CN100334448C - Drug screening system using tetinol X receptor as target spot - Google Patents

Abstract

The present invention discloses a method for filtering medicine molecules using a retinol X receptor (RXR) and an orphan receptor (TR3) as medicine target points, and also an applying method for a medical composition filtered with the former method to treat various diseases such as cancer, cardio-cerebrovascular diseases, diabetes, etc.

Description

With the retinol X receptor is the medicament sifting motion system of target spot

Technical field

The present invention relates to the drug screening field.More specifically, the invention provides a kind of with retinol X receptor (RXR) and orphan receptor (TR3) method as the new screening of medicaments molecule of drug target.The present invention also provides the pharmaceutical composition that screens in this way to be used for the treatment of the method for multiple diseases such as cancer, cardiovascular and cerebrovascular diseases, diabetes.

Background technology

Application of compound technology with retinol sample activity is well-known, all records to some extent on the patent of world many countries and science publication.Retinol sample reactive compound has been widely used in treating the multiple disease of the mankind and animal, comprise and be used for prevention and treatment cancer and precancerous lesion, as breast cancer, cutaneum carcinoma, prostate cancer, cervical carcinoma, the cancer of the uterus, colon cancer, carcinoma of urinary bladder, cancer of the esophagus, cancer of the stomach, lung cancer, laryngocarcinoma, carcinoma of mouth, blood and lymphsystem tumor, be used for the treatment of and organize atypical hyperplasia, maldevelopment, leukoplakia, mucous membrane papilloma and Kaposi sarcoma.Retinol sample reactive compound also participates in the adjusting to metabolic disorder, is applied to the treatment to type ii diabetes, hyperlipidemia and arteriosclerosis.

The effect of retinol sample reactive compound is receptor-mediated by two nucleoids, and these two receptor families are called after retinoic acid receptors (RAR) and retinol X receptor (RXR) respectively, and every kind of acceptor has α, β and three kinds of hypotypes of γ again.RAR and RXR two albuminoids are in conjunction with the C district high conservative of DNA, and the key distinction is the amino acid sequence difference of part in conjunction with the E district, and homology is lower than 27%; Difference mainly is the amino acid sequence difference in A/B district between the various hypotypes.

RXR is except that can forming homodimer (Homodimer), also can form heterodimer with I type nuclear receptor member, comprise vitamin D receptor (VDR), peroxisome propagation activated receptor (PPAR), Thyroid Hormone Receptors (TR) and multiple orphan receptor, as liver X receptor (LXR), embryo X acceptor (PXR), sustained activation acceptor (CAR) and orphan receptor TR3/Nur77/NGFI-B (Kastner, P., Cell, 1995,83,859-869; Mangelsdorf, D.J., etal., Cell, 1995,83,841-850) form heterodimer (Heterodimer) with RAR, pth receptor (TR), vit D3 receptor nuclear receptors such as (VD3R), combine with target gene with dimeric form, activate or suppress the transcriptional activity of specific gene, formation is the transcription factor storehouse of regulatory gene transcriptional activity extensively.

They can regulate and control some identical genetic transcriptions the DBD district height homology explanation of RXR and RAR, but the different biologically actives that their ligands specific produces may be regulated different genes with RXR-RXR, RAR-RXR and be expressed relevant, especially RXR can form heterodimer with the nuclear receptor protein that other adjusting is grown, the extensively expression activity of regulation and control karyogene, thereby the activation of RXR may be as regulating the important link that cell grows in the body.

The mechanism of action of the transcriptional activity of its allos marriage chain of RXR regulation and control is widely studied (Kastner, P., Cell, 1995,83,859-869; Mangelsdorf, D.J., etal., Cell, 1995,83,841-850; Zhang, X.K., et al., Nature, 1992b, 358,587-591).Impressive progress to RXR research is to have found that RXR can move out from nucleus combine the formation heterodimer with other nuclear receptors after in recent years, navigate on the cytoplasmic mitochondria, cause that cromoci discharges, participate in the pair cell regulation of apoptosis, this discovery has great importance, and may have to can be applicable to treat the multiple disease relevant with apoptosis.

Orphan receptor TR3 (also claiming nur77 and NGFI) is another kind of nuclear receptor, TR3 and its close relative family member NOT-1 (also claiming Nurr1 and RNR-1) and NOR-1 (also claiming MINOR and TEC) form a noticeable subfamily (Chang in nuclear family, C., etal.J Steroid Biochem, 1989,34,391-395; Zhang, X.K., et al., Nature, 1992b, 358,587-591; Hedvat, C.V., et al., Mol Endocrinol, 1995,9,1692-1700; Kastner, P., Cell, 1995,83,859-869; Mangelsdorf, D.J., et al., Cell, 1995,83,841-850).TR3 is a kind of instantaneous primary-response gene, its expression can be induced rapidly by many extracellular stimulus factors such as growth factor and Fo Bo ester etc., and act on special DNA response element (NBRE or NurRE) regulation and control target gene expression, participate in regulating cell propagation and differentiation (Wilson, T.E., et al., Science, 1991,252,1296-1300; Phillips, A., et al., Mol Cell Biol, 1997,17,5946-5951).Another important discovery in recent years is that TR3 also can move out from nucleus, regulates multiple important different biological function, for example, handles PC chromaffin cell oncocyte with NGF, and TR3 can transfer to the tenuigenin from nucleus, helps the PC12 cell differentiation that NGF induces.In other cells, TR3 transfers to tenuigenin from nucleus and is positioned mitochondria, but inducing cell pigment C discharges, thereby promote Apoptosis (Li, H., etal.Science, 2000,289,1159-1164), prompting TR3 may be again molecule (Maruyama, K., et al. before a kind of potential apoptosis, Cancer Lett, 1995,96,117-122).Find that further TR3 can form heterodimer (Forman, B.M., et al.Cell, 1995,81,541-550 with RXR; Perlmann and Jasson, Gene Dev, 1995,9,769-782), and can with orphan receptor COUP-TF (Wu et al., Embo J, 1997b, 16,1656-1669) interact.Because COUP-TF combines with RAR β promoter, can make RAR β efficiently express (Lin et al., Mol Cell Biol, 2000,20,957-970), so, by interacting with RXR and COUP-TF, TR3 scalable RAR β expression also changes reaction (Wu et al., Embo J., the 1997b of cell to retinol, 16,1656-1669).

RXR and TR3 transfer to the tenuigenin from nucleus and participate in the regulating cell apoptosis important biological significance is arranged, the discovery of the action pathway that this is new, make RXR and the TR3 might be as important drug target, its molecular model can be used for screening new medicines such as treatment apoptosis-associated diseases such as tumour, thereby the molecular model of setting up RXR and TR3 new role approach has significant values.Therefore, new RXR approach need be sought in this area, so that discovery and developmental function are in the therapeutic agent of the approach of RXR mediation.

Summary of the invention

Purpose of the present invention just provides a kind of with retinol X receptor (RXR) and orphan receptor (TR3) method as the screening of medicaments molecule of drug target.

Another object of the present invention provides the pharmaceutical composition that contains the compound that filters out, and these compound combinations can be applicable to treat and apoptosis-related disease.

In a first aspect of the present invention, a kind of method of screening compounds is provided, described compound regulation and control retinol X receptor (RXR) and orphan receptor TR3 are moved to following position from nucleus: tenuigenin, cell membrane, mitochondria, golgiosome and/or endoplasmic reticulum, and it comprises step:

(a) provide a kind of cell, this cell contains RXR and TR3 in nucleus;

(b) there is or do not exist the described cell of cultivation under the test compounds situation; With

(c) select and regulate RXR and TR3 from the nucleus of described cell compound: tenuigenin, cell membrane, mitochondria, golgiosome and/or endoplasmic reticulum to the migration of following position.

In another preference, described compound increases the migration of RXR to described cell matter.

In another preference, described compound weakens the migration of RXR to described cell matter.

In another preference, described compound is selected from following material: peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrates, fat, synthetic compound, natural products, antibody or antibody fragment, organic molecule, inorganic molecules and nucleotide sequence.

In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it contains compound and the pharmaceutically acceptable carrier that useful said method of the present invention filters out, and described pharmaceutical composition influences apoptosis from nucleus to mitochondrial subcellular fraction migration by regulation and control RXR.

In a third aspect of the present invention, a kind of method of screening compounds is provided, the formation of described compound regulation and control RXR/TR3 II type heterodimer, thus influence natural death of cerebral cells, the method comprising the steps of:

(a) provide a kind of cell, described cell contains the Bcl-2 family protein relevant with mitochondria, and described cell also contains can form I type and II type RXR/TR3 heterodimer;

(b) there is or do not exist the described cell of cultivation under the test compounds situation; With

(c) select the compound that selective induction II type heterodimer forms.

In another preference, this method also comprises selects regulation and control II type heterodimer from the compound of nucleus to mitochondrial migration.

In another preference, this method also comprises the compound of selecting regulation and control II type heterodimer to combine with the Bcl-2 family protein.

In another preference, the formation of heterodimer is differentiated with the method that is selected from down group:

(a) differentiated according to the configuration difference of described heterodimer;

(b) according to the Subcellular Localization of described heterodimer and recognized; Or

(c) to the specific reporter gene identification of II type heterodimer.

In fourth aspect present invention, a kind of pharmaceutical composition is provided, it contains compound and the pharmaceutically acceptable carrier that useful said method of the present invention filters out, and described pharmaceutical composition influences apoptosis from nucleus to mitochondrial subcellular fraction migration by regulation and control II type RXR/TR3 heterodimer.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 shows the analysis result of Laser Scanning Confocal Microscope, shows that RXR when drug-induced Apoptosis, is positioned mitochondria.

Fig. 2 is the Laser Scanning Confocal Microscope image of a series of immunostaining cells, shows not have 15d-PGJ ₂The time, RXR α transfection enters H460 lung carcinoma cell or the neural eucaryon oncocyte of SY5Y.Yet as cell 15d-PGJ ₂After the processing, a large amount of RXR α appear in the tenuigenin.The expression vector of Flag-RXR α is transfected advance described clone after, use 15d-PGJ ₂(5 μ M) handled cell 3 hours, dyeed with the antibody mediated immunity of Flag then and detected RXR α, with Laser Scanning Confocal Microscope observation Flag-RXR α.

Fig. 3 is the dose response analysis result, shows 15d-PGJ with the competition binding analysis ₂Adding the back combines with RXR α is efficient.15d-PGJ ₂Substituted combine with the RXR ligand binding domain [ ³H] 9-cis-RA, Ki is 1.8 μ M.Bacteria purification RXR alpha ligands land albumen is being with or without the 15d-PGJ of described concentration ₂When existing, with 10nM[ ³H] 9-cis-RA together cultivates.By Sephadex G-25 desalting column wash-out, bond is with after radioactivity is separated, and the radioactivity that washes away is with liquid scientillation count measurement.

Fig. 4 is the Laser Scanning Confocal Microscope image of a series of immunostaining cells, shows that non-steroid anti-inflammation drugs indocin (indomethacin) and voltaren see diclofenac (diclofenac) can induce RXR α to move to tenuigenin from nucleus.GFP-RXR α and Mito-RFP transfection are entered the cos7 cell, with or need not 10 ^-4M indocin and voltaren see diclofenac were handled cell 4 hours, observed the distribution of GFP-RXR α and Mito-RFP with Laser Scanning Confocal Microscope.

Fig. 5 shows the analysis result of Laser Scanning Confocal Microscope, shows RXR and TR3 when drug-induced Apoptosis, and co is in mitochondria.

Fig. 6 is the Laser Scanning Confocal Microscope image of the PC12 cell of a series of immunostainings, shows 15d-PGJ ₂Move to tenuigenin with RXR α zygotic induction RXR α homodimer/strand or TR3/RXR α heterodimer from nucleus.The PC12 cell is advanced in the common or independent transfection of the expression vector of Flag-RXR α and GFP-TR3, uses 15d-PGJ ₂(5 μ M) handled cell 3 hours, dyeed with the antibody mediated immunity of Flag and detected RXR α, with Laser Scanning Confocal Microscope observation Flag-RXR α and GFP-TR3, the image overlap of the two.

Fig. 7 shows the analysis result of Laser Scanning Confocal Microscope, shows RXR and TR3 when drug-induced Apoptosis, and co is complementary in mitochondria.

Fig. 8 is the western blot figure that GST pull-down analyzes, and shows that TR3 and RXR form a heterodimer and interact.

Fig. 9 is a western blot figure, Calu-6 lung cancer and ZR75-1 breast cancer cell with or without trans-RA (10 ^-6M) or 15d-PGJ ₂(5 μ M) or they combine and handled 24 hours, detect the expression of RXR β with Western.

Figure 10 is a western blot figure, shows that RXR α/TR3 heterodimer is at external and 15d-PGJ ₂Cultivate together and fully phased out RXR α/TR3 heterodimer and combine with β RARE's.In vitro translated RXR α and TR3 and 15d-PGJ ₂(10 ^-6M) be with or without anti-TR3 or anti-RXRR antibody in the presence of common the cultivation 15 minutes, cultivate with the isotope-labeled β RARE of 32P-again, use the EMSA analytical reactions.

Embodiment

The applicant with multiple apoptotic stimulus factor (as Buddhist ripple ester (12-O-myristoyl phorbol-13-ethyl ester, TPA), indocin or diclofenac: known clinically anticarcinogen such as 5-Fu and unknown compound SR11247) the different clone (LNCap prostate gland cancer cell, Cos7 cell, H460 lung carcinoma cell, SY5Y oncocyte, PC-12 cell, SMMC7721 hepatoma carcinoma cell) of processing, find that these medicines can combine and induce RXR to move to tenuigenin from nucleus with RXR, these medicines are by depending on the migration cell death inducing (Fig. 1) of RXR.

The applicant also finds the metabolic product 15-deoxidation-12 of cyclopentane prostaglandin, 14-prostaglandin J ₂(15d-PGJ ₂) new mechanism of action.15d-PGJ ₂In the regulation and control inflammatory process, have vital role, comprise the activation that suppresses NF-KB.15d-PGJ in addition ₂Many tumor cell lines all there is the effect of antiproliferative effect and apoptosis induction, but its mechanism of action and unclear.The 15d-PGJ that the report micro-molar concentration is arranged recently ₂In conjunction with and activate PPAR γ, prompting 15d-PGJ ₂May work by PPAR γ.Yet the various 15d-PGJ that studies show that ₂May also play a role with the mechanism that does not rely on PPAR γ.The applicant finds 15d-PGJ ₂Regulate and control the apoptosis (Fig. 2,3) of multiple cancer cell by the new action pathway of RXR.

The applicant further finds can interact with many cytoplasm proteins after RXR moves out from nucleus, the interaction energy cell death inducing of RXR and tenuigenin target spot, and its effect does not rely on the transcriptional control effect (Fig. 4) of RXR.

The applicant finds that also the formation of certain heterodimer can induce RXR to move to tenuigenin from nucleus, and as under the effect of apoptotic stimulus factor, the RXR/TR3 heterodimer of formation can be moved to tenuigenin from nucleus.The applicant also finds, TR3 moves from nucleus that to come out be that migration with RXR α is closely-related.For example with TPA handled cotransfection FlagRXR α and GFP TR3 cell, RXR α and TR3 all come across and are spot distribution in the tenuigenin, and to combine albumen overlapped with mitochondria, and control group the two all be positioned at nucleus.The applicant further suppresses RXR α or the expression of TR3 in the LNCap cell respectively with the siRNA technology, handle cell with TPA again, no matter be that R all can not move from nucleus with R α or TR3, prompting is when drug-induced Apoptosis, and it is complementary (Fig. 5～8) that RXR α and TR3 are moved to tenuigenin from nucleus.Because the crystal structure of TR3/NGFI-B shows that TR3/NGFI-B does not contain the part binding pocket, therefore, the regulating action of the DNA of external TR3/RXR heteroduplex can not owing to the combining of TR3.The applicant has further investigated 15d-PGJ ₂With the possibility that RXR α directly combines, use [ ³H9]-the competition analysis result of cis-RA and RXR alpha ligands land shows 15d-PGJ ₂Replace [ ³H9]-cis-RA is attached to the ligand binding domain of RXR, and Ki is 1.8 μ m, shows 15d-PGJ ₂Can directly combine with RXR α.

The applicant also finds, the RXR/TR3 heterodimer from nucleus moves to down drug-induced that to act on the RXR/TR3 heterodimer that the DNA regulatory gene transcribes in plastidogenetic structural shape and its nuclear different.

The applicant further finds, the RXR/TR3 heterodimer that the apoptosis factor stimulates the back to form is moved to tenuigenin by the way of output that relies on CRM1 from nucleus, this output is that the nuclear output sequence (NES) of spiral 7 of the C-end of a kind of RXR of being positioned at causes, and this output sequence only can cause just when the RXR/TR3 heterodimer is the apoptosis induction configuration that the nucleus that CRM1 relies on exports.

The applicant finds that further the nucleus output sequence (NES) of RXR α is positioned at the LBD spiral 7 of its C end.The crystal structure of RXR α discloses, and RXR α forms homodimer or with RAR and PPAR heterodimer or when forming triple strand structure, remarkable different change of configuration (Bourguet, W., et al.MolCell, 2000:5,289-298 have taken place spiral 7; Gampe, R, T., etal.Mol Cell, 2000a:5,545-555; Gampe, R.T., et al.Genes Dev, 2000a:14,2229-2241).The monomer structure of the spiral 7 of RXR α comprises a αLuo Xuanjiegou (Bourguet, W., et al.Nature, 1995:375,377-382), because the existence of the glutamic acid in the middle of the spiral 7, when RXR α formed homodimer or forms heterodimer or three and form triple strand structure with RAR or PPAR, the α spiral became π spiral (Bourguet, W., et al.MolCell, 2000:5,289-298; Gampe R.T., et al.Mol Cell, 2000a:5,545-555; Gampe, R.T., et al.Genes Dev, 2000a:14,2229-2241), when the applicant finds that RXR α and TR3 form heterodimer, α spiral (I type can take place equally, be positioned nucleus) to π spiral (the II type can cause that heterodimer moves to tenuigenin) conversion, and find that further the Subcellular Localization of heterodimer and its function are closely related.

The applicant only find in RXR α/TR3 heterodimer RXR α NES by the RXR part in conjunction with regulate, part (rexinoids) as some RXR, 9cis-RA for example, effectively suppress RXR α/TR3 heterodimer and act on mitochondria, thereby inhibition apoptosis, and some other RXR part such as 15d-PGJ ₂Then promote the generation of apoptosis.

The applicant also finds 15d-PGJ ₂Can effectively suppress the expression of the retinoic acid receptors β (RXR β) of multiple cancer cell, but 15d-PGJ ₂This effect and the activity of PPAR γ irrelevant.By gel mobility shift assay, the applicant finds 15d-PGJ ₂Influenced TR3/RXR α heteroduplex combine (Fig. 9,10), prompting 15d-PGJ with β RARE ₂It is the activity that acts on TR3/RXR α heteroduplex and suppressed this heteroduplex.

The present invention is based on the new RXR action pathway that the applicant finds, this approach relate to RXR α from nucleus to cytoplasmic migration, in conjunction with understanding to this new RXR approach molecular basis, with suitable screening technique, can find that selective regulation is subjected to the compound of the one or more functions of RXR approach adjusting.

The applicant can regulate and control a kind of formation of RXR/TR3 heterodimer of uniqueness based on some new compounds, and make this heterodimer move to mitochondria from nucleus, act on mitochondria, cause apoptosis, further invented new screening technique and found out this new compound, the RXR/TR3 heterodimer of therefore predict that these regulating compounds can activate, strengthen, inhibition, minimizing or alternate manner being regulated a kind of uniqueness is moved to the method for mitochondrial compound from nucleus.

The applicant finds that RXR can be induced from nucleus moves to the tenuigenin, and interacts with the tenuigenin target spot, and this can be used for developing the screening technique that screening can be regulated and control the compound of this migration.The applicant finds that the non-genomic of the novelty of RXR is guided function into can be by acting on the mitochondria modulating apoptosis, and this can be used for developing the screening technique of the compound of modulating apoptosis.Similar, the applicant finds that the RXR part can be by formation and the nucleus output modulating apoptosis that acts on RXR α/TR3 heterodimer, this can be used for screening the compound of modulating apoptosis.These are found the test compounds that is used for modulating apoptosis and all can be used as medicine group and composition, are used for the treatment of, prevent and other is used to regulate and control the method for pathological state such as apoptosis.

Concretely, this invention comprise find out can induce RXR from nucleus to tenuigenin migration and make RXR and the interactional medicine of tenuigenin target.

This invention comprise find out can activate, strengthen, weaken, the method for the compound of the formation of heterodimer that inhibition, reverse, destruction or alternate manner are regulated and control a kind of uniqueness.

This invention comprises to be found out and can activate, and strengthens, weakens, inhibition, reverse, destruction or alternate manner regulate and control a kind of heterodimer of uniqueness from the method for nucleus to the compound of tenuigenin migration.

The present invention utilize it screening compounds method and use the method for this regulating compound, provide regulating compound that is used for the treatment of apoptosis relevant abnormalities/morbid state and the pharmaceutical composition that contains this compound, so RXR may represent a kind of desirable molecular target that is used for the developing cancer medicine.RXR/TR3 apoptosis pathway heterodimer is represented the another kind of desirable target that is used to develop treatment cancer and disease medicament.

The applicant finds that RXR can drug-inducedly be moved out from nucleus by many, and this migration be with tenuigenin be target as mitochondria.In case RXR is positioned at outside the nucleus, just interact with many various cellular pathways such as apoptotic cells matter targets of inducing.These tenuigenin effects of RXR are different with the traditional role of DNA combination regulate gene expression by it.

The applicant finds that further rxr receptor and TR3 acceptor form a kind of heterodimer of uniqueness in nucleus when drug-induced Apoptosis.This heterodimer is exported from nucleus, acts on mitochondria, promptly causes apoptosis.This discovery causes the screening technique invented, considers the discovery of the compound that is used to regulate described discovery and then processing and prevention correlation circumstance thus, identification and its dosage form.

The applicant finds that RXR combines the adjusting cell biological function and has two kinds of heterodimer forms at least with TR3, be I type and II type RXR/TR3 heterodimer, the DNA response element that the former is special in nucleus, transcribing of regulatory gene, the main propagation that participates in cell, the latter then can be under the inducing of certain compound moves out from nucleus and is positioned the mitochondria organelle, and the cell that is acted on better is to get mammal cell line, be more preferably cancerous cell line, preferably LNCaP clone.

The applicant also finds the Subcellular Localization with the technical regulation RXR in the document, and migration all is certain.And the routine techniques in those documents will be used the marrow that many these and other known technologies obtain this invention, the applicant better uses immunostaining, immunoblotting assay, detectable marker, and/or the natural fluoresence albumen and the micro-visible technology that are connected with destination protein matter and peptide practicality.

Definition

The used singulative " " of this literary composition, " with ", " this " remove clear indicate all comprise plural corresponding things.For example " compound " refers to one or more this compounds, and " this enzyme " comprises that a special enzyme is also included within other family member well known and equivalent in the document.

In general, the experimental procedure of used subsequently buzzword and following described cellular incubation, molecular genetics, nucleic acid chemistry, hybridization all be those be familiar with commonly used in the literature.Recombinant nucleic acid method, polynucleotide are synthetic, cellular incubation, transgenosis merge (for example electroporation, microinjection, lipofection) and all use standard technique.General enzyme reaction, oligonucleotide synthesize and purification step all carries out according to the description of product.As technology and the step 1 all according to the various conventional reference book that provides in conventional method in the document and the document, i.e. Maniatis et al., Molecular Cloning:A Laboratory Manual (1989), 2nd Ed., ColdSpring Harbor, N.Y.; And Berger and Kimmel, Methords in Enzymology, Volume152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif., this patent reference these data.Oligonucleotide can be synthetic with applying biological system oligonucleotide compositor according to product description.These steps all are commonly used in the document, and the reader easily obtains.All these documents are incorporated herein by reference.

Here " antisenses " of being mentioned refer to strand, two strands, three chain oligonucleotides and the peptide nucleotide (PNAS) that combines with rna transcription thing or DNA.Deriving from the oligonucleotide of-10 to+10 section of initial position of gene transcription such as initiation site, is a special example.Three chains that form antisense can combine with double-stranded DNA and suppressor is transcribed.Antisense molecule general 100% and the complementation of meaning chain, but also may part complementary (be less than 100% complementation, for example 95%, 90%, 80%, 70% in addition still less).Antisense molecule can obtain (for example solid phase phosphoramidite is synthetic) by genetic transcription method or chemosynthesis.Antisense oligonucleotide may comprise L-or D-form, also can suitably be modified for the opposing degraded when giving patient's medication.Special case comprises 5 ' and 3 ' chain, they can resist each tissue or the endonuclease of body fluid and the effect of exonuclease in the animal body of being present in.For reducing expression of gene, antisense nucleotide does not require that expressing control element plays a role in vivo.This antisense molecule can be absorbed by cell or by the passive cell that diffuses into, and also available support transfered cell is as viral vectors.Yet, to such an extent as to antisense molecule may be translated by nucleotide coding, even the nucleotide of this antisense molecule of encoding may be effectively with express control element and be connected, make this translate molecule in cell or the extracellular expression kept or improved.

Term " snoop tag " refers to can the selected any part that detects in screening is analyzed.As unrestrictedly, (for example then comprise isotope labeling ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), affinity labeling (biological example element/avidin, chain enzyme antibiotin, paratope, metal knot and position, epi-position mark, FLASH binding site---referring to United States Patent (USP) 6,451,569; 6,054,271; 6,008,378 and 5,932,474---glutathione or maltose binding site), fluorescence and cold light part (for example fluorescence and derivant thereof, GFP, rhodamine and derivant thereof, lanthanum etc.) and enzyme part (for example horseradish peroxidase, beta galactosidase, beta-lactamase, luciferase, alkaline phosphatase).This snoop tag can carry out in position.For example use the main antibody of non-marked, and it can be detected by an inferior antibody that has snoop tag.

Term " DNA binding site " or " DBD " refer to the protein domain that can combine with a special dna sequence dna comprise that at least one zinc refers to sequence.

Here the term of being mentioned " division " comprises the test compounds that causes the RXR-TR3 heterodimer virtually completely to separate (for example above 90% separation).Term " fully division " comprises and causes at least 50% test compounds of separating with acceptor in conjunction with cofactor.

Here the term of being mentioned " expression on the function " refers to a coded sequence, and it can be transcribed, translation, posttranslational modification (if meaningful), and places cell, brings into play its function.About a report box, functional expression refers generally to the production of this coded cell surface receptor protein sufficient quantity, reports a result that transcribes who reports oligonucleotide so that a detectable signal with statistical significance to be provided.

Here the term of being mentioned " LBD " or " ligand binding domain " refer to the protein structure domain of a nuclear receptor, as the steroids superfamily receptors of being mentioned here or other suitable nuclear receptor, they combine with a physiology part, configuration changes, take place with a protein-bonded intermolecular interaction or do not change, based on the functional areas that connected in having provided a detectable activity.

Here the term of being mentioned " regulator " refers to the test compounds of wide scope, not only comprise natural, also comprise synthetic and semi-synthetic organic molecule, protein, oligonucleotides antisense molecule and RNAi, promptly influence RXR indirectly or directly and move to cytoplasmic material from nucleus.

And the precursor of regulator (if can change into the compound of regulator) also is considered to regulator.Similarly, transforming precursor is that the compound of regulator also is considered to regulator.

" natural fluoresence albumen " refers to form height fluorescence, endogenous chromophoric albumen, and the formation of these albumen is the cyclisation of the interior exogenous nucleotide by protein and oxidation or because as the adding of fluorescence cofactor enzyme.General this chromophore spectrum comes from hypofluorescence amino acid for example tryptophane and tyrosine.Endogenous fluorescin separates from a large amount of marine species and the clone comes out, comprise ocean pansy Renilla reniformis, R.kollikeri and R.mullerei, sea pens Ptilosarcus, Stylatula and Acanthoptilum also have the Pacific Northwest jellyfish Aequorea Victoria; (SPIE conference 1999 such as Szent-Gyorgyi; D.C.Prasher et al., Gene, 1992,111:229-233), DsRed and yellow fluorescin have also obtained from coral.Obtained deriving from the many mutant of GFP of Aequorea victoria, compared with natural GFP, they have unique spectral quality, fluorescence strengthens, and expresses and improves, (GreenFluorescent Proteins collapsible in mammal, Chapter 2, pages 19-47, edited Sullivan and Kay, AcademicPress, U.S.patent Nos:5,625,048 to Tsien et al., essued April 29,1997; 5,777,079 to Tsien et al., issuedJuly 7,1998; And U.S.Patent No.5,804,387 toCormack et al., issued September 8,1998).These functional fluorescins have remarkable spectrum characteristic than wild-type protein in many cases, and this invents with it as reporter gene.If unrestricted, mentioned natural fluoresence albumen comprises GFP, EGFP, YFP, RFP and DS red.

Here the term of being mentioned " the practical connection " refers to the connection on the oligonucleotide element functional relationship.When a nucleotide is practical the connection during with other nucleotide sequence generation functional relationship.For example, if promoter or enhancer have influenced transcribing of coded sequence, it just therewith coded sequence practical connection has taken place.The practical connection means that connected dna sequence dna generally is continuous, must also need be in reading frame in conjunction with two protein-coding regions.Yet, general and promoter several Kb at interval when enhancer plays a role, intron sequences length also is different, so some oligonucleotide sequences may be practical connections but are not continuous.In fact have identical time and identical apoptosis-specific sexual norm with the gene of natural generation with the i.e. expression of the practical structural gene (as the HSVtk gene) that connects of transcription regulating nucleotide sequence of an endogenous gene of an oligonucleotide sequence.

Here mentioned term " nonsteroid anti-inflammatory drugs " refers to that those suppress the compound of inflammation by the generation that suppresses prostaglandin.These compounds have anti-inflammatory, analgesia, analgesic effect.With those sterids (for example hydrocortisone or prednisone) that have only antiinflammatory action as reference.Nonsteroid anti-inflammatory drugs is a for example indocin of COX2 inhibitor, voltaren see diclofenac, celecoxib naproxen, brufen and rofecxib.

Here mentioned term " nucleotide " and " nucleotide sequences " comprise the antisense nucleoside sequence, RNA molecule or aptamer sequence.For example nucleotide sequence such as aptamer can combine with the one or more components of RXR or RXR/TR3 or RXR/Agent compound, regulate the level and the stability of this compound.Aptamer is the nucleotide sequence with the three-dimensional structure picture that can combine with the micromolecule target, comprise metallic ion, organic dyestuff, medicine, amino acid, cofactor, aminoglycoside, microbiotic, nucleoside base analog, nucleotide and pdef polypeptide (Jayasena, S.D., Clinical Chemistry, 1999,45:9,1628-1650).Be listed in stability in the cell in order to increase these nucleotides sequences, can be modified it with the several method of being referred in the document.

Here mentioned term " orphan receptor " is the nuclear receptor superfamily member, and its native ligand (hormone) does not also find.

Here mentioned term " plan peptide " refers to non-peptide compound, with corresponding polypeptide be topological analog.For example this plan peptide still keeps the amino acid whose part or all of functional group that has critical function in the polypeptide.For example this plan peptide also can partly or entirely be made up of non-peptide main chain, also so designs other plan peptide such as glucose support, pyrrolidone support, steroids support, phenylpropyl alcohol diazine support or the like in the literature.Intend the advantage that Toplink provides each peptide species not have, can stablize by alimentary canal during owing to administration, so Orally-administrable.Intend Toplink in addition and be designed to better pass through blood-brain barrier.

Here mentioned term " RNAi " refers to disturb with the method for RNA interference the micromolecule of double-stranded RNA.It is to make the process of specific sequence gene silencing by transcribing back RNA degraded that RNA disturbs, and this is because double-stranded RNA (dsRNA) homologue has caused the generation of cryptiogene sequence.A special example of double-stranded RNA comprise corresponding to the sense strand that about 21 continuous nucleotide are arranged and the antisense strand of the target gene of 19 RNA nucleotide hybridization, hang two nucleotide (Elbashir et al. at every chain 3 ' end, Nature, 2001,411:494-498; Zamore, Nat.Struct.Biol., 2001,8:746-750).Approximately the dsRNAs of 25-30 nucleotide be used to RNAi (Karabinos et al., Proc.Natl.Acad.Sci.USA, 2001,98:7863-7868).DsRNA can be external synthetic and lead as cell by method described in the document.The translation of target polypeptide reduces in this way.

Reporter gene comprises any direct or indirect generation specificity reporter gene product, and the medicine that detectable marker, the part of enzyme or cytogene type if can monitoring gene be transcribed relies the gene of being subjected to property.Mentioned reporter gene includes the albumen of enzymatic activity, and the amplification of the enzyme of gene expression is provided, as beta-lactamase, luciferase, beta galactosidase, catalytic antibody and alkaline phosphatase.Other reporter gene comprises protein such as natural fluoresence albumen or homologue, and the natural or modified forms of cell surface protein or endogenous gene has existed or remains development in the future for the specific process of analyzing these albumen.

Here " selective induction " mentioned refers to the conversion of cell mass from non-apoptotic state to apoptotic state.Conversion is the increase that can detect apoptosis preferably, is more preferably the increase of apoptosis 10%, and best is that apoptosis is 50% increase.

Here " processing " mentioned or " disposal " comprise the processing of the morbid state relevant with apoptotic effect, and be specific as follows:

A) generation of the morbid state that blocking-up is relevant with apoptotic effect;

B) suppress the morbid state relevant with apoptotic effect: as stop its development; Or

C) reduce the morbid state relevant with apoptotic effect: as state is taken a turn for the better.

Here mentioned term " transcriptional activation domain " refers to strengthen protein or the albumen territory that the structure sequence introne is transcribed.Strengthen the ability transcribe and to influence that gene is derivable transcribes or the foundation level of gene is transcribed or both.For example report that polypeptide may comprise the promoter of the minimum of transcribing of the sequence of ordering about a reporter gene of coding for one.This report polypeptide can change the nuclear receptor sensitive cell line over to, is used to set up improved host cell.In the presence of the apoptosis activator, make the cloned sequence of the reporter gene expression silence of cultured cell be also included within (for example reduce basal transcription and guarantee detectable inductivity).The specificity example of many other transcriptional regulatory elements such as specificity minimal promoter and report element are all reported in the literature to some extent, can select to be used for inventive method and oligonucleotide structure based on the required application of pursuer.When the suitable transcriptional regulatory element of selecting to be used for this invention and other 26S Proteasome Structure and Function sequence, with reference to the patent document of document resource and announcement, Genbank and other sequence information data resource.If desired, can make up a transcriptional regulatory element by oligonucleotides synthetic (with being connected) according to available sequence information (as U.S. Genebank sequence, response element, minimal promoter etc.).

Term " I type and/or II type " refers to the variation of RXR:TR3 heterodimer configuration.The configuration of I type heterodimer makes it be positioned nucleus, can combine with the DNA response element, and the configuration of II type heterodimer makes it move to tenuigenin from nucleus, combines with mitochondria, triggers apoptosis.

Here the term of being mentioned " system " refers to complete organism and based on the system of cell, comprises and be used for the various compositions of analytical reactions in the RXR-TR3 of the test compounds of mentioning heterodimer II type cellular pathways here.

As for polypeptide, term " actual equate " mean two peptide optimum ratios to the time, for example when omitting gap parameters and carry out the best comparison, have at least 90% sequence equal by program GAP or BESTFIT, better have at least 95% sequence equal.Better, residue position inequality is different with conservative aminoacid replacement.Conservative aminoacid replacement is meant the residue mutual alternative of similar side chain.For example, the amino acid of aliphatic lateral chain is arranged is aminoacetic acid, alanine, valerian propylhomoserin, leucine and isoleucine to a group; It is serine and alpha-amino-beta-hydroxybutyric acid that a group has the amino acid of aliphatics-hydroxyl side chain; The amino acid that a group contains amino side chain is asparagine and glutamine; It is phenylalanine, tyrosine and tryptophane that a group has the amino acid of aromatic series side chain; It is lysine that a group has the amino acid of basic side chain, arginine and histidine; With a group the amino acid of sulfur-containing side chain being arranged is cystine and MET.Better the alternative group of conserved amino acid is valerian propylhomoserin-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-leucine, glutamic acid-asparagus fern amino acid and asparagine-glutamine.

Because technology and scientific terminology can not be enumerated out fully, any its meaning of term that does not limit can be regarded as the meaning in this invention citing document.May comprise that about " restriction enzyme " or " high-fidelity enzyme " this kind of enzyme and any other are fit to the enzyme of described standard, perhaps then comprise one or more methods of acquisition cDNA sequence known in the list of references or the method that in this explanation, will read about method.

Major advantage of the present invention is:

(a) the present invention has found its different biological function of the inferior location decision of cell of RXR and TR3, and this new discovery has caused the aspect of new medicament sifting motion system;

(b) a second aspect of the present invention is successfully to design and green fluorescent protein GFP is connected to target gene aptly, does not influence the function of target gene, and green fluorescence can be convenient to observe the inferior location of cell of target gene simultaneously;

(c) a third aspect of the present invention is that red fluorescent protein RFP is connected to (RFP-mito) on the mitochondria specific proteins, utilizes Laser Scanning Confocal Microscope can observe gene of interest easily and moves out from nucleus and be positioned on the mitochondria;

(d) utilize screening technique of the present invention, can efficiently screen acquisition apace is the compound of target spot with RXR and/or TR3, utilize fluorescence location can find clearly that the heteroduplex of these compounds regulation and control RXR and/or TR3 moves to the tenuigenin from nucleus, thus the regulating cell apoptosis;

(e) this screening technique has been successfully applied to various kinds of cell system, and screens and can cause apoptotic known and unknown compound.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

In first kind of screening technique, handle cell with various test compounds, and analyze the nucleus output of its RXR α.

LNCaP (ATCC, Cat.No.CRL-1740) or other similar cell, as H460 lung carcinoma cell, SY5Y neuroma cell and COS-7 cell incubated overnight in 96 holes, 384 holes, 1536 holes or other tissue culturing plate of having bought easily at first.

Screening can be adopted following two kinds of methods.First method is the influence that detection compound is moved the endogenous RXR α that expresses in the suitable cell.Second method relates to the transfection of the RXR α cDNAs of mark or unmarked GFP, FLAG or myc.Can use the antibody test endogenous receptor of RXR α, and the acceptor of transfection can be used the antibody of RXR α or the antibody test of mark.With standard program receptor plasmid such as GFP-RXR alpha fusion expression plasmid (or other mark merges) transfection are advanced cell.According to Li, H.et al. (Science, 2000:298,1159-64) preparation GFP fusion expression plasmid.After the transfection 16 hours, handle cell with following either party's method: (1) is 0.5% hyclone (FBS) only; (2) in 0.5%FBS, add a kind of apoptotic stimulus (as to LNCaP cell TPA); (3) in 0.5%FBS, add test compounds.The reaction plate cumulative volume: 96 orifice plates, 100 μ l, 24 orifice plate 1ml, every kind of compound addition is 1-5 μ l, and TPA is 100ng/ml, and all the other predictive compound working concentration scopes are 10 ^-5～10 ^-7Mol/L.Behind treated 3 hours of the cell, wash with phosphate-buffered salt (PBS), fix with 4% paraformaldehyde solution, with suitable antibody cellular immunity is dyeed then, observe the Subcellular Localization (BioRad) of endogenous RXR α with the MRC-1024MP confocal laser scanning microscope.The cell receptor distributed image that comparison process is crossed and the Subcellular Localization of control cells.Immunostaining with associated protein and tissue detects whether RXR α is moved to tenuigenin and mitochondria.DAPI dyeing is used to detect nucleus, and mitochondria detects with the Hsp60 immunostaining, and endoplasmic reticulum detects with the calprotectin of dyeing.

Embodiment 2

In second kind of screening technique, handle cell with various test compounds, and analyze RXR α/TR3 heterodimer mitochondrial effect.

At first with LNCaP (ATCC, Cat.No.CRL-1740) or other similar cell, as H460 lung carcinoma cell, SY5Y neuroma cell and COS-7 cell incubated overnight in 96 holes, 384 holes, 1536 holes or other tissue culturing plate of having bought easily.

Screening can be adopted following two kinds of methods.First method is the influence that detection compound is moved the endogenous RXR α/TR3 heterodimer of expressing in the suitable cell.Second method relates to mark or unmarked GFP, the RXR α of FLAG or myc and the transfection of TR3 cDNAs.Can use the antibody test endogenous receptor of RXR α and TR3, and the acceptor of transfection can be used RXR α and the antibody of TR3 or the antibody test of mark.With standard program receptor plasmid such as Flag-RXR alpha fusion expression plasmid (or suitable labeled receptor merges) and GFP-TR3 fusion expression plasmid (or the warm plasmid of mark that suits) transfection are advanced cell.According to Li, H.et al. (Science, 2000:298,1159-64) preparation GFP fusion expression plasmid.After the transfection 16 hours, handle cell with following either party's method: (1) is 0.5% hyclone (FBS) only; (2) in 0.5%FBS, add a kind of apoptotic stimulus (as to LNCaP cell TPA); (3) [the reaction plate cumulative volume is to be 100 μ l for 96 orifice plates, is 1ml for 24 orifice plates to add test compounds in 0.5%FBS.Every kind of compound volume is 1-5 μ l, and TPA is 100ng/ml, and all the other test compounds working concentration scopes are 10 ^-5～10 ^-7Mol/L.Cultivate cellular control unit and added the cell 3 hours of test compounds, use phosphate-buffered salt (PBS) washing then, and be fixed among paraformaldehyde solution T * PBS of 4%.With suitable antibody cellular immunity is dyeed then and detect RXR α and TR3, observe endogenous or the RXR α of transfection and the Subcellular Localization (BioRad) of TR3 with the MRC-1024MP confocal laser scanning microscope.The cell receptor distributed image that comparison process is crossed and the Subcellular Localization of control cells.Detect with the immunostaining of associated protein and tissue whether RXR α and TR3 are moved to tenuigenin and mitochondria.Detect nucleus with DAPI dyeing, and mitochondria detects with the Hsp60 immunostaining, endoplasmic reticulum detects with the calprotectin of dyeing.

Embodiment 3

In another screening technique, existing or not existing under the situation of RXR alpha inhibitor, handle cell with various test compounds, analyze the influence of its pair cell growth and apoptosis then.These inhibitor comprise but are not limited only to siRNA that its effect is to suppress RXR α, perhaps can use antisense TR3.In this example, with RXR α siRNA.

With containing 0.5%FBS nutrient solution cultured cell in tissue culturing plate, transient transfection GFP-RXR α makes up as described in example 1 above.Then with one of the following cell of handling: (1) is 0.5% hyclone (FBS) only; (2) TPA; (3) TPA among the 0.5%FBS and RXR α siRNA; (4) test compounds or; (5) test compounds and RXR α siRNA.With MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-hexichol-tetrazole bromide) and DAPI (4,6-diamides-2-benzene indoles) analyzing and testing cell enlargement and apoptosis.

Compare with non-transfected cells, transfection the cell forfeiture growth inhibited effect of RXR α siRNA, show that the growth inhibited effect of compound needs RXR α.

Embodiment 4

In further screening technique, LMB is used to suppress the nucleus output of the dependent RXR α of CRM1 and RXR α/TR3 heterodimer.Therefore existing and not existing under the situation of LMB, handle cell, detect RXR α or the RXR α/Subcellular Localization of TR3 heterodimer and the growth inhibited of compound and apoptotic effect with various test compounds.

Cultured cell in tissue culturing plate, transient transfection or not transfection RXR α and TR3 expression vector make up as described in example 1 above.Then with one of the following cell of handling: (1) is 0.5% hyclone (FBS) only; (2) in 0.5%FBS, add a kind of apoptotic stimulus (as to LNCaP cell TPA); (3) in 0.5%FBS, add a kind of apoptotic stimulus (as to LNCaP cell TPA) and LMB; (4) test compounds and the LMB among the test compounds among the 0.5%FBS or (5) 0.5%FBS.

With culture plate cell dyeing and observation, decide mode according to embodiment 1 as the subcellular fraction of embodiment 1 described each albumen of detection.Method with embodiment 3 detects growth inhibited and apoptosis.

Under the situation without LMB, the tenuigenin locator meams shows whether testing drug causes the nucleus output of RXR α or RXR α/TR3 heterodimer.If contain the nucleus location that the culture plate of LMB shows RXR α or RXR α/TR3 heterodimer, show that nucleus output CRM1 is dependent.Show to lack growth inhibited and apoptosis if contain the culture plate of LMB, show that then test compounds passes through the tenuigenin location cell growth inhibiting of RXR α or RXR α/TR3 heterodimer and apoptosis-induced.

Embodiment 5

In another screening technique, handle RXR α or TR3 albumen external or that in bacterium, synthesize with various test compounds, with the formation of gel mobility shift assay RXR α/TR3 heterodimer.This analysis will detect the formation which kind of test compounds can be regulated RXR α/TR3 heterodimer.There is or do not exist cultivation RXR α and TR3 albumen under the test compounds situation.Subsequently with potpourri with ²³The β retinoic acid reaction sequence (β RARE) of P mark is together cultivated, with the combination of gel mobility shift assay RXR α/TR3 heterodimer.9-cis-RA can strengthen RXR α/TR3 heterodimer and combine with DNA's, can be used as positive control, and 15d-PGJ ₂Can stop the combination of heterodimer, can be used as negative control.

The combination that certain compound strengthens heterodimer will show the RXR α/effect of TR3 heterodimer in nucleus of withing a hook at the end of this compound, LBD dimer surface that may be by RXR α/TR3 heterodimer induce the transcriptional activity that causes heterodimer.The combination that similar certain compound suppresses heterodimer may show that the nucleus that causes heterodimer of inducing on the LBD dimer surface that this compound may be by RXR α/TR3 heterodimer exports.

Embodiment 6

In another screening technique, handle cell with various test compounds and also analyze mitochondrial effect.In this example, existing and not existing under the situation of RXR-NES antibody, handle cell with all cpds.Whether this analysis can detect those compounds that cause apoptosis by RXR α/its effect of TR3 heterodimer approach.

At first with LNCaP or other similar cell, as the H460 lung carcinoma cell, SY5Y neuroma cell and COS-7 cell are with incubated overnight in 96 holes, 384 holes, 1536 holes or other tissue culturing plate of having bought easily.Handle cell with one of following reaction mixture then: the test compounds among (1) 0.5%FBS; (2) test compounds and RXR-NES antibody.Control board contains only FBS in addition, FBS and a kind of known apoptosis induction medicine such as TPA, FBS and RXR-NES antibody, or FBS, RXR-NES antibody and TPA.Cultivate potpourri then, with the PBS washing, and be fixed in the paraformaldehyde solution, as described in embodiment 1.

Use earlier the Hsp60 IgG antibody, again with Cy3 in conjunction with IgG antibody to the protein Hsp 60 of mitochondria combination dye (Pharmingen, Cat.No.).With two similar step antibody techniques RXR or RXR/TR3 are dyeed.Collect fluoroscopic image, analyze the doubling of the image with the MRC-1024MP laser scanning co-focusing microscope.

The Subcellular Localization that compares heterodimer and Hsp60.In the culture plate that only contains test compounds, RXR α or RXR α/TR3 show the test compounds activation or strengthen RXR α to regulation of apoptosis with overlapping of Gsp60.In the culture plate that contains test compounds and RXR-NES antibody, RXR α or the location of RXR α/TR3 in nucleus, the apoptotic stimulus effect that shows test compounds is suppressed by the migration of the heterodimer that stops NES and regulate.

Embodiment 7

In another screening experiment, to inducing RXR α or RXR α/TR3 heterodimer from nucleus organ such as mitochondria in cytoplasma membrane and various cell, the compound of golgiosome and endoplasmic reticulum migration carries out high flux screening with FRET (fluorescence resonance energy transfer) (FRET).Whether acceptor is moved to cytoplasma membrane in order to study, and a kind of cytoplasma membrane target sequence is merged as the CAAX box of the C end that contains K-ras and blue fluorescent protein (BFP) or suitable fluorescin.Be research mitochondria target spot, a kind of mitochondria target sequence such as fruit bat outer lines plastochondria memebrane protein Mas70p striden film district and BFP or suitable fluorescin fusion.For research mitochondria target spot,, combine with BFP or suitable fluorescin with the ER target sequence in the ER specificity isoform of a kind of ER specific target sequence such as cytochrome b5.Be research golgiosome target spot, with a kind of Gorky's specific target sequence and BFP or suitable fluorescin fusion of taking from the ST3Gal transferase I.Fusion plasmid and GFP-RXR α or GFP-TR3 will be stabilized transfection and advance COS-7 cell or other suitable cell.Stably express GFP-RXR α or GFP-TR3 and the clone that BFP merges will utilize previously described FRET technology, be used for the compound that high flux screening is induced RXR α or TR3 organ migration in cytoplasma membrane or other cell.

Pharmaceutical composition

The using method of compound and pharmaceutical composition in this invention is provided here.The method comprise with a kind of mode that depends on the specific cell type change the compound of nuclear receptor activity noted earlier and pharmaceutical composition in vivo with external using method.

The method of saying specifically just comprise the discovery and the use of apoptosis regulation compound.

Be modulator in case certain compound is differentiated by screening technique of the present invention, this compound just can be made into pharmaceutically acceptable form so, as using Remington ' s Pharmaceutical Science, 18 ^ThEd., MackPublishing Co., Easton, the described method of PA (1990), thus produce the pharmaceutical composition that can be used for treating disease and pathological state.

Give patient medication with the medicine that the method for being told about is found out here, medication separately also can be made into potpourri, adds suitable carrier or excipient, has the effective dose of medicine thing to patient.The drug dose that effective dose has guided improvement of patient's symptom or life to prolong.

Medicine also can be made into the acceptable salt of pharmacy.Pharmaceutically acceptable salt comprises the salt that contains hydrochloride, sulfate, phosphate, acetate, citrate, lactate, tartrate, methyl alcohol sulfonate, ethane sulfonate, benzene sulfonate, p-toluene sulfonate, cyclohexyl sulfonate and quininic acid as those.This salt may be to make as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, lactic acid, tartrate, methyl alcohol sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, p-toluenesulfonic acid, cyclohexyl sulfonic acid and quininic acid with acid.

Prepare the acceptable salt of pharmacy with standard technique.For example, the free base form of medicine at first is dissolved in the suitable solvent such as water or water-methanol solution that contains suitable acid.Evaporating liquid is with separated salt then.In another example, the method with free base of reaction and acid in organic solution prepares salt.

Carrier or excipient help drug administration, for example, increase the solubleness of medicine.The example of carrier and excipient comprises lime carbonate, calcium phosphate, various sugar and various forms of starch, cellulose, animal glue, vegetable oil, tygon and the physiology solution that suits.

The available standards method of pharmacy is as detecting the toxicity and the result of treatment of these medicines, as detecting LD with cellular incubation or animal used as test ₅₀(50% dead dosage) and ED ₅₀(50% effective dose).The ratio of toxicity and effective dose is a therapeutic index, available LD ₅₀/ ED ₅₀Expression.Medicine with higher therapeutic index is relatively good.The data that drawn from these cell culture assays and zooscopy can be released the dosage range that is used for the people.The dosage of these medicines preferably is present in and comprises little ED ₅₀Or do not have in the scope of toxicity.Dosage can change to some extent according to dosage that is provided and used method of administration.

For the medicine found out of the useful method of being told about here, the estimation of effective dose may all come from cell culture assays at the beginning.For example, dosage can form in animal model, to obtain to comprise IC ₅₀Circulating plasma concentration, in cellular incubation, detecting (obtain half dosage of protein complex maximal destruction as detection of drugs, or in the compound cell dosage of level and/or active maximum half that suppresses).This information can be used for detecting more accurately human effective dose.Can detect blood plasma level, as use HPLC.

The doctor can select suitable pharmaceutical composition, method of administration and dosage (Seee.g.Finglet al., in The Pharmacologocal Basis of Therapeutics, Ch.1p.1 (1975)) according to patient.The doctor should finish, interrupt petty the time or adjust dosage according to decision such as toxicity, organ dysfunction is assorted.Opposite, if when clinical dosage is not enough (avoiding toxicity), the doctor also will be appreciated that and regulates dosage to higher level.The dosage scope should change to some extent with disease severity and the method for administration handled.And dosage and possible administration frequency also will be adjusted to some extent according to age, body weight, concrete patient's response situation.Also can be used for the animal doctor with the similar program of above-mentioned discussion.

According to the special circumstances of handling, these medicines can be administered systemically or topical, and medication can be with reference to Remington ' s Pharmaceutical Sciences, 18 ^ThEd., Mack Publishing Co., Easton, PA (1990).Suitable route comprises oral cavity, rectum, through skin, vagina, administration in mucous membrane or intestines: outside the intestines approach comprise in intramuscular, subcutaneous, intramedullary injection and the sheath, directly in the ventricle, in the intravenous, peritonaeum, in the nose or intraocular injection.

The medicine that is used to inject can be made into aqueous solution, the suitable damping fluid of the most handy physiology such as Hanks solution, Ringer solution or physiological saline.To through the mucosal route administration, preparation should adopt the mode that penetrates of appropriate carrier infiltration, and these penetrate and generally are found in document.

Attention will be used the pharmacy acceptable carrier, to make the medicine that contains the dosage that is suitable for being administered systemically.Select suitable carrier and production decision.These medicines, particularly those medicines that exist with the solution form, but intestines external administration are as by intravenous injection.Can accept carrier with pharmacy common in the document, these medicines can be made the dosage that is suitable for oral administration easily.These carriers can make the medicine of this invention make the preparation of patients' energy oral absorption such as tablet, capsule, pill, liquid, gel, syrup, slurries, suspension.

But the technology of using always in the medicine list of references of administration in the cell.For example, these medicines can wrap up into liposome, then, and by the above administration.Liposome is the globular adiponectin duplicature, and inside is water wettability.All molecules when having liposome to form in the aqueous solution all wrap up into hydrophilic inside, and fat wrappage and outside microenvironment are isolated, because liposome and cell membrane merge, therefore, the liposome thing can effectively enter tenuigenin.In addition, because their hydrophobicity, little organic molecule can directly inject in the cell.

The medicine that is suitable for this invention is formed, and comprises the composition of the active ingredient of the effective dose that can obtain intended purposes.But definite list of references of effective dose especially can be described with reference to this literary composition.Except active component, these medicines are formed also can contain suitable drug acceptable carrier, wherein comprises the excipient and the assistant that help active medicine to be prepared into the process of the patent medicine that pharmacy can use.Be used for oral patent medicine and can be made into tablet, dragee, capsule or solution.This invents described medicine composition and can generate by known way, as by conventional mixing, dissolving, granulating, makes dragee, suspension, emulsification, makes capsule and pill, or freeze-drying process.

The system that is used for the intestines external administration comprises the aqueous solution of the active medicine that exists with the aqueous solution form, in addition, the suspending liquid of active medicine can be made into suitable oily injectable suspensions, the lipophilic solvent or the excipient that are fit to comprise fat oil such as sesame oil, or Acrawax, as ethyl oleate or triglyceride or liposome.Water injection suspending liquid may comprise the material that can increase suspension viscosity, and as carboxycellulose sodium, sorbierite or glucosan also can comprise suitable stabilizing agent and medicine, increases drug solubility to make the solution of high concentration.

Be used for oral patent medicine by making in conjunction with active medicine and solid excipient, this potpourri can be milled into acceptable degree, also can be made into the potpourri of particulate.If desired, after adding suitable assistant, can be made into tablet and sugar-coated preparation.Specifically, suitable excipient is some fill such as sugar, comprises lactose, sucrose, mannose or sweet pears alcohol; The natural gum of cellulose patent medicine such as cornstarch, wheaten starch, rice starch, potato starch, animal glue, system acacia gum, methylcellulose, hydroxy-methyl cellulose, sodium carboxymethyl cellulose and/or polymerising ethylene pyrrolidone (PVP).If desired, can add disintegrant such as cross-linked polymeric vinylpyrrolidone, agar or alginic acid or salt such as alginates.

Dragee can wrap up suitable shell, and therefore available concentrated sugar solution contains gum arabic, talcum, polymerising ethylene pyrrolidone, tygon hexanediol, and/or titania, lacquer solution and suitable organic solvent or solvent mixture.Dyestuff or coating can be added into the various combination that is used to discern or indicate active compound doses in tablet or the sugar-coat.

Can be used for the cooperation insertion capsule that oral patent medicine comprises that animal glue is made, but the soft seal capsule that animal glue is made and plastic cement such as glycerine or sorbierite.Cooperate to insert capsule and contain active component, and with filling material such as lactose, binder such as starch, and/or mixing such as lubricant such as talcum or dolomol and stabilizing agent.In soft capsule, active medicine solubilized or be suspended in the suitable liquid, as fat oil, whiteruss or liquid polymeric ethylene glycol.Can add stabilizing agent in addition.

More available method of sending comprises:

A. be encapsulated in the liposome;

B. use the reverse transcription carrier transduction;

C. utilize the nucleus target spot that on many nucleus albumen, finds to navigate to nucleus;

D. carry out cell transfecting with the transplanting again or the administration of transfected cell in the body;

E.DNA transportation system

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (4)

1. the method for a screening compounds, described compound regulation and control retinol X receptor RXR and orphan receptor TR3 are moved to following position from nucleus: tenuigenin, cell membrane, mitochondria, golgiosome and/or endoplasmic reticulum, it is characterized in that, comprise step:

(a) provide a kind of cell, this cell contains RXR and TR3 in nucleus;

(b) there is or do not exist the described cell of cultivation under the test compounds situation; With

(c) select and regulate RXR and TR3 from the nucleus of described cell compound: tenuigenin, cell membrane, mitochondria, golgiosome and/or endoplasmic reticulum to the migration of following position.

2. the method for claim 1 is characterized in that, described compound increases the migration of RXR to described cell matter.

3. the method for claim 1 is characterized in that, described compound weakens the migration of RXR to described cell matter.

4. the described method of claim 1, it is characterized in that described compound is selected from following material: peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrates, fat, synthetic compound, natural products, antibody or antibody fragment, organic molecule, inorganic molecules and nucleotide sequence.

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