) ligand binding domain, and several (iv) transcriptional activation functions. This modular construction is conserved with other members of the nuclear receptor superfamily. The receptors recognize certain DNA sequences, which they then bind to. After binding to DNA, the receptor is thought to interact with components of the basal transcriptional machinery and with sequence specific transcription factors. Various ligands, such as hormones, are believed to affect these interactions (For a complete discussion, see Steroid Hormone Receptors: Many Actors in Search of a Plot, Cell, Vol. 83, pp. 851 -857, December, 1 995) . In accordance with the methodology of the present invention, measurements of such interactions, taken in real time and kinetic pattern, allow one to identify and characterize ligands that are able to affect a receptor's interaction with its binding element. "Co-activators" or "co- repressors" may also be present in various tissues on a tissue selective basis. They bind to nuclear receptors and can modify their interactions with DNA. Co-activators are generally responsible for enhancement of transcriptional activation, while co-repressors generally repress transcription. Accordingly, the present methodology allows one to evaluate the effect of any of a variety of substances directly on such interaction, or indirectly in the presence of such co-activators or co- repressors with an ultimate effect on the receptor/binding element interaction. Hence, the present invention provides a useful tool for the elucidation and development of ligands that affect transcription of target genes, either directly or indirectly, and, if desired, on a tissue selective basis. This has far-reaching implications in many fields, e.g., in the field of reproductive hormone research. For example, prior to the present discovery, it was not known that estradiol effects DNA interaction with estrogen receptor. Based on this discovery utilizing the techniques described herein, it is now possible to assay for substances that can mimic the effect of estradiol, or inhibit its affect, or in some other way can modulate this phenomenon. This may be entertained on a tissue specific basis. For example, one may desire to substitute estradiol with another agonist that is more potent, or that upregulates gene expression in bone but not uterine tissue, or down regulates gene expression in uterine but not bone tissue. This would thereby be useful in hormone replacement therapies to reduce osteoporosis without side effects that often manifest themselves through estrogen receptor stimulation in uterine tissue. The methodology of the present invention also has applicability in many other therapeutic fields, or indeed, wherever the kinetics of element interactions may be usefully measured. Using the methodology of the invention, one may obtain and identify agents capable of binding to or otherwise interacting with the binding elements or receptor, or the binding complex. The screened substances in such assays can be, but are not limited to, proteins, peptides, peptidomimetics, carbohydrates, vitamin derivatives, compounds, or other pharmaceutical agents or any mixtures thereof . The substances can be selected and screened at random or rationally selected or designed using protein modeling techniques. As used herein, a substance is said to be "rationally selected or designed" when the substance is chosen based on the configuration of the particular binding element, protein or complex. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., "Application of Synthetic Peptides: Antisense Peptides, " In Synthetic Peptides, A User's Guide, W. H. Freeman, N.Y., 289-307 ( 1 992), and Kaspczak et a/. , Biochemistry 28, 9230-8 (1 989). Pharmaceutical agents and the like may be similarly generated using techniques known to the art. In certain preferred embodiments, a screening capability is provided which enables the identification of agonists and antagonists, with the binding elements or binding complex used as targets for novel human therapeutics. For example, in the cardiovascular area, the claimed methodology may be of use in the discovery of new agents for the treatment of atπal and ventricular arrhythmias, heart failure including associated arrhythmias and cardiac ischemia. The action of such agents would possibly be effected through the modulation of the kinetics duration of the cardiac action potential. Thus, in yet further preferred embodiments, methods of modulating cellular activity to provide therapeutic value are provided, by applying to a patient in need of such modulation, a substance capable of interacting with the binding elements contained in the relevant cells of such patient and modulating the activity of same (a good example of which are cardiac cells, useful for cardiac modulation purposes). Application of such substances may take the form of in vitro, ex vivo, or in vivo application, each in a formulation suitable to deliver the substance to the cell membrane and to sustain such delivery for a time sufficient to allow the substance to interact with the membrane. Appropriate formulations, concentrations of substances, application time, and other relevant parameters may be established by utilizing routine methods in the art. Such compositions may comprise conventional delivery/carrier systems, e.g., posome or phosphohpid encapsulation, water or saline solutions, polymeric compositions, and the like. Another suitable endpoint one skilled in the art may utilize in optimizing these parameters is "cell death. " Such assays may be performed in vitro and extrapolated to in vivo conditions, or in some cases may be easily established directly in vivo. The field of insecticides is instructive for this purpose. For example, by applying the substance directly to a test sample comprising the target insect pest (whole organism) and noting the appropriate parameters at which an acceptable per cent of insect death is attained. In the methodology of the present invention, the determination of the kinetic pattern of the interaction may be carried out in a variety of ways, as long as one may measure the association and disassociation rates of the interaction of the receptor binding partner with its corresponding binding element. Hence, any assay suitable to measure the kinetics of this interaction may be utilized, such as fluorometry assays, light scattering assays and the like. Kersten, S. , Dawson, M.L., Lewis, B.A. , Noy, N. ( 1 996), Biochemiestry 35( 1 2):381 6-3824; Wen, J. , Azakawa, F , Philo, J.S. ( 1 996), Analytical Biochemistry 240, 1 55-1 66. In certain embodiments, it is preferred that the first binding element be immobilized. For example, in certain particularly preferred embodiments, surface plasmon resonance technology is utilized to study DNA binding by a nuclear receptor. The receptor-DNA complex formation is measured in real time, as a direct measurement of the interaction as it is actually occurring and clearly in a dynamic sense. This information can then be fit into a mathematical model describing this interaction, in order to obtain kinetic and thermodynamic rates of the receptor-DNA association and dissociation In accordance with this preferred embodiment, a solid phase sensor device has a binding element immobilized onto it. The solid phase may comprise a glass, plastic, or other suitable support material with a thin metallic film deposited thereon. This metallic layer is preferably gold because it can be chemically modified and the binding of non-specific proteins is generally low. The binding element is immobilized in any manner suitable to its chemical composition and that will allow the binding assay to be conducted without a substantial loss of such attached binding element. Covalent attachment is typically utilized, with a carboxylated dextran matrix often suitable for this purpose. In those instances wherein covalent attachment interferes with the steπc conformation of the substance that is attached or in some other way adversely affects normal ligand binding, the immobilized element may be attached with an "extender" portion. These may take the form of spacers such as streptavidin/biotm complexes, antibodies, and the like, which serve to free up the ligand so immobilized, to allow it to bind in a normal fashion to its binding partner The immobilized first binding element may be selected from a variety of binding elements that play a role in transcription regulation. These include consensus DNA-binding response elements and other response elements. For non-consensus response elements, a description of hormone dependent transcriptional enhancers may be found in Norπs, J. et a/. , J. of Biol. Chem. 270, 22777-22782, 1 995. A second article, Schwabe, J. et al. , Structure 3:201 -21 3, 1 995 details other elements, including consensus response elements from various promoters such as VITA2, VITBK2), VITBK 1 ) , VIT1 1 , PS2, and c-fos. The immobilized binding partner is contacted with a nuclear receptor binding partner for a time suitable to allow a binding complex to be formed. By "binding complex" is generally meant a protein-protein, protein-DNA sequence, or nucleotide sequence-nucleotide sequence complex. Any receptor or portion derived therefrom or synthetically constructed therefrom may be utilized in this methodology in accordance with one's desire to study its interactions with a particular binding element, such as DNA. For example, sequences derived from mtracellular hormone or hormone-like response element sequences may be utilized, such as those derived from or based upon specificity of the glucocorticoid, mineralcorticoid, retenoid, androgen, progesterone, or estrogen receptors, including, without limitation, ER B . The nuclear receptors or their derivatives may be obtained in any of a variety of ways, including extraction from biological sources, via synthesis of novel r sequences, the construction of chimeπcs and other hybrids, for example, a hormone receptor ligand binding domain attached to a DNA binding domain of another transcription factor, and the like. In certain embodiments, one may chose to use pure receptor to elucidate the nature of the complexes that could be formed by the receptor and its hormone response element or other binding partner interaction. Commercially available recombmant expression systems that express receptor may be used, and if desired, the expressed protein may be further purified. To determine the level of purity of the receptor under study, one can use routine methodology such as SDS gel eiectrophoresis. In a particularly preferred embodiment, a kinetics analysis of the binding interaction may be conducted utilizing a surface plasmon resonance detector - "BIAcore" to measure the effect of various ligands on nuclear receptor interaction with DNA. A commercially available instrument, the BIAcore™ biosensor system is suited to carry out the methodology of the present invention and may be obtained from BIACORE, Inc., Piscataway, N.J., U.S.A. This instrument permits the monitoring of macro-molecular interactions in real-time. The detection system uses surface plasmon resonance (SPR), a quantum mechanical phenomenon which detects changes in the refractive index at the surface of sensor chip (39). The binding of a soluble ligand to the immobilized one leads to an increase in the ligand concentration at the sensor surface, with a corresponding increase in the refractive index. This refractive index change alters the SPR which can be detected optically (40) . BIAcore 2000 allows the simultaneously detection of the interaction events on four different spots, located in diffei ent flow cells (FC), on the sensor surface. The flow system allows the sample to be addressed to individual FCs or serially to predefined combinations of the four FCs. Exemplary of the methodologies claimed herein, one skilled in the art may utilize the BIAcore™ system. To obtain kinetic rates of the nuclear receptor-hormone response element interaction, gradient surfaces of immobilized DNA are used. One skilled in the art will understand that when the immobilized DNA can be saturated with the nuclear receptor, this is an implication that the interaction studied is specific. Based on the signal at equilibrium the stoichiometry between the nuclear receptor and the hormone response element may be calculated. Using real time interaction analysis reveals that ligand binding dramatically affects the kinetics of hER interaction with the Vit. A 2 response element. Binding of estradiol induces rapid formation of a unstable ER-ERE complex, and furthermore, binding of "pure" antagonist such as ICI- 1 82,780 results in a slow formation (k a is approximately 1 00 times lower) and a very stable receptor-DNA complex (k d is almost 1 00 times lower. Most importantly, the present invention demonstrates that there is a good correlation between the kinetics of hER-ERE interaction induced by a hormone and its biological effect. For example, stability of the ER-Vιt.A 2 complex, which can be characterized by its dissociation constant (kd), is decreasing from E 2 (pure agonist) « 17α-Ethιnyl Estradiol > 4(OH)Tamoxιfen (partial agonist) > Raloxifene (partial antagonist) > ICI 1 82,780 ("Pure" antagonist). It is interesting that this order corresponds to the increase in the antagonistic activity of these compounds on consensus ERE. Differences in the kinetics of receptor-DNA interaction induced by the binding of the ligand could be related to the behavior of the steroid hormone receptor in vivo. Binding of estradiol accelerates the ER-turnover more then 50-fold, compared with unhganded ER, and more than 1000- fold compared to ER liganded with ICI-182,780. Therefore, ligand binding, inducing conformational changes, is not just modulating a receptor's interaction with other transcriptional factors. ( 1 1 -1 3) . One skilled in the art will appreciate that these changes might be affecting the kinetics of receptor-DNA binding which regulates the frequency of receptor-DNA complex formation. Thus, a correlation may exist between the rate of gene transcription and the frequency of receptor-DNA complex formation. Various manifestations of the ligand binding including: dissociation from hsp90 and p59, modulation of receptor dimeπzation status, modification of the kinetics of receptor-DNA interaction, effects on receptor interaction with TIF'S, represent different levels at which ligands of steroid receptors may affect transcriptional regulation The present invention is further exemplified as detailed below, which is not meant to be interpreted as limitative thereof. MATERIALS AND METHODS Equipment and Reagents - The BIAcore system, sensor chips CM 5 (certified), Tween-20, the amine coupling kit containing Nhydroxysuccinimide, N-ethyl-N'-(3-diethylaminopropyl) carbodiimide (EDC) and ethanolamine hydrochloride were all obtained from Pharmacia Biosensor AB. The buffer used for ail experiments was 50 mM Tris-HCI, 1 50 mM NaCI, 1 0 mM MgCI 2 , 0.05% Tween-20, pH 7.5. 1 7-β-Estradiol, 1 7α-Ethynyl Estradiol, 4-hydroxytamoxifen and tamoxifen were obtained from - Sigma, 3hydroxytamoxifen was obtained from RBI, raloxifene was synthesized according to the methods of Jones, CD., Jevnikar, M.G., Pike, A.J., Peters, M.K. , Black, L.J., Thompson, A.R., Falcone, J.F., Clements, J.A., J. Med. Chem. ( 1 984) 27: 1057-1066. ICI-1 82,780 was provided by Zeneca Pharmaceuticals. [ 3 Hj- 1 7-β-Estradiol was from DuPont, NEN. Biotinylation of Oligonucleotide - Several potential ERE's were synthesized as a self-annealing oligonucleotides that form hairpin duplex constructs upon heating and rapid cooling. A 75 bp oligonucleotide (Vit.A 2 ) containing a specific binding site for ER was derived from the Vitellogenin A 2 gene of Xenopus laevis response promoter. The second oligo - (DR) was designed as two directly repeated half-sites. The third oligo (C3) was derived from a mouse complement component C3 gene promoter (29). Following annealing, the oligonucleotides were biotinylated by incorporation of biotin-dATP with Klenow enzyme and purified from unincorporated biotinylated nucleotides by gel filtration on a Chromaspin 10 column (Clontech). Preparation of the Sensor Chip - The BIAcore™ - biosensor system (Pharmacia Biosensor, Uppsala, Sweden) was utilized for these experiments. To immobilize specific DNA, the surface of a CM 5 sensor chip (certified) was first modified with streptavidin according to instructions from the manufacturer. The surface was activated by injection of a solution containing 0.2M N-ethyl-N-(3-dιethylamιnopropyl) carbodiimide (EDC) and 0.05M N-hydroxysuccmimide (NBS). Four different surfaces with 291 8, 1 922, 1 363 and 908 resonance units (RU) of streptavidm were obtained by injecting Streptavidin at 1 0 μg/ml and varying a contact time from 1 to 6 minutes. Gradient surfaces with 1 830, 1 300, 900 and 600 RU of Vιt.A 2 -DNA was then obtained by injection of 50 μl of biotinylated oligonucleotide solution at 33 ng/μl. After each protein injection, the surface was regenerated with one 10 μl injection of 0.1 % SDS solution. One cycle of regeneration was sufficient to remove bound ER. Binding assay and data analysis — Each binding cycle was performed with a constant flow of 50 mM Tπs-HCI, 1 50 mM NaCI, 10 mM MgCI 2 , 0.05% Tween-20, pH 7.5 buffer of 5 μl/mm. Samples of ER were injected across the surface via a sample loop. Once the injection plug had passed the surface, the formed complexes were washed with the buffer for an additional 500- 1000 sec. All experiments were performed at 25°C Data were collected at 1 Hz, and analyzed using the BIAEvaluation program 2.1 (Pharmacia Biosensor AB) on a Compaq PC. This program uses a nonlinear least squares analysis method for the determination of rate binding constants for macromolecular interactions. The dissociation kinetics of the ER-ERE complexes can be described by a double exponential decay: A.B^ A^ B,. One of these complexes dissociates from the surface with apparent rate of k d1 = 0.01 sec 1 which corresponds to a complex half-life of several minutes and represents only 5 to 10% of total bound protein. The second complex is much more stable. Its apparent dissociation rate - k d2 = 1 0 3 to 10 5 sec 1 , depending on the nature of ligand, it represents approximately 90 to 95% of total bound protein. Association kinetics can be described by a model which uses two analytes (A, and A 2 ) interacting with the same binding site: A 1 + A 2 = A 1 B + A 2 B (28). The apparent equilibrium dissociation constants for the second complex, which represent the majority of bound material, were calculated from the two rate constants (K D = k d2 /k a2 ) . Purification of ER. - Partially purified recombmant human estrogen receptor was obtained from PanVera Corp. (about 80% pure) and purified to homogeneity as assessed by visualization on a Coomassie-stamed SDS gel by gel- iltration on Superdex-200 column (Pharmacia) and chromatography on Nι 2 + -affιnιty column (Pharmacia). Receptor was eluted from Nι 2 + -affιnιty column with a linear gradient of imidazole from 0 to 0.5M in 50mM Tπs-HCI, 500 mM NaCI, pH 7.5. Gel Shift Assays - VιtA 2 , DR and C3 oligonucleotides were labeled by incorporation of [α- 32 P]-dATP (6000 Ci/mmol) with Klenow enzyme and purified from unincorporated nucleotides by gel filtration on a Chromaspin 1 0 column (Clontech) Probes had a specific activity of 20x10 7 cpm/μg. DNA binding reaction were carried out in buffer containing 2 ng of labeled DNA, 20 mM Tπs-HCI, pH 7.9,0. 1 mM EDTA, 5mM MgCI 2 , 50 mM KCI, 0.5 μg/μl poly d(l-C), 10% Glycerol, 1 mM DTT, and 0.5-3.0 μg of purified ER in a total volume of 20 μl at 25°C or 37°C The prote -DNA complex was separated on 8% (75: 1 , acrylamide to bisacrylamide) polyacrylamide gels. The gels were dried and subjected to autoradiography. Gel quantitation was performed using Phosphorlmager SI (Molecular Dynamics) . Gel Electrophoresis and Western Blotting - Purified ER was prepared by boiling for 5 minutes in SDS-polyacrylamide gel electrophoresis sample buffer, separated in 1 2% SDS polyacrylamide gels (34) using the PhastSystem (Pharmacia) and either stained with Coomassie Brilliant Blue G250 or transferred to nitrocellulose membrane (Hybond-ECL, Amersham) for Western blotting. After the transfer, the membranes were blocked, incubated with primary anti-hER antibody, and then developed with secondary antibody linked to Hydrogen Peroxide Catalase (Amersham) as described (30) The anti-human polyclonal antibody against the AB region of the estrogen receptor was produced at Wyeth-Ayerst Research. Gel filtration chromatography - A Superdex 200 (30/10) column was obtained from Pharmacia. The column was equilibrated with 50 mM TrisHCI, 1 50 mM NaCI, 10 mM MgCI 2 , 0.05 % Tween-20, pH 7.5 and calibrated with thyroglobuhn (669 kDa), ferπtin (440 kDa), catalase (232 kDa) aldolase ( 1 58 kDa), BSA (67 kDa), ovalbumin (43 kDa) and RNase ( 1 3.7 kDa), all at 1 mg/ml. To analyze ER-DNA complexes by gel- filtration, 35 [ng of purified ER in 50 μl 50 mM Tπs-HCI, 1 50 mM NaCI, 10 mM MgCI 2 , 0.05 % Tween-20, pH 7.5. were incubated with 20 ng of [ 32 PHabeled ohgonucleotide in presence or absence of [ 3 H]-Estradιol (10 6 M) at room temperature for 30 mm. The sample was then applied on to the Superdex 200 column and eluted in the buffer described above (flow rate 0.25 ml/mtn.). Radioactivity in fractions (0.25 ml) from the column was detected by liquid scintillation spectrometry RESULTS Purification of hER - Commercially available partially purified recombmant r baculovirus-infected Sf9 cells hER was used for this study. This protein was further purified to homogeneity using gel-filtration and chromatography on a Nι 2 + - affinity column. As described previously (31 ), metal-affinity chromatography can be applied successfully for hER purification. hER binds to a Nι 2 + - resin without additional poly-His fusion. The affinity of this interaction is relatively high, receptor can be eluted from the column with 1 00 mM imidazole. Purified ER is detected as one band on a Coomassie-stained SDS-gel. The Apparent Molecular Weight of the purified receptor is 67 kDa. The hER was separated in 1 2% SDS polyacrylamide gels and transferred to nitrocellulose membrane (Hybond- ECL, Amersham) for Western blotting. After transfer, the membrane was blocked, incubated with primary antibody, and then developed with secondary antibody linked to Hydrogen Peroxide Catalase (Amersham). Western Blot analyses demonstrates that this protein can be recognized by an anti-hER antibody. (Data not shown.) Purified hER specifically interacts with DNA - In vitro, purified receptor binds with high affinity to a palindromic inverted repeat element containing the sequence AGGTCA spaced by 3 bp derived from the Vitellogenin A 2 gene (Vit. A 2 ) promoter of Xenopus laevis (32). In Gel Retardation Assays (GRA) bound complex typically yield a major band, and a more slowly migrating minor band. Both of these bands are supershifted by an anti-hER antibody, which implies that they represent different ER-ERE complexes. (Data not shown.) Two other oligonucleotides were also used in this work. One contains two directly- repeated AGGTCA elements spaced by 3 bp (DR) which represents a high affinity binding site for the vitamin D 3 receptor (VDR) (35). The other oligonucleotide, derived from the mouse complement component C3 gene promoter (C3), which is regulated by estradiol (29). It contains a perfect AGGTCA and one element - AGTCTA different from consensus, positioned as an inverted repeat with a spacing of 3 bp. It has been demonstrated that minimal activity is associated with a single copy of the C3 enhancer element when this oligo is inserted 5' to a thymidine kinase promoter and cotransfected with human ER in to Ishikawa cells (36). GSA demonstrates that the amount of shifted DR or C3 is more than 100 times lower than that for Vit.A 2 . This result implies that the affinity of the ER interaction with these oligonucleotides is significantly reduced compared to the Vit.A 2 . (Data not shown.) Analysis of ER-ERE complexes by gel-filtration - To clarify the nature of the various ER-DNA complexes, they were examined using analytical gel-filtration chromatography. This technique is very useful for the evaluation of the apparent molecular weight and stoichiometry of different protein-DNA complexes. Purified hER elutes from this column as a major peak with apparent MW of 1 40 kDa and several minor peaks with apparent MW of 70 kDa, 300 kDa and 540 kDa (data not shown). This result suggests that the major molecular form of hER in solution at this concentration is a dimer. The fact that other complexes besides monomers and dimers were found implies that hER has a tendency to aggregate. hER bound to [ 3 H]-estradιol elutes as two complexes, with apparent MW of 70 and 140 kDa (data not shown) with a dimer being the major molecular form of ER under these conditions. To analyze ER-DNA complexes by gel-filtration, the receptor was incubated with [ 3 H]-estradιol overnight and then mixed with [ 32 P]-labeled Vit. A 2 ohgonucleotide before this mixture was applied on Superdex 200 column. For a hganded and unhganded ER, major ER-ERE complex, approximately 95-98%, as detected by peaks integration, elutes from this column as a peak with apparent mass of 140 kDa. A small amount, approximately several percent of the total DNA loaded, was eluted as a high molecular weight ER-ERE complex (Fig. 1 ). Analysis of the ligand affect on ER binding to a specific ERE by Gel Shift Assays (GSA) - Brown and Sharp (37) reported that in the presence of Mg 2 + HEO, mutated hER ( 1 4), binds the ERE in hormone-dependent r fashion at 20°C - 37°C Later Metzger et al (38) demonstrated that for a wild type hER (HEGO) in the presence of E 2 or 4(OH)tamoxιfen, premcubatioπ at 4°C or at 37° C had little effect on DNA binding, whereas in the absence of ligand, or in the presence of ICI-1 82,780, incubation at 37°C resulted in greatly reduced DNA binding. To further examine if hER demonstrates a ligand dependency for ER-ERE complex formation detectable by GSA, the receptor was incubated at 25 °C or at 37 °C in the absence or the presence of E 2 , 4- hydroxytamoxifen, raloxifene or ICI-1 82,780. Labeled DNA was then added and incubation was continued at the same temperature. Complexes formed were analyzed by GSA (data not shown) our results demonstrate that E 2 binding had little or no effect on the amount of ER- ERE complex formed at 25 ° C, as detected by GSA. A similar result was obtained with 4-hydroxytamoxιfen, raloxifene and ICI-1 82,780. If, however, premcubation was performed at 37°C, significant reduction of ER-ERE complex formation was observed in the absence of ligand or in presence of the antagonist, ICI-1 82,780. Therefore, the purified hER exhibits the same ligand dependency for DNA binding as wild type hER (HEGO) (38), which mean that the results of the interaction measurement may be extrapolated to in situ mechanisms. Analysis of ER-ERE interaction by SPR-detector - BIAcore. Absence of detectable differences in the amount of formed ER-ERE complex by ganded or un ganded ER at 25 °C may also reflect the inability of GSA to detect differences in affinity of hgand-induced ER-ERE interaction. On the other hand, it may be due to the fact that ligand binding affects the kinetics of ER-ERE interaction while having a minimal effect on affinity The methodology of the present invention conveniently addresses these possibilities, by utilization of real time analysis, to study ligand effects on the kinetics of ER-ERE complex formation at 25 °C Utilization of the BIAcore system for the determination of a kinetic pattern usually starts from the equilibration of the surface with running buffer (Fig. 2). There is an "injection jump" at the beginning and end of each injection due to the difference in the refractive index between the running and sample buffers (presumably due to small changes in salt concentration) . Protein is injected during the "wash-in" phase. During the "wash-out" phase, the running buffer is injected across the flow cells. After approximately 1 5 seconds from the start or the end of injection, the data generated can be used for the kinetic analysis. 1094 RU of DR o gonucleotide in FC 1 , 943 RU of Vιt.A 2 - ohgonulceotides in FC 2 and 1063 RU of C3-ohgonucleotιdes in FC 3 were immobilized on the surface of a sensor chip (see Materials and Methods). Human ER bound with E 2 was injected over the surfaces coated with these different oligonucleotides (Fig. 2) . High affinity interaction was detected in the flow cell with Vιt.A 2 immobilized This is indicated by a fast increase of the refractive index, detectable after the "injection jump" in the beginning of injection (see Fig 2, Vιt.A 2 ) . At the end of injection, significant amount of ER is still bound to the surface, which can be detected as a difference between the refractive index of the injection over the surface with immobilized Vιt.A 2 and the base line. At the same time, binding of hER to immobilized streptavidin (data not shown), DR or C3 surfaces was very low. Thus, the mass increase on the surface of the sensor chip in response to the injection of hER is due to specific receptor binding to DNA. Injection of ER over the surface with no DNA immobilized results in no detectable binding (data not shown). To study how different ligands affect ER-ERE interactions, surface with a gradient of immobilized Vιt.A 2 was created. First a gradient of immobilized streptavidm in different flow cells was obtained by varying the streptavidm injection time as illustrated in Fig. 3A. As a result, surface with 291 8 RU, 1 922 RU, 1 363 RU and 908 RU of streptavidin in FCs 1 , 2, 3 and 4 respectively, was obtained. Injection of 50 μl of biotinylated Vιt.A 2 at 2 ng/μl resulted in a surface with 1 817 RU, 1 290 RU, 91 7 RU and 605 RU of immobilized Vιt.A 2 in FCs 1 to 4, respectively (Fig. 3B). Serial injections of unhganded ER and ER incubated overnight with 1 0 6 M of E 2 , 4(OH)tamoxιfen, raloxifene, and ICI- 1 82,780 at protein concentrations ranging from 35 nM to 270 nM were run over the sensor chip with immobilized gradient of the Vιt.A 2 . Fig. 4 demonstrates overlaid sensograms of the injections of the hER at different protein concentrations, hganded with E 2 , over the gradient of immobilized Vιt.A2 Clear correlation between the change of refractive index and the level of immobilized DNA was obtained. Saturation of immobilized ERE with injected hER was reached for the hER hganded with 4(OH)tamoxιfen and E 2 at 270 nM hER. There was a linear relationship between the surface density (amount of immobilized DNA) and the saturation response (refractive index at the end of injection) obtained with 270 nM injection of hER in the interval of the DNA densities used for this experiment (data not shown) . Calculated stoichiometry for the formed ER-ERE complex (42), was 1 : 1 .92 ± 0.1 1 . This result confirms that hER binds specific oligo- Vιt.A2 as a homodimer. Fig. 5A shows overlaid sensograms obtained by injections of 50 μl of un ganded hER and hER hganded with E 2 , 4(OH)tamoxιfen, raloxifene, and ICI-1 82,780 at receptor concentration of 89.5 nM over the FC with 900 RU of VιtA2 immobilized. It can be seen that the different ligands significantly affect the ER-ERE interaction. Relative to unhganded ER or ER hganded with ICI-1 82,780, much more of the ER-ERE complex is formed when ER is hganded with E 2 or 4(OH)tamoxιfen. It can also be seen that this complex is less stable then the complex induced by ICI- 1 82,780 and raloxifene. Sensograms of the injections of hganded and unhganded hER over the gradient surface with immobilized Vιt.A2 were analyzed using the BIAEvaluation 2.1 program, as described (28). Values of apparent dissociation, association and affinity rate constants determined for ER-ERE interaction in the absence and presence of different ligands are summarized in Table 1 . E 2 -bιndιng (pure agonist) induced fast formation (k, = 9.62x10 4 M ' sec 1 ) of unstable ER-ERE complex (kd = 1 .86x10 3 sec-1 ), at the same time ICI-1 82,780 binding (pure antagonist) induced slow formation (k,, = 1 .48x10 3 m-i sec ' ) of a very stable ER-ERE complex (k d = 6.35x 10 5 sec 1 ) . Additional experimentation was conducted to determine if derivatives of the same compound may have a different effect on ER-ERE interactions. hER was premcubated with analogs of 4(OH)tamoxιfen at 10 6 M (tamoxifen, 3{OH)tamoxιfen, 4(OH)tamoxιfen, zuclomiphen and toremifene) overnight, and then used for serial injections at different protein concentrations over the surface of a sensor chip carrying immobilized Vιt.A2. Effect of these compounds on the kinetics of hER interaction with the ERE was examined as already described (28). Values of the apparent association and dissociation rates are summarized in Table 1. Fig.5B presents overlaid sensograms of hER liganded with the analogs of 4(OH)tamoxιfen, injected over a surface with ERE immobilized. It is clear that even small chemical modification of the ligand (position of OH group in the molecule of tamoxifen), presumably modulating the receptor's conformation, may affect receptor-DNA interactions. It can be seen that the stability of the ER-ERE complex induced by the 4(OH)tamoxιfen is much lower (see Table 1), than that of the complexes induced by 3(OH)tamoxifen and tamoxifen. On consensus ERE, 4(OH)tamoxιfen is a more potent agonist then other analogs of tamoxifen used in this study. Table 1. Effect of Different Compounds on the Kinetics of ERE-ER Interaction Compound kd (sec-1) ka (M-I*sec-1) KD (M) No hgaπd 5.06 ±0.13 x 10 4 8.17 ± 1.26 x 10 3 6.19x10-8 17P-Estradιol 1.86±006x 10 3 9.62 + 0.04 x 10 4 1.93x10 β 17oc-Ethιnyl Estradiol 1.94 + 0.03 x 10 3 1.05 ± 0.04 x 10 5 184x10 β 17ot-Estradιol 1.96 ±0.02 x 10 3 8.78 ± 0.05 x 10 4 2.23x10 β t 4(OH)Tamoxιfen 1.05 ±0.02 x 10 3 7.50 ± 0.03 x 10" 1.40x10 8 3(OH)Tamoxιfen 7.42 + 0.14 x 10 4 5.38 ± 0.02 x 10 4 1.38x10" Tamoxifen 8.66 ±0.22 x 10 4 6.60 ± 0.03 x 10 4 1.31x10 β Clomiphene 4.17 ±0.13 x 10 4 3.12± 0.03 x 10 4 1.33x10 β Enclomiphen 4.72 ±0.09 x 10 4 5.35 + 0.01 x 10 4 8.82x10 9 Zuclomiophen 4.79 ±0.04 xlO-4 5.68 ± 0.01 x 10" 8.43x10 9 Raloxifen 1.71 ±0.04 xlO 4 1.45 ± 0.05 x10 4 1.17x10 s ICI-182,780 6.35± 0.15 xlO 5 1.48 ± 0.16 x10 3 4.29x10 β REFERENCES 1. Evans, R.M. (1988) Science 240, 889-895 2. Green, S., Shambon, P. (1988) Trends Genet, 4, 309-314 3. Beato, M. (1989) Cell, 56, 335-344 4. 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Claims: What is Claimed is: 1 . A method for determining a kinetic pattern of the interaction of a binding element with all or a portion of a nuclear receptor binding partner comprising the steps of: (a) providing a first binding element and means to measure ligand binding interactions in real time; (b) contacting said first binding element with a receptor binding partner and allowing a binding complex to be formed; (c) contacting the binding complex with a substance whose effect on such complex formation it is desired to determine; (d) measuring the interaction with the nuclear receptor binding partner in real time; and (e) determining the ligand interaction in real time as a measure of a kinetic pattern of the interaction. 2. The method of Claim 1 wherein said first binding element is immobilized onto a solid phase sensor device. 3. The method of Claim 2 wherein the solid phase sensor device comprises a surface plasmon resonance defector. 4. The method of Claim 1 , 2, or 3 wherein the binding element is a response element. 5. The method of Claim 4 wherein the response element is a consensus response element. 6. The method of Claim 1 , 2, 3,4, or 5 wherein the nuclear receptor binding partner is derived from tissues selected from the group consisting essentially of bone, cardiovascular, thyroid, brain, mammory gland, and reproductive tissues. 7. The method of Claim 6 wherein the nuclear binding partner is derived from reproductive tissue selected from the group consisting of uterus, testes, or ovarian tissue. 8. The method of Claim 6 wherein the nuclear receptor binding partner is an oligonucleotide sequence derived from said nuclear receptor from any of said tissues. 9. The method of Claim 7 or 8 wherein the first binding element is the estrogen response element. 1 0. The method of Claim 1 , 2, 3, 4, 5, 6, 7, 8, or 9 further comprising the addition of a co-activator. 1 1 . The method of Claim 1 , 2, 3, 4, 5, 6, 7, 8, or 9 further comprising the addition of a co-repressor. 1 2. The method of Claim 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 wherein the substance is an antagonist. 1 3. The method of Claim 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 wherein the substance is an agonist. 14. The method of Claim 1 or 2 wherein the receptor binding partner is derived from a nuclear receptor. 1 5. The method of Claim 14 wherein the nuclear receptor is selected from the group consisting of androgen, progesterone, and estrogen receptors. r 1 6. The method of Claim 1 5 wherein the steroid receptor is an estrogen receptor. 1 7. The method of Claim 1 6 wherein the substance is an agonist. 1 8. The method of Claim 1 7 wherein the agonist is estradiol.

Inventors: CHESKIS BORRIS Y, LYTTLE C RICHARD, CHESKIS, BORRIS, Y., LYTTLE, C., RICHARD
Applicants: AMERICAN HOME PROD, AMERICAN HOME PRODUCTS CORPORATION
Application year: 1998
Main classification: G01N33
IPC codes: C12M1, C12N15, C12Q1, G01N33