EP0912896A1 - Identification d'inhibiteurs de l'ultraspiracle - Google Patents

Identification d'inhibiteurs de l'ultraspiracle

Info

Publication number
EP0912896A1
EP0912896A1 EP97928958A EP97928958A EP0912896A1 EP 0912896 A1 EP0912896 A1 EP 0912896A1 EP 97928958 A EP97928958 A EP 97928958A EP 97928958 A EP97928958 A EP 97928958A EP 0912896 A1 EP0912896 A1 EP 0912896A1
Authority
EP
European Patent Office
Prior art keywords
usp
reporter gene
receptor
test
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97928958A
Other languages
German (de)
English (en)
Other versions
EP0912896A4 (fr
Inventor
Julia N. Heinrich
Fernando Dela Cruz
Donald R. Kirsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
American Cyanamid Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by American Cyanamid Co filed Critical American Cyanamid Co
Publication of EP0912896A1 publication Critical patent/EP0912896A1/fr
Publication of EP0912896A4 publication Critical patent/EP0912896A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43569—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from flies
    • G01N2333/43573—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from flies from Drosophila
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention relates to the identification of inhibitors of "orphan" nuclear receptors (i.e. , receptors for which no natural ligand is known).
  • the invention relates to the Ultraspiracle protein (Usp) of Drosophila melanogaster and homologues thereof in other insect species.
  • the invention provides methods for identifying compounds, variant nuclear proteins, and other auxiliary proteins that interfere with Usp function. USP inhibitory compounds are useful as insecticides or as lead compounds for the development of insecticides.
  • the ultraspiracle (Usp) gene of Drosophila melanogaster encodes a protein that is required throughout the development of flies (Oro et al. , Develop. 115:449, 1992). It was first identified by the phenotype of mutants that died in the molting process between the first and second instar stages and thus had two sets of spiracles (Perrimon et al. , Genetics 111:23, 1985). The behavior of these mutants suggested that Usp functions in the ecdysone (Ec) response pathway of metamorphoses and imaginal disk formation. It has subsequently been found that Usp also functions in female reproduction and eye mo ⁇ hogenesis, and may participate in Ec-dependent and Ec-independent processes in the fly. (Garen et al.,
  • Usp can functionally substitute for RXR in dimerizing with the vertebrate nuclear receptors thyroid hormone receptor (TR), vitamin D receptor (VDR), and peroxisomeproliferator-activated receptor (PPAR) (Yao et al., Cell 71:63, 1992); (ii) Usp cannot bind or respond to retinoic acid ligands (Yao, 1992, supra; (iii) Usp heterodimerizes with the ecdysone receptor (EcR) and thereby confers on EcR the ability to mediate ecdysone responses (Oro et al. , Develop.
  • TR thyroid hormone receptor
  • VDR vitamin D receptor
  • PPAR peroxisomeproliferator-activated receptor
  • the present invention provides a method for identifying mediators of the transcriptional activity of "o ⁇ han” nuclear receptors, i.e., receptors for which no natural ligand has been identified.
  • the invention provides a method for identifying compounds that inhibit the function of Drosophila ultraspiracle protein (Usp). The method comprises:
  • a S. cerevisiae cell that expresses Usp and a Usp binding partner comprising mAR ⁇ EcR-CDEF.
  • the cell also contains a reporter gene comprising an ecdysone response element operatively linked to DNA encoding arginine permease (the CAN1 gene product).
  • the reporter gene is transcriptionally activated only in the presence of a functional complex between Usp and mAR ⁇ EcR-CDEF; as a consequence, the cell expresses CAN1 and is sensitive to the toxic (growth-inhibitory) action of canavanine (an arginine analogue).
  • modulators of transcriptional activity of a nuclear receptor are identified according to the present invention by contacting the receptor with a binding partner with which it forms a heterodimer, wherein the heterodimer interacts with, and transcriptionally activates, a known DNA response element in a hormone-independent manner.
  • modulators e.g., compounds, variant receptors, auxiliary proteins
  • FIG. 1 is a schematic representation of native and recombinant nuclear receptors: Ultraspiracle protein (Usp); Ecdysone receptor (EcR); mammalian androgen receptor (mAR); N-terminal truncated EcR ⁇ A/B; and recombinant chimeras.
  • the recombinant proteins are depicted according to the standard designation of functional domains A through F and are aligned by domain C.
  • FIG. 2 is a schematic representation of the method for identifying Usp inhibitors according to the present invention.
  • a canl yeast strain contains three plasmids: Y ⁇ pcUsp expresses Usp; YBpmAR ⁇ EcR-CDEF expresses mAR ⁇ EcR-CDEF; and YEpCANl expresses canavanine permease (CAN1) under the control of the ecdysone response element (EcRE 2 ).
  • EcRE 2 ecdysone response element
  • co-expression of Usp and mAR ⁇ EcR- CDEF results in the expression of CAN1 and growth repression by canavanine.
  • an inhibitor (I) of Usp interferes with the expression of canavanine permease and allows growth even in the presence of canavanine.
  • the present invention includes methods and compositions for identifying compounds that inhibit the functions of Drosophila ultraspiracle protein (Usp) as well as those of Usp homologues in other insect species.
  • a yeast cell comprises: (a) a Usp binding partner; (b) Usp or derivatives thereof capable of forming a functional complex with the binding partner; and (c) a reporter gene, which requires for its expression a functional Usp-Usp binding partner complex.
  • the yeast cell is incubated in the absence and presence of test compounds that are being evaluated for their ability to interfere with Usp function, and the resulting cultures are monitored for expression of the reporter gene.
  • Usp-inhibitory compounds are identified as those compounds that reduce the expression of the reporter gene in treated cultures relative to control cultures (including untreated cultures and wild-type cultures).
  • the methods of the present invention can be used to identify compounds that inhibit the function of a nuclear hormone receptor, even when the natural ligand of the receptor is not known (i.e., "o ⁇ han” receptors).
  • a cell preferably a yeast cell, which expresses the receptor and a suitable reporter gene and in which the reporter gene is transcriptionally activated by the receptor in a ligand- independent manner. If required to achieve ligand-independent transcriptional activation of the reporter gene, a heterodimeric binding partner of the receptor is co-expressed in the same cell.
  • Usp is encoded by an open reading frame of 1527 nucleotides and is a polypeptide of 508 amino acids having a molecular mass of 55,252 daltons (Henrich et al. , Nuc.Acids.Res. 18:4143, 1990; Shea et al. , Genes Dev. 4: 1128, 1990; Oro et al., Nature 347:298, 1990).
  • Usp has an apparent domain structure typical of the nuclear steroid receptor family, including an A/B (transactivation) domain, a C (DNA binding/dimerization/transactivation) domain, a D (nuclear localization) domain, and an E (dimerization) domain.
  • any derivative of Usp may be used that is capable of forming a functional complex with a Usp binding partner.
  • a "functional Usp-Usp binding partner complex” as used herein is a complex that interacts productively with a cognate DNA transcriptional activation sequence (specified by the Usp binding partner) so as to activate transcription of DNA sequences located downstream of the transcriptional activation sequence.
  • Useful Usp derivatives may include Usp polypeptides in which one or more amino acids have been added or deleted relative to the wild-type sequence, or in which one or more amino acids have been replaced with different amino acids that do not affect the formation of a functional Usp-Usp binding- partner complex.
  • the methods of the present invention can be used to screen Usp mutants or derivatives to identify those that retain their ability to form a functional Usp-Usp binding partner complex.
  • Binding partners of Usp or of o ⁇ han receptors encompass native and recombinant polypeptides that form functional complexes with Usp or with other o ⁇ han receptors.
  • Usp binding partners according to the present invention include without limitation ecdysone receptor (EcR), which comprises three isoforms: EcR-A, EcR-Bl , and EcR-B2 (Koelle et al., Cell 67:59, 1990; Talbot et al. , Cell 73: 1323, 1993); and the vertebrate receptors TR, VDR, and PPAR (Yao et al., Cell 71:63, 1992).
  • EcR ecdysone receptor
  • Useful binding partner derivatives include those in which one or more amino acids have been added or deleted relative to the wild-type sequence, or in which one or more amino acids have been replaced with different amino acids that do not affect the formation of a functional Usp- Usp binding-partner complex. Chimeras between different nuclear receptor proteins, such as, for example, between Drosophila nuclear receptor proteins known to bind Usp and other, non-Drosophila members of the steroid receptor polypeptide family, are also included.
  • the Usp binding partner comprises a chimera comprising the A/B domain of a mammalian androgen receptor and the C,D,E, and F domains of EcR.
  • Reporter genes useful in practicing the present invention include genes (i) that are transcriptionally activated by a Usp-Usp binding partner complex; and (ii) whose expression in yeast is readily detectable.
  • a reporter gene comprises at least two DNA sequence components, which are operably linked to each other: (i) a 5' regulatory region, including promoter elements and elements responsive to the particular Usp-Usp binding partner complex employed; and (ii) a 3' protein-coding region encoding a reporter polypeptide. These two sequence components may additionally be separated by sequences encoding a 5 '-untranslated region of the messenger RNA, including sequences that function in initiation of protein synthesis.
  • the protein-coding sequence of the reporter polypeptide may be flanked on its 3' terminus by a polyadenylation consensus sequence, transcription termination sequence, and the like.
  • suitable regulatory regions include without limitation those comprising an ecdysone response element (ERE), androgen response element (ARE), Vitamin D response element (VRE), retinoic acid response element (RRE), thyroid hormone response element (THRE), chicken ovalbumin upstream transcription factor response element (COUP-TE), and peroxisome proliferator-activated response element (PPAP-RE) .
  • sequence of a particular response element may be truncated, multimerized, combined with other response elements, mutated, covalently modified, placed at varying distances upstream of the reporter polypeptide coding sequence, and otherwise manipulated, so long as the resulting response element sequence as a whole confers on the reporter gene the capacity to be transcriptionally activated by the particular Usp-Usp binding partner complex employed, i.e. , the reporter gene comprises a functional response element.
  • reporter polypeptides include without limitation: /3-galactosidase derived from E. coli (LacZ); arginine permease derived from S. cerevisiae (CANl); polypeptides involved in nucleoside and amino acid metabolism, such as the products of the URA3, LEU2, LYS2, HIS3, HIS4, TRP1, and ARG4 genes; polypeptides that confer resistance to drugs such as hygromycin, tunicamycin, cycloheximide, and neomycin; and green fluorescence protein (GFP) (Guthrie et al. , Meth.Enzymol. Vol. 194, 1991; Prasher, Trends Gen. 11:320, 1995).
  • GFP green fluorescence protein
  • Detection of expression of the reporter gene may be achieved using any means known in the art, including without limitation enzymatic assays, growth assays, immunoassays, ligand binding assays, drug resistance assays, fluorescence assays, and the like.
  • transcription of the reporter gene occurs at a level that is low enough so that it is either undetectable in the assay employed, or can be readily distinguished in such an assay from the transcription that occurs in the presence of Usp-Usp binding partner complex.
  • Reporter gene expression in the presence of a functional Usp-Usp binding partner complex may confer a positive or negative trait on the transformed yeast cell.
  • yeast comprising both a functional Usp-Usp binding partner complex and an appropriate reporter gene exhibit poor growth and growth is restored in the presence of an inhibitor of Usp function.
  • any suitable recombinant cloning vectors may be used for introducing into yeast DNA sequences encoding Usp and Usp binding partners, as well as DNA sequences comprising reporter genes.
  • Such vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. prototrophy or antibiotic resistance, and one or more expression cassettes.
  • the inserted sequences may be synthesized by standard methods or isolated from natural sources. Suitable vectors include without limitation YEp and Yip vectors (Hill et al., Yeast 2: 163, 1986).
  • Non-limiting examples of yeast promoters that may be present in these vectors to direct the expression of Usp and Usp binding partners include metallothionein promoter (CUP1), triosephosphate dehydrogenase promoter (TDH3), 3-phosphoglycerate kinase promoter (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.
  • CUP1 metallothionein promoter
  • TDH3 triosephosphate dehydrogenase promoter
  • PGK 3-phosphoglycerate kinase promoter
  • GAP1 galactokinase
  • GAL1 galactokinase
  • ADH alcohol dehydrogenase
  • Host yeast cells may be transformed by any suitable method, including without limitation methods that employ calcium phosphate, lithium salts, electroporation, and spheroplast formation (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1982). Suitable host cells include without limitation Saccharomyces cerevisiae and Schizosaccharomyces pombe. Any host cell in which ligand-independent activity of a Usp-Usp binding partner can be measured may be used in practicing the invention. Host cells may also be modified or manipulated with respected to their ability to produce different types of covalent modification of proteins, such as, e.g. , phosphorylation (Bai et al., Vitamins Horm. 51:289, 1995).
  • Yeast cells comprising Usp, a Usp-binding partner, and an appropriate reporter gene are used in an assay to identify compounds that interfere with Usp function.
  • the cells are incubated under conditions in which Usp and a Usp binding partner are expressed and form functional complexes, resulting in expression of the reporter polypeptide.
  • Cultures expressing Usp, Usp binding partner and reporter polypeptides are incubated in the presence of test compounds to form test cultures, and in the absence of test compounds to form control cultures. Incubation is allowed to proceed for a sufficient time and under appropriate conditions to allow for interference with Usp function and turnover of pre-existing reporter polypeptides.
  • an assay is performed to monitor the level and/or activity of the reporter polypeptide. Additional controls, with respect to both culture samples and assay samples, are also included, such as, for example, wild-type yeast and yeast expressing a functional CANl gene product. Usp inhibitory compounds are identified as those that reduce the expression of the reporter gene in the test cultures relative to the control cultures.
  • useful Usp inhibitory compounds identified by the methods of the present invention will be those that interfere with any of the following: (a) the formation of a functional Usp-Usp binding partner complex; (b) the interaction of an agonist with the Usp-Usp binding partner complex; or (c) the functional interaction of the Usp-Usp binding partner complex with the cognate DNA response element that is normally transcriptionally activated by a native Usp-Usp binding partner complex.
  • the methods of the present invention are adapted to a high- throughput screen, allowing a multiplicity of compounds to be tested in a single assay.
  • inhibitory compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall,
  • a compound may be tested for Usp inhibitory activity in a cell expressing, in addition to Usp, a different Usp binding partner and reporter gene than was present in the cell in which the inhibitory activity of the compound was originally detected.
  • Inhibitory activity may be tested in vitro by monitoring the ability of the compound to inhibit heterodimeric complex formation between Usp and the Usp binding partner and/or complex formation between the Usp-Usp binding partner complex and DNA containing the appropriate response element; this may be achieved using, e.g. , glycerol gradient fractionation, gel shift assays, and protease protection assays.
  • Usp inhibitors may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in commercial insecticide applications, etc. These modifications are achieved and tested using methods well-known in the art.
  • IGRs insect growth regulators
  • insecticide activity of Usp inhibitors identified using the methods of the present invention is tested using techniques well-known in the art. For example, formulations of each identified compound (see below) may be sprayed on a plant to which insect larvae are then applied; after an appropriate time, the degree of plant destruction by the larvae is quantified.
  • Usp inhibitory compounds are formulated in a biologically acceptable carrier.
  • suitable biologically acceptable carriers include, but are not limited to, phosphate-buffered saline, saline, deionized water, or the like.
  • Preferred biologically acceptable carriers are physiologically or pharmacologically acceptable carriers.
  • the insecticide compositions include an insecticide effective amount of active agent.
  • Insecticide effect amounts are those quantities of the insecticide agents of the present invention that afford prophylactic protection against insect infestation in plants and animals, and which result in amelioration or cure of an existing insect infestation in plants or animals.
  • This insecticide effective amount will depend upon the target insect, the agent, and the host. The amount can be determined by experimentation known in the art, such as by establishing a matrix of dosages and frequencies and comparing a group of experimental units or subjects to each point in the matrix.
  • the insecticide active agents or compositions can be formed into dosage unit forms such as, for example, emulsifiable concentrates (EC), suspension concentrates (SC), and water dispersable granules (WDG).
  • the insecticide active agents or compositions can be formed into dosage unit forms such as for example, creams, ointments, lotions, powders, liquids, tablets, capsules, sprays, or the like. If the insecticide composition is formulated into a dosage unit form, the dosage unit form may contain an insecticide effective amount of active agent. Alternatively, the dosage unit form may include less than such an amount if multiple dosage unit forms or multiple dosages are to be used to administer a total dosage of the active agent.
  • Dosage unit forms can include, in addition, one or more excipient(s), diluent(s), disintegrant(s) , lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), abso ⁇ tion enhancer(s), stabilize ⁇ s), bactericide(s), or the like.
  • the insecticide agents and compositions of the present invention are useful for preventing or treating insect infestations in plants and animals. Prevention methods inco ⁇ orate a prophylactically effective amount of an insecticide agent or composition.
  • a prophylactically effective amount is an amount effective to prevent infestation and will depend upon the insect, the agent, and the host. These amounts can be determined experimentally by methods known in the art and as described above. Treatment methods inco ⁇ orate a therapeutically effective amount of an insecticide agent or composition. A therapeutically effective amount is an amount sufficient to reduce an insect infestation.
  • This amount also depends upon the target insect, the agent, and the host, and can be determined as explain above.
  • prophylactically and/or therapeutically effective amounts can be administered in one admimstration or over repeated administrations. Therapeutic administration can be followed by prophylactic administration, once the initial insect infestation has been resolved.
  • the insecticide agents and compositions can be applied to plants topically or non-topically, i.e. , systemically.
  • Topical application is preferably by spraying onto the plant.
  • Systemic admimstration is preferably by foliar application or by application to the soil and subsequent abso ⁇ tion by the roots of the plant.
  • N-terminal of the full length nuclear receptors and chimeras described below were fused in frame to the aminoterminus of the seventy-six amino acid ubiquitin (Ub ⁇ ) in high-copy
  • YEp yeast expression vectors (McDonnell et al., Mol. Cell.Biol. 9:3517, 1989; Mak et al., J.Biol. Chem. 264:21613, 1989; Mak et al. Rec.Prog.Horm.Res. 49:347, 1994).
  • a recombinant fusion protein is expressed, which is cleaved by an endogenous protease to release the mature nuclear protein or chimera shown in Figure 1.
  • the YEpcUsp plasmid was constructed by amplifying the Usp gene in the pCFI plasmid (Shea et al., Genes Dev. 4: 1128, 1990) as two PCR fragments and inserting them into the Eag I and Dra in sites of the multiple cloning sites, of the YEpcMCS.
  • the 5' half of Usp was amplified with the sense primer 5'-ACTTCACGGCCGATGGACAACTGCGACCAGGACGCCA-3' containing at the 5' end Eag I and the antisense primer 5'-CACCTGGGCAAAGTGCGGCATCAT-3' containing at the 3' end BssH TJ.
  • the 3' half of Usp was amplified with the sense primer 5'-CAAACAGCTCTTCCAGATGGTCGA-3' containing at the 5' end a BssR U site, and the antisense primer 5'-ACTACTCAAACAGTGCTACTCCAGTTTCATCGCCAGGCC-3' containing a Dra HI site at the 3' end.
  • the two PCR fragments and YEpcmcs were cleaved with Eag I and Dra HI site, mixed and ligated together to obtain YEpcUSP.
  • YEpcLW the copper-responsive yeast metallothionein promoter (CUP1) regulates the expression of Usp, and leucine (LEU) is the selectable marker.
  • CUP1 copper-responsive yeast metallothionein promoter
  • LEU leucine
  • the YEpEcRBl plasmid was made in three steps. First, YEp was modified at the Afl U-Kpn I site by the addition of a double- stranded linker 5
  • PCR polymerase chain reaction
  • pMK-EcR was digested first with BspE I and then with EcoN I, the digest was resolved on a 1 % agarose gel, and the 2.6 kb fragment of the EcR was excised, gel purified, and inserted into the EcoN I and BspE I sites of YEpBspE to obtain YEpEcR.
  • YEp£cR the expression of EcR is regulated by the yeast constitutive promoter triosephosphate dehydrogenase (TDH3), and tryptophan (TRP) is the selectable marker.
  • Plasmids YEpEcR-A and YEpEcR-B2 were made from YEpEcR-Bl by excising from YEpEcR-Bl an Afl II and Asc I fragment, and inserting in its place a PCR fragment that was identical to the original except for containing the unique sequences of EcR-A and EcR-B2:
  • the templates were pWT57 and WT56 (Talbot et al. , Cell, 73: 1323 (1993), respectively.
  • the sense primers were 5'-CTTGTCTTAAGACTAAGAGGTGGCATGGATACTTGTGGATTAGT-3 ' and 5 ' -CTTGTCTTAAG ACTAAG AGGTGGC ATGTTGACG ACGAGTGG AC A-3 ' , respectively.
  • the antisense primer 5'-GCACTCCTGACACTTTCGCCTCAT-3' was used for both reactions.
  • the YEpEcR ⁇ A/B plasmid was made by excising from YEpEcR-Bl a Bam ⁇ I and BspE I fragment containing the 7DH3 promoter, the Ubi gene and the EcRAA/B; and replacing it with both a fragment containing the TDH3 promoter, Ubi gene, and a novel Dra UL site.
  • the Dra JH site resulted in an additional 9 bases downstream of the EcRAA/B from the closest BspE I site.
  • the reinserted fragment was made from two PCR fragments, the first PCR reaction amplified the TDH 3 -Ubi portion of YEpecr with the sense primer 5'-ATGTGTCAGAGGTTTTCACCG-3' that included at the 5' end a BamH I site and the antisense primer 5 '-TC ATCAC ACGTGGTTGGCC AAGAC AAG-3 ' which included at the 3' end a Dra m site; and the second PCR reaction amplified sequences downstream of the A/B domain of the EcR to BspEl site with the sense primer 5'-TCATCACACCACGTGGAGCTGTGCCTGGTTTGCGGCGAC-3 ⁇ which contains a Dra III site at the 5 ' end , an d th e anti sen se pri mer S'-CTCTCTTCAACCCACCAAAGGCCA-S', which contains Bam ⁇ I site at the 3' end.
  • the YEpmAR5 plasmid (Mak et al., Rec.Prog.Horm.Res. 49:347, 1994) and either YEpEcR-Bl or YEpEcRAA/B plasmids were used to make six chimeras ( Figure 1).
  • the name of the chimeras begins with the nuclear receptor which contributes the N-terminal domain(s), followed by a slash " ⁇ " indicating the junction between two nuclear receptors, and ends with the name of the second nuclear receptor plus the domains which it contributes. All DNA base numbers are based on the numbers used in the gene sequences of EcR (Koelle et al.
  • base number 1 is not the A in the initiation codon of the ORF.
  • + 1 of the A in the ATG of the ORF subtract 1067 from the EcR sequences and 32 from the mAR sequences.
  • the first chimera, YEpEcR ⁇ mAR-DE was made in two steps. First, the EcRBl gene was digested with Sac I and BspE I to excise bases 2151 to 3706 and retain the sequences composing the A/B and C regions and one-third of the D region. Second, the sense primer
  • 5'-CATCATGAGCTCTCGTAAGCTGAAGAAACTTGGAAATCT-3' and the antisense primer 5'-CATCTTCTCCGGATCACTGTGTGTGGAAATAGATGGGCT-3' were used (with the mAR5 gene as a template) to amplify the androgen receptor gene from bases 1856 to 2732, representing regions D and E, and a 0.9 kb fragment having Sac I and BspE I ends was obtained. After digestion with the respective restriction enzymes, the mAR fragment was cloned into the Sac I and BspE I sites of YEp£cJ? prepared previously.
  • the second chimera, YEpmARXEcR-DEF ' was made with Dra TH and BssH ⁇ linkers inserted into the Sac I and BspE I sites, respectively, of YEpEcRBI.
  • the resulting plasmid was then cut with Dra HI and BssH U to release the 1.8 kb DNA fragment from bases 2151 to 3706 encoding two-thirds of the D region and all of the E and F regions.
  • This fragment was isolated and cloned into the Dra Ul and BssH ⁇ sites of YEpmAR5 (Mak et al. Rec.Prog.Horm.Res. 49:347, 1994) which contain bases 33 to 1850 of the mAR. Both sites are present at the 3' end of the truncated mAR.
  • the resulting protein expressed contains the A, B and C regions of the mAR and the D, E and F regions of EcR.
  • the third chimera, YEpmAR ⁇ EcR-EF was made by amplifying from the EcRBl bases 2358 to 2306 with the sense primer 5 ' -C ATGATC ACACAGTGC AGO ATGTATG AGC AGCC ATCT-3 ' and antisense primer 5'-GATCTAGCGCGCCTATGCAGTCGTCGAGTGCTCCGA-3 ⁇ obtaining a 1.3 kb fragment encoding the E and F regions of EcR and including Dra UL and BssH ⁇ ends, respectively, digesting the PCR product with the appropriate enzymes, and cloning it into the Dra HI and BssH U sites of YEpmAR ⁇ .
  • YEpmAR ⁇ contains a truncated mAR gene from bases 33 to 2072 and expresses a portion of the androgen receptor encoding the A, B, C and D regions.
  • the sixth chimera, YEpmAR ⁇ EcR-CDEF was made by inserting into the Dra HI site of YEpEcR ⁇ A/B a PCR product made from YEpmAR using the sense primer 5'-TCATCACACCACGTGATGGAGGTGCAGTTAGGGCTGGGA-3' containing from the 5' end a Dra HI site followed by the mAR gene from the first translational start ATG site; and the antisense primer 5'-TCATCACACGTGGTGGGTCTTCTGGGGTGGAAAGTAATA-3', that encodes a Dra HI site at the 3' end along with sequences of the A/B domain mAR gene.
  • the plasmid YEpV3 is disclosed in McDonnell et al., Md. Cell Biol, 9:3517, 1989; and the plasmid YEpRXRa is disclosed in Mak et al. , Gene, 145: 129, 1994.
  • Expression of Usp and RXR ⁇ is driven by the copper-responsive yeast metallothionein promoter (CUP1), and the selectable markers are leucine (LEU2) and tryptophane (TRPl), respectively.
  • CUP1 copper-responsive yeast metallothionein promoter
  • LEU2 leucine
  • TRPl tryptophane
  • Expression of the EcR's, the chimeras, and VDR are regulated by the yeast constitutive triosephosphate dehydrogenase (TDH3) and tryptophane (TRPl) serves as a selectable marker.
  • TDH3 yeast constitutive triosephosphate dehydrogenase
  • TRPl tryptophane
  • the Usp reporter plasmid YEp-UspRE 2 -LacZ was constructed by replacing in YEpEcRE 2 -LacZ the EcR response element (EcREJ with two copies of a putative Usp response element (UspRE 2 ).
  • the UspRE 2 corresponded to the sequence from -64 to -44 of the chorion sl5 promoter (Shea et al. , Genes Dev. 4:1128, 1990;Khoury Christianson et al., Proc.Natl.Acad.Sci.
  • the EcR response reporter plasmid (Koelle et al., Cell 67:59, 1990) YE ⁇ EcRE 2 -LacZ was made as described (Mak et al., /. Biol. Chem. , 264:21613, 1989) and contained two copies of the EcRE in the Drosophila heat shock promoter 27 (hsp27), which were inserted into the Xho I site of pC2 and upstream of the yeast iso-1-cytochrome c promoter (CYCl) fused to the structural gene of E. coli LacZ (EcRE 2 -LacZ) , and has URA3 as the selectable marker (Mak et al., J. Biol. Chem.
  • the EcRE2 was constructed by synthesizing two oligonucleotides: 5'- T C G A G G A C A A G T G C A T T G A A C C T G T C T C C C G G G C - 3 ' a n d 3'-CTG ⁇ CACGTAACTTGGGAACAGAGGGCCCGAGCT-5 ⁇ which contained the 23 bases of hsp27 followed by a Sma I site and ending at the 5' ends four nucleotides of compatible Xho I overhangs.
  • the oligonucleotides were kinased, annealed together, and ligated into the reporter vector pC2 which had been previously digested with Xho I and dephosphorylated with calf intestinal alkaline phosphate.
  • Both UspRE 2 and EcRE 2 were subjected to digestion with Sma I to show that they were inserted into the Xho I site, and to DNA sequencing analysis (Sequenase kit, Stratagene) to verify the presence of two copies of the element.
  • the YEp- VDRE 2 reporter plasmid was made by excising an Xho I fragment from YEpEcRE 2 -LacZ and reinserting two copies of a 25 base pair sequence present in the human osteocalcin promoter (hOC) that was made from the oligonucleotide pair 5 TCGAGCTTACCGGGTGAACGGGGGCATTAC 3' 3 CGAAGGCCCACTTGCCCCCGTAATGAGCT 5' .
  • hOC human osteocalcin promoter
  • the mAR response reporter plasmid YRpA 2 is disclosed in Mak et al. , Recent Prog. Horm. Res. , 49:347, 1994.
  • the canavanine (CANl) response reporter YEp£cR£ 2 -C4Ni was constructed from YEpEcRE 2 -LacZ by excising the BamH I and Sac I portion containing the LacZ and reinserting a PCR product of CANl.
  • the PCR reaction contained as template genomic DNA from the wild type yeast strain S288C and primers made according to the sequence of CANl (Hoffmann, J.Biol.Chem. , 260:11831, 1985).
  • the sense primer 5'-GTGCTCGGATCCATGACAAATTCAAAAGAAGACG-3' encodes a BamH I site followed by the 5' and of CANl
  • the antisense primer 5'-TGGTGGGAGCTCCTATGCTACAACATTCC-3' encodes the 3' and of the CANl gene followed by a Sac I site.
  • Plasmid YEpUsp-LacZ was used in S. cerevisiae strain BJ2168, which has the genotype MAT ⁇ leu2 trpl ura3-52 prbl-1122 pep4-3 prcl-407 gal2. Plasmids YEpEcRE 2 -CANl , YEpEcRE 2 -LacZ and YRpA 2 were used with the canl deleted S. cerevisiae yeast strain CGY44(DC45), which has the genotype MATor stell-Al his4-519 leu2 trpl ura3 canl -101.
  • yeast were pretreated with 10 ⁇ M muristerone A or 1 ⁇ M testosterone.
  • cytosolic extracts cells were harvested by centrifugation, washed twice in water, and resuspended in Z buffer (60 mM Na 2 HP0 4 , 40 mM NaH 2 P0 4 , 10 mM KC1, 1 mM MgS0 4 , 50 mM /?-mercaptoethanol, pH 7.0). The cells were then lysed with 0.5 mm glass beads (Braun Instruments) by 5 cycles of vortexing (1 min each) followed by 1 min incubation on ice.
  • the lysates were subjected to high-speed centrifugation to separate the paniculate and supernatant fractions.
  • the protein concentration of the supernatant (cytosokc extract) as determined by the Bradford assay (BioRad) was typically 5-10 mg/ml.
  • jS-galactosidase activity was measured in a 1-ml reaction containing 800 ⁇ l
  • nucleotides -64 and -44 of Drosophila follicular-specific chorion gene sl5 can function as a cis-binding element for Usp
  • a duplicate copy of this sequence was inserted into a reporter plasmid that contains proximal promoter elements and the LacZ gene, and the resulting reporter plasmid YEpUspRE 2 -LacZ was tested for jS-galactosidase expression.
  • Table 1 shows that all cytosolic extracts from BJ2168 yeast containing the reporter plasmid YEp-UspRE 2 -LacZ had significant and similar levels of ⁇ -galactosidase activity, as compared to those with the control plasmid YEpcL ⁇ cZ, which had undetectable levels (range of 1500 MU/mg compared with O MU/mg, respectively).
  • the presence of Cu 2+ which induces expression of Usp from the copper-responsive metallothionein promoter (CUP1) copper promoter in YEpc Usp, had no effect.
  • CUP1 copper-responsive metallothionein promoter
  • EcR-A or EcR-B2 The transcriptional activity of EcR-A or EcR-B2 was then tested using the repo ⁇ er plasmid YEpEcRE : -LacZ.
  • the EcR isoforms differ in their N-terminal sequence; therefore, by domain swapping, the YEpEcR-Bl plasmid was modified to give zYEpEcR-A plasmid and a YEpEcR-B2 plasmid.
  • the yeast strains were transformed with the appropriate combination of plasmids and tested for /J-galactosidase activity.
  • Table 2 shows that cytosolic extracts containing YEpEcRE 2 -LacZ in the absence or presence of Usp had either undetectable or insignificant levels of /3-galactos ⁇ dase activities. These levels ranged from 0 to 132 MU/mg, and their mean (58) was defined as basal activity and used to determine the fold induction of activity. Cytosolic extracts containing an EcR isoform in addition to YEp-EcREyLacZ exhibited significant levels of /3-galactosidase activity, and the level was further increased for EcR-Bl and EcR- A containing cells by co-expression of Usp.
  • the three isoforms share the carboxyl terminal 36 ammo acids of their A/B domain and have different sequences at their N-terminal. being composed of 226 197 and 17 amino acids for EcR-Bl , EcR-A and EcR-B2. respectively
  • the results also show a decrease in both the level of the EcRs' constitutive activity, 4103 ⁇ 709 Mu/mg, 2523 ⁇ 710 MU/mg, and 480 ⁇ 150 MU/mg, respectively, and their enhancement by Usp, 4 4 ⁇ 0 3. 3 0 ⁇ 1 5.
  • EcR-Bl and mAR which include 1) an EcR truncated of its N- terminal or A/B domain (EcR ⁇ A/B), 2) an EcR containing its own A/B, C and part of D domains with the remaining portion of domain D and domain E derived from the mAR (EcR ⁇ mAR-DE), 3) an EcR in which domains A/B and C come from mAR and D,E and F are from the EcR (mAR ⁇ ecr-DEF), 4) and EcR similar to mAR ⁇ EcR-DEF except in which domain D is also from the mAR (mAR ⁇ EcR-EF), 5) an EcR similar to mAR ⁇ EcR- EF but lacing domain F (mAR ⁇ EcR-E) 6) and EcR similar to mAR ⁇ EcR-DEF but in which domain D from the mAR was retained (mAR-D ⁇ EcR-DEF), and 7) an EcR in which the
  • domain C specifies the cis-DNA binding domain, and reporter genes were selected based on the origin of the domain C present in the chimeric receptor. That is, chimeras with domain C from EcR were tested with YEp-EcRE 2 -LacZ, while chimeras with domain C from mAR were tested with YRpA 2 -L ⁇ cZ.
  • mAR ⁇ EcR-DEF and mAR ⁇ EcR-EF and mAR-D ⁇ EcR- DEF have similar low constitutive activities of about 300 Mu/mg.
  • Co-expression of Usp with the above chimeras decreases the activity of the first chimera and induces the other three to about 2000 MU/mg.
  • the inability to induce mAR ⁇ EcR- DEF but the ability to induce mAR-D ⁇ EcR-DEF suggests that the D region from the mAR is an important region for the function of the DNA binding domain. This speculation is consistent with observations made previously with a truncated mAR.
  • Canavanine/arginine permease is a membrane transporter for arginine and is the only means of entry of the toxic arginine analog, canavanine (Hoffmann, J .Biol. Chem. , 260: 11831, 1985).
  • Yeast containing CANl fail to grow on appropriate concentrations of canavanine, while yeast deleted for canl are resistant and viable; the sensitivity is semidominant since CANlcanl cells are sensitive (Broach et al., Gene 8:121, 1979). This property has been exploited as a selectable marker for the isolation of mutant strains and cloned genes.
  • the following experiment describes the use of CANl as an inducible reporter gene.
  • FIG. 2 is a schematic representation of the transcriptionally regulated CANl system for monitoring rescue of cell growth and the reduction to practice of this system.
  • the schematic shows that canl yeast cells transfected with YEpcUsp, YEpmAR ⁇ EcR-CDEF (designated "YEpCH8") and YEpEcRE-CANl accumulate the canavanine permease on their plasma membrane and canavanine intracellularly, leading to cell toxicity (Panel A). However, in the presence of an inhibitor (I) to Usp transcription, CANl expression is inhibited (X) and cells growth occurs (Panel B).
  • I MATERIALS AND METHODS A.
  • Cupric Sulfate (CuS0 4 ) (Sigma cat. #C- 1297) 100 mM was prepared by dissolving 1.6 g per 100 ml dH 2 0, filter sterilizing, and storing at RT protected from bight.
  • Canavanine Stock (Can) (Sigma cat.#C-9758) was prepared by dissolving 100 mg per ml, filter sterilizing and storing at 4°C.
  • Arginine Stock (Arg) was prepared by dissolving 100 mg per ml, filter sterilizing and storing at 4°C.
  • Liquid Medium contained the following components: 12 g dextrose 60 ml 10X YNB 110 ml Gold Concentrate
  • Agar Medium contained the following components:
  • the yeast strain CGY44: YEpmAR ⁇ EcR-CDEFIYEpcUSP/YEpEcRE 2 -CANl was grown overnight from an individual clone in 5 ml liquid Gold media with shaking at 30°C. A 200 ⁇ l aliquot of the overnight culture was transferred to 50 ml liquid Gold medium and shaken at 30 C C overnight. The OD 600 of the culture was measured (approximately 1.0). A 6 ml aliquot of cells at an OD 600 of 1 was removed and mixed with 150 ml Gold Mix Medium Agar that had been precooled to 50°C. The mixture was poured into a large Sumilon screening dish. After the agar solidified, the test samples were applied onto the plate.
  • l A inch filter disks containing 50 and 100 ⁇ g arginine were used as a positive control. The plate was incubated in a 30°C incubator overnight and the plates were analyzed the next day. The arginine disks showed a zone of growth visible after 16 h of incubation (and more robust growth after 24 hours).
  • the assay was performed independently five times within a three week period. Each experiment used newly grown yeast cells and newly prepared media. Filter disks containing samples listed in Table 4 were assayed on each plate. Only the disks containing arginine produced a zone of growth, with the 100 ⁇ g arginine giving the largest zone and 10 ⁇ g giving the smallest zone.
  • Yeast cells containing mAR ⁇ EcR-CDEF, YEpEcRE 2 -CANl and Usp respond to the presence of canavanine (spotted on a filter disk) by producing larger zones of growth inhibition, as compared to comparable cells not containing Usp which have no detectable inhibitor zones (Panel A Usp column vs. Vector column).
  • canavanine spotted on a filter disk
  • YEpecRE 2 -CANl and Usp respond to the presence of canavanine (spotted on a filter disk) by producing larger zones of growth inhibition, as compared to comparable cells not containing Usp which have no detectable inhibitor zones (Panel A Usp column vs. Vector column).
  • Non-specific leads have included the antifungal compound nystatin, compounds that block the arginine permease, and tryptophane, which caused the loss of the YEpmAR ⁇ EcR-CDEF plasmid which has TRPl as the selectable marker. This observation may indicate that the co-expression of the EcR and Usp is essential for cell death.
  • strains containing either YEpEcR-Bl, YEpEcR-A, YEpEcR-B2, or the YEpEcR ⁇ mAR-DE, and YEpEcRE 2 -CANl , with or without YEpcUsp can be used.
  • Their sensitivity to canavanine is shown in Table 7.
  • the EcR isoforms all produce a zone of no growth surrounding a filter disk containing 100 ⁇ g of canavanine (34, 17, and 17 mm, respectively) as compared to their absence (12 mm).
  • Yeast containing YEpEcRE 2 -CANl in the absence or presence of either Usp or RXR ⁇ do not show sensitivity to canavanine, i.e. exhibited no significant zone surrounding a filter disk spotted with 100 ⁇ g canavanine (15, 13, and 12 mm, respectively).
  • Yeast containing YEpEcRE 2 -CANl and EcR ⁇ A/B show a zone of no growth (22 mm) which is not affected by the additional presence of Usp (20 mm) but which is enlarged by the presence of RXR ⁇ (29 mm).
  • yeast containing YEpEcRE 2 -CANl and mAR/EcR-CDEF show no sensitivity to canavanine and the additional presence of RXR ⁇ has no effect (15 mm zone), but the additional presence of Usp produces a larger zone (26 mm).
  • a set of natural products (6,500 samples) with known and diverse activities was assayed by transferring 10 to 20 ⁇ g of each compound onto the agar test plate using a 96-well replica plate.
  • Three compounds produced a zone of killing surrounded by a zone of growth, and have been identified as potential lead compounds.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne l'identification d'inhibiteurs de récepteurs nucléaires 'orphelins', ou de récepteurs pour lesquels on ne connaît aucun ligand naturel. L'invention concerne la protéine Ultraspiracle, ou Usp, de Drosophila mélanogaster et ses homologues dans d'autres espèces d'insectes. L'invention permet d'identifier des composés, des protéines nucléaires variantes, et d'autres protéines auxiliaires qui interfèrent avec la fonction Usp. Des composés inhibiteurs de la fonction Usp sont utiles comme insecticides ou comme composés principaux pour le développement d'insecticides.
EP97928958A 1996-05-31 1997-05-30 Identification d'inhibiteurs de l'ultraspiracle Withdrawn EP0912896A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US1881796P 1996-05-31 1996-05-31
US18817P 1996-05-31
PCT/US1997/010212 WO1997045737A1 (fr) 1996-05-31 1997-05-30 Identification d'inhibiteurs de l'ultraspiracle

Publications (2)

Publication Number Publication Date
EP0912896A1 true EP0912896A1 (fr) 1999-05-06
EP0912896A4 EP0912896A4 (fr) 2004-08-18

Family

ID=21789922

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97928958A Withdrawn EP0912896A4 (fr) 1996-05-31 1997-05-30 Identification d'inhibiteurs de l'ultraspiracle

Country Status (4)

Country Link
EP (1) EP0912896A4 (fr)
JP (1) JP2000516085A (fr)
CA (1) CA2257110A1 (fr)
WO (1) WO1997045737A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2337329B (en) * 1997-04-30 2001-09-05 Imp Cancer Res Tech Inhibitors of nuclear protein/nuclear receptor interaction
GB9708676D0 (en) * 1997-04-30 1997-06-18 Imp Cancer Res Tech Chemical compounds
AUPP135698A0 (en) 1998-01-15 1998-02-05 Commonwealth Scientific And Industrial Research Organisation Insectical modalities
WO2001061350A1 (fr) * 2000-02-18 2001-08-23 Lifesensors, Inc. Procedes et compositions d'identification de ligands de recepteurs nucleaires
US7037656B2 (en) 2000-02-17 2006-05-02 Lifesensors, Inc. Methods and compositions for identifying ligands for nuclear receptors
GB0403966D0 (en) * 2004-02-23 2004-03-24 Syngenta Ltd Methods

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7492291A (en) * 1990-02-26 1991-09-18 Board Of Trustees Of The Leland Stanford Junior University Identification and expression of insect steroid receptor dna sequences
US5688691A (en) * 1990-03-22 1997-11-18 The Salk Institute For Biological Studies Insect retinoid-like receptor compositions and methods
AU1173995A (en) * 1993-11-10 1995-05-29 Arch Development Corporation Ubiquitous nuclear receptor: compositions and methods

Also Published As

Publication number Publication date
JP2000516085A (ja) 2000-12-05
WO1997045737A1 (fr) 1997-12-04
EP0912896A4 (fr) 2004-08-18
CA2257110A1 (fr) 1997-12-04

Similar Documents

Publication Publication Date Title
Leydon et al. Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex
DE69731960T2 (de) Verfahren und zusammensetzungen für die identifizierung von receptor effectoren
Krüger et al. Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis
DE69831083T2 (de) Reprimiertes trans-aktivatorsystem zur charakterisierung von protein-protein interaktionen
AU686685B2 (en) Methods for screening for antimycotics
US7319009B2 (en) Methods and compositions for identifying receptor effectors
US5700682A (en) Mechanism based screen for retinoid X receptor agonists and antagonists
US20030235857A1 (en) Regulation of fungal gene expression
Schaerer-Brodbeck et al. Functional interactions between the p35 subunit of the Arp2/3 complex and calmodulin in yeast
Schröder et al. The unfolded protein response represses differentiation through the RPD3-SIN3 histone deacetylase
US6110698A (en) Screen for ultraspiracle inhibitors
EP0912896A1 (fr) Identification d'inhibiteurs de l'ultraspiracle
WO1997045737A9 (fr) Identification d'inhibiteurs de l'ultraspiracle
Del Sorbo et al. Cloning and functional characterization of BcatrA, a gene encoding an ABC transporter of the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea)
DE60216721T2 (de) Verfahren zum Nachweis von Protein-Proteininteraktionen in Membranen
US6887661B1 (en) Recombinant bHLH-PAS/JHR polypeptide and its use to screen potential insecticides
WO1997045731A1 (fr) Identification de ligands de recepteurs de ecdysones
WO1997045731A9 (fr) Identification de ligands de recepteurs de ecdysones
Chen et al. A highly efficient and sensitive screening method for trans-activation activity of estrogen receptors
JP4546827B2 (ja) 物質を同定する方法
DE102008064553B4 (de) Verfahren zum Nachweis und/oder zur Identifizierung hormonell wirksamer Substanzen
US20030007956A1 (en) Proteins that interact with betaTrCP
Kirsch et al. Molecular Genetic Screen Design for Agricultural and Pharmaceutical Product Discovery
CA2559867A1 (fr) Procede de criblage d'agents modulant l'ubiquitination de la proteine ikb.alpha. et moyens destines a la mise en oeuvre dudit procede
AU2003202416B2 (en) Regulation of fungal gene expression

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19981202

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: BASF AKTIENGESELLSCHAFT

A4 Supplementary search report drawn up and despatched

Effective date: 20040706

RIC1 Information provided on ipc code assigned before grant

Ipc: 7G 01N 33/68 B

Ipc: 7G 01N 33/569 B

Ipc: 7G 01N 33/53 A

17Q First examination report despatched

Effective date: 20050301

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20051112