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WO1998048281A1 - Methode d'identification et de caracterisation des ligands des recepteurs nucleaires - Google Patents

Methode d'identification et de caracterisation des ligands des recepteurs nucleaires Download PDF

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Publication number
WO1998048281A1
WO1998048281A1 PCT/US1998/006672 US9806672W WO9848281A1 WO 1998048281 A1 WO1998048281 A1 WO 1998048281A1 US 9806672 W US9806672 W US 9806672W WO 9848281 A1 WO9848281 A1 WO 9848281A1
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binding
receptor
interaction
dna
nuclear receptor
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PCT/US1998/006672
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English (en)
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Borris Y. Cheskis
C. Richard Lyttle
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American Home Products Corporation
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Priority to JP54607398A priority Critical patent/JP2002507276A/ja
Priority to EP98914486A priority patent/EP0977994A1/fr
Priority to CA002286761A priority patent/CA2286761A1/fr
Priority to AU68831/98A priority patent/AU6883198A/en
Publication of WO1998048281A1 publication Critical patent/WO1998048281A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones

Definitions

  • 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.
  • 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 7 ⁇ -estrad ⁇ ol (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), dime ⁇ zation (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) .
  • TIFS transc ⁇ ptional intermediary factors
  • the ligand is generally accepted as playing a key role in the initiation of this cascade of events.
  • estrogen affects the receptor's ability to bind specific DNA (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 dime ⁇ zation 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.
  • SPR surface plasmon resonance
  • 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.
  • 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.
  • 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 chime ⁇ c 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 transc ⁇ ptional 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.
  • FIG. 1 Gel-filtration of ER-ERE complexes. 35 ⁇ g of purified hER in 50 ⁇ l of 50 mM T ⁇ s-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]-Estrad ⁇ ol 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.
  • FIG. 1 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 V ⁇ t.A2, and 1063 RU of C3 (see Materials and Methods) were obtained by injection of 50 ⁇ l of biotinylated ohgonucleotide solution at 33 ng/ ⁇ l over the surface with immobilized Streptavidm. 30 ⁇ l 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 ⁇ l/min.
  • the flow path was first set to allow serial injections through FC 1 to 4.
  • EDC/NHS 1
  • the Streptavidm was injected at 10 ⁇ g/ml (2) and at first directed only to FC 1 .
  • 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.
  • 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 ⁇ l of 0.1 % SDS, to remove Streptavidm that was not bound covalently.
  • 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.
  • 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.
  • Estrogen receptor ligands modulate it's interaction with DNA.
  • 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.
  • 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) .
  • 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.
  • 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.
  • estradiol effects DNA interaction with estrogen receptor.
  • estradiol 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.
  • 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.
  • 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.
  • a screening capability which enables the identification of agonists and antagonists, with the binding elements or binding complex used as targets for novel human therapeutics.
  • the claimed methodology may be of use in the discovery of new agents for the treatment of at ⁇ al 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.
  • 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.
  • 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.
  • 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.
  • any assay suitable to measure the kinetics of this interaction may be utilized, such as fluorometry assays, light scattering assays and the like.
  • the first binding element be immobilized.
  • 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
  • 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.
  • 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 Nor ⁇ s, 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.
  • 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.
  • 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 chime ⁇ cs and other hybrids, for example, a hormone receptor ligand binding domain attached to a DNA binding domain of another transcription factor, and the like.
  • Commercially available recombmant expression systems that express receptor may be used, and if desired, the expressed protein may be further purified.
  • routine methodology such as SDS gel eiectrophoresis.
  • 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.
  • BIAcoreTM 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).
  • SPR surface plasmon resonance
  • 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.
  • FC diffei ent flow cells
  • stability of the ER-V ⁇ t.A 2 complex which can be characterized by its dissociation constant (kd), is decreasing from E 2 (pure agonist) « 17 ⁇ -Eth ⁇ nyl Estradiol > 4(OH)Tamox ⁇ fen (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.
  • ligand binding including: dissociation from hsp90 and p59, modulation of receptor dime ⁇ zation 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
  • 1 7- ⁇ -Estradiol, 1 7 ⁇ -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- ⁇ -Estradiol was from DuPont, NEN. Biotinylation of Oligonucleotide -
  • 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).
  • 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).
  • 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-d ⁇ ethylam ⁇ nopropyl) carbodiimide (EDC) and 0.05M N-hydroxysuccmimide (NBS).
  • EDC N-ethyl-N-(3-d ⁇ ethylam ⁇ nopropyl) carbodiimide
  • NBS N-hydroxysuccmimide
  • Binding assay and data analysis Each binding cycle was performed with a constant flow of 50 mM T ⁇ s-HCI, 1 50 mM NaCI, 10 mM MgCI 2 , 0.05% Tween-20, pH 7.5 buffer of 5 ⁇ l/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,.
  • 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.
  • ER 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 N ⁇ 2 + -aff ⁇ n ⁇ ty column (Pharmacia). Receptor was eluted from N ⁇ 2 + -aff ⁇ n ⁇ ty column with a linear gradient of imidazole from 0 to 0.5M in 50mM T ⁇ s-HCI, 500 mM NaCI, pH 7.5.
  • 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) .
  • hER Protein 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 N ⁇ 2 + - affinity column. As described previously (31 ), metal-affinity chromatography can be applied successfully for hER purification. hER binds to a N ⁇ 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).
  • GAA Gel Retardation Assays
  • 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 .
  • hER has a tendency to aggregate.
  • hER bound to [ 3 H]-estrad ⁇ ol 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.
  • ER-DNA complexes were incubated with [ 3 H]-estrad ⁇ ol overnight and then mixed with [ 32 P]-labeled Vit. A 2 ohgonucleotide before this mixture was applied on Superdex 200 column.
  • 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.
  • 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-hydroxytamox ⁇ fen, raloxifene and ICI-1 82,780.
  • 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.
  • 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.
  • 5A shows overlaid sensograms obtained by injections of 50 ⁇ l of un ganded hER and hER hganded with E 2 , 4(OH)tamox ⁇ fen, raloxifene, and ICI-1 82,780 at receptor concentration of 89.5 nM over the FC with 900 RU of V ⁇ tA2 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)tamox ⁇ fen. It can also be seen that this complex is less stable then the complex induced by ICI- 1 82,780 and raloxifene.
  • Fig.5B presents overlaid sensograms of hER liganded with the analogs of 4(OH)tamox ⁇ fen, 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)tamox ⁇ fen is much lower (see Table 1), than that of the complexes induced by 3(OH)tamoxifen and tamoxifen. On consensus ERE, 4(OH)tamox ⁇ fen is a more potent agonist then other analogs of tamoxifen used in this study.

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Abstract

L'invention concerne une méthodologie qui permet d'étudier l'effet de ligands pouvant moduler les fonctions des récepteurs nucléaires, notamment leur interaction avec un ADN spécifique ou d'autres facteurs de transcription. Ces récepteurs jouent un rôle majeur dans la cascade des phénomènes physiologiques et biochimiques intercellulaires et intracellulaires. Ils ont généralement une structure modulaire, qui leur permet de reconnaître certaines séquences d'ADN ou éléments de liaison, tels que des éléments de réponse hormonale, et de s'y fixer. Divers ligands, tels que les hormones, affectent cette interaction. Selon la méthodologie présentée ici, les mesures de ces interactions sont effectuées en temps réel et concernent leur cinétique, ce qui permet à l'utilisateur d'identifier et de caractériser les ligands capables d'affecter l'interaction d'un récepteur avec son élément de liaison. La présente invention constitue donc un outil utile pour élucider et développer des ligands affectant, directement ou indirectement, la transcription de gènes cibles.
PCT/US1998/006672 1997-04-24 1998-04-06 Methode d'identification et de caracterisation des ligands des recepteurs nucleaires WO1998048281A1 (fr)

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JP54607398A JP2002507276A (ja) 1997-04-24 1998-04-06 核内受容体リガンドの同定及び特性化のための方法
EP98914486A EP0977994A1 (fr) 1997-04-24 1998-04-06 Methode d'identification et de caracterisation des ligands des recepteurs nucleaires
CA002286761A CA2286761A1 (fr) 1997-04-24 1998-04-06 Methode d'identification et de caracterisation des ligands des recepteurs nucleaires
AU68831/98A AU6883198A (en) 1997-04-24 1998-04-06 Method for the identification and characterization of nuclear receptor ligands

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Citations (2)

* Cited by examiner, † Cited by third party
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WO1990005303A1 (fr) * 1988-11-10 1990-05-17 Pharmacia Ab Surfaces de captage capables d'interactions biomoleculaires selectives, a utiliser dans des systemes de biocapteurs
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