WO1998041866A1 - Dosage a haute capacite dans lequel des proteines de fusion sont utilisees - Google Patents

Dosage a haute capacite dans lequel des proteines de fusion sont utilisees Download PDF

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Publication number
WO1998041866A1
WO1998041866A1 PCT/US1998/004610 US9804610W WO9841866A1 WO 1998041866 A1 WO1998041866 A1 WO 1998041866A1 US 9804610 W US9804610 W US 9804610W WO 9841866 A1 WO9841866 A1 WO 9841866A1
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Prior art keywords
screening
recited
compounds capable
binding
fusion protein
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Jeffrey D. Hermes
Scott P. Salowe
Peter J. Sinclair
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Merck and Co Inc
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Merck and Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • HTRF Homogeneous Time-Resolved Fluorescence
  • Eu(K) has a long-lived fluorescent signal which facilitates the homogeneous nature of the assay. A time delay in reading the signal eliminates the principal difficulty in applying fluorescence to screening formats. This technology is useful in both binding and protease assays.
  • the instant invention relates to a method of screening that can be used to determine if compounds are capable of binding to a protein or are capable of blocking ligand-protein or protein-protein interactions.
  • the instant invention covers a method of screening for compounds capable of binding to a fusion protein which comprises combining a test compound, a biotinylated ligand, a fusion protein (target protein, peptide linker and FK506-binding protein), a donor- labeled ligand, and acceptor-labeled streptavidin, and then measuring the fluorescence attributable to the binding of the biotinylated ligand to the fusion protein in the presence of the test compound relative to a control assay in the absence of the test compound, so as to determine the effect the test compound has on the binding of the biotinylated ligand.
  • the instant invention also covers a method of screening for compounds which inhibit a protease which comprises combining a test compound, a fusion protein substrate (reporter protein, peptide linker and FK506- binding protein), a protease, a donor-labeled ligand, and acceptor- labeled streptavidin, and then measuring the fluorescence attributable to the binding of the intact fusion protein substrate in the presence of the test compound relative to a control assay in the absence of the test compound, so as to determine the effect the test compound has on proteolytic activity.
  • a fusion protein substrate reporter protein, peptide linker and FK506- binding protein
  • protease a donor-labeled ligand
  • acceptor- labeled streptavidin acceptor- labeled streptavidin
  • This invention provides an immediate means of making use of HTRF technology for the functional assay of either ligand binding to a single or multiple signal transduction domain(s), tyrosine phosphatases, nuclear receptors or bacterial tRNA synthetases in a signal increase assay or proteolytic activity in a signal decrease assay.
  • the present invention is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors.
  • FIGURE 1 Binding of the donor-labeled ligand (Eu(K) labeled ligand for FKBP), fusion protein (FKBP:SH2), biotinylated ligand and the acceptor-labeled streptavidin (SA-XL665), which involves a fluorescent energy transfer that can be measured.
  • Eu(K) labeled ligand for FKBP fusion protein
  • FKBP:SH2 fusion protein
  • SA-XL665 acceptor-labeled streptavidin
  • FIGURE 2 Cleaving of donor-labeled ligand (Eu(K) labeled ligand for FKBP), fusion protein substrate (FKBP:SH2:ITAM:B) and the acceptor-labeled streptavidin (SA- XL665).
  • FIGURE 3 Cleaving of donor-labeled ligand (Eu(K) labeled ligand for FKBP), fusion protein substrate (FKBP:acetyl- CoA:B) and the acceptor-labeled streptavidin (SA-XL665).
  • an embodiment of this invention is a method of screening for compounds capable of binding to a fusion protein which comprises the steps of: a) mixing a test compound, a biotinylated ligand, the fusion protein, a donor-labeled ligand and acceptor-labeled streptavidin; b) incubating the mixture for an appropriate time; c) measuring the time-resolved fluorescence attributable to the binding of the biotinylated ligand to the fusion protein in the presence of the test compound; and d) determining the binding of the biotinylated ligand to the fusion protein in the presence of the test compound relative to a control assay run in the absence of the test compound.
  • another embodiment of this invention is a method of screening for compounds capable of inhibiting a protease which comprises the steps of: a) mixing a test compound, a fusion protein substrate, a donor-labeled ligand, acceptor-labeled streptavidin, and a protease; b) incubating the mixture for an appropriate time; c) measuring the time-resolved fluorescence attributable to the binding of the intact fusion protein substrate to the acceptor-labeled streptavidin in the presence of the test compound; and d) determining the binding of the intact fusion protein substrate to the acceptor-labeled streptavidin in the presence of the test compound relative to a control assay run in the absence of the test compound.
  • FKBP FK506 Binding Protein
  • fusion protein refers to a "target protein” fused to an "FK506-binding protein” (FKBP), the two proteins being separated by a "peptide linker".
  • FKBP FK506-binding protein
  • fusion protein substrate refers to a "reporter protein” fused to an FKBP, the two proteins being separated by a peptide linker.
  • a "peptide linker” may consist of a sequence containing from about 1 to about 20 amino acids, which may or may not include the sequence for a protease cleavage site.
  • An example of a peptide linker which is a protease cleavage site is represented by the amino acid sequence GLVPRGS. (SEQ. ID. NO. 1).
  • proteases refers to an enzyme that catalyzes the hydrolytic breakdown of proteins and/or peptides.
  • proteases may include, but are not limited to, thrombin, Human Immunodeficiency Virus (HIV) protease and Tumor Necrosis Factor (TNF) converting enzyme.
  • TNF Tumor Necrosis Factor
  • target protein refers to any protein that has a defined ligand.
  • Types of target proteins include, but are not limited to, tyrosine phosphatases (FKBP-phosphatase chimera as receptor and biotinylated- peptide containing a phosphonomethylene isostere as ligand), nuclear receptors (FKBP-receptor and biotinylated DNA) and bacterial tRNA synthetases (FKBP-tRNA synthetase and biotinylated tRNA).
  • FKBP-phosphatase chimera as receptor and biotinylated- peptide containing a phosphonomethylene isostere as ligand
  • nuclear receptors FKBP-receptor and biotinylated DNA
  • FKBP-tRNA synthetase and biotinylated tRNA bacterial tRNA synthetases
  • target protein also included within this definition of target protein are single and multiple signal transduction domains, such as, but not limited to, Src homology 1 (SHI), Src homology 2 (SH2), Src homology 3 (SH3), and pleckstrin homology (PH) domains [Hanks & Hunter, FASEB J., 9, 576-596
  • SHI domain refers to a family of homologous protein domains that bind ATP and catalyze tyrosine phosphorylation of peptide and protein substrates.
  • SH2 domain refers to a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts.
  • SH3 domain refers to a family of homologous protein domains that share the common property of recognizing polyproline type II helices.
  • PH domain refers to a family of homologous protein domains that mediate both protein- protein and protein-lipid interactions.
  • SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 domains present in the tyrosine kinases ZAP70, Syk and Lck. The DNA sequences were obtained from GenBank, National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894.
  • the Accession Numbers for the sequences are: human ZAP70 (L05148) human Syk (L28824) and human Lck (XI 3529).
  • the sequences for ZAP70, Syk and Lck are disclosed in the sequence listing as follows: the isolated DNA encoding for a fusion protein containing ZAP70 is (SEQ. ID. NO. 2); the isolated DNA encoding for a fusion protein containing Syk is (SEQ. ID. NO. 3); the isolated DNA encoding for a fusion protein containing Lck is (SEQ. ID. NO. 4); the sequence for the FKBP-ZAP70:SH2 fusion protein is (SEQ. ID. NO. 5); the sequence for the FKBP-Syk:SH2 fusion protein is (SEQ. ID. NO. 6); and the sequence for the FKBP-Lck:SH2 fusion protein is (SEQ. ID. NO. 7).
  • reporter protein refers to a protein containing covalent biotin or a noncovalently bound biotinylated ligand.
  • proteins containing covalent biotin are the C-terminal 87 residues of the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli and the biotin-carrier subunit of transcarboxylase from Propionibacterium shermannii.
  • a protein containing a noncovalently bound biotinylated ligand is the tandem SH2 domains of ZAP70 bound to a biotinylated phosphopeptide derived from an Immunoreceptor Tyrosine-based Activation Motif (IT AM) sequence of the human T-cell receptor, the B-cell receptor or a high affinity Immunoglobulin E. (IgE) receptor.
  • IT AM Immunoreceptor Tyrosine-based Activation Motif
  • IgE Immunoglobulin E.
  • Zeta ( ⁇ ) 1 sequence of the human T-cell receptor may be utilized.
  • SEQ. ID. NO. 8 California Peptide Research Inc., 918 Enterprise Way, Suite 1, Napa, CA 94558
  • donor-labeled ligand refers to an organic cryptate-containing molecule loaded with a fluorescent lanthanide metal which binds to the FKBP.
  • fluorescent lanthanide metal is europium.
  • the europium-cryptate molecule was obtained from CIS Bio International, Subsidiary of Compagnie ORIS INDUSTRIE SA, Boite Postale 175, F30203 Bagnols sur Ceze Cedex, France, is depicted below.
  • acceptor-labeled streptavidin refers to streptavidin coupled to one or more light harvesting molecules.
  • An example of a light harvesting molecule is allophycocyanin and an example of a acceptor-labeled streptavidin useful in the instant invention is SA-XL665. (CIS Bio International, Subsidiary of Compagnie ORIS INDUSTRIE SA, Boite Postale 175, F30203 Bagnols sur Ceze Cedex, France)
  • control assay refers to the assay when performed in the presence of the donor-labeled ligand, acceptor-labeled streptavidin, and either fusion protein substrate or fusion protein plus biotinylated ligand, but in the absence of the test compound.
  • FK506-binding proteins may include, but are not limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention.
  • a variety of host cells used to produce the FKBP chimeras may be used in this invention, which include, but are not limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes in a variety of host cells, such as, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • vectors suitable for FKBP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pT7 (USB), pET (Novagen), pIBI (IBI), pProEX-1 (Gibco/BRL), pBluescript II (Stratagene), pTZ18R and pTZ19R (USB), pSE420 (Invitrogen), pVL1392 (Invitrogen), pBlueBac (Invitrogen), pBAcPAK (Clontech), pHIL (Invitrogen), pYES2 (Invitrogen), pCDNA (Invitrogen), pREP (Invitrogen) or the like.
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
  • E. coli containing an expression plasmid with the gene for the target or reporter protein fused to FKBP are grown and appropriately induced. The cells are then pelleted and resuspended in a suitable buffer.
  • FKBP- 12 lacks sequences that specifically direct it to the periplasm, FKBP fusions may be located there and can be released by a standard freeze/thaw treatment of the cell pellet.
  • the resulting supernatant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods.
  • the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly.
  • a fusion protein which contains a single or multiple SH2 domain(s) may be purified by preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support.
  • a freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the affinity matrix. The desired fusion protein is then specifically eluted with phenyl phosphate.
  • the biotinylated ligand is mixed with the FKBP fusion protein in a suitable buffer in the presence of the donor-labeled ligand in the well of a black microplate. After a suitable incubation period to allow complex formation to occur, acceptor-labeled streptavidin is added to capture the tagged ligand and any bound fusion protein. The plate is incubated for a sufficient period to allow the capture to go to completion and then the time resolved fluorescent signal is measured.
  • Screening for agonists/antagonists/inhibitors is carried out by performing the initial incubation prior to the acceptor-labeled streptavidin capture step in the presence of a test compound(s) to determine whether they have an effect upon the binding of the biotinylated ligand to the fusion protein. This principle is illustrated by Figure 1.
  • a FKBP fusion protein substrate is combined with the protease in a suitable buffer in the presence of the donor-labeled ligand and acceptor-labeled streptavidin in the well of a black microplate. After a suitable incubation period, the time resolved fluorescent signal of the remaining intact fusion protein substrate is measured. Screening for inhibitors is carried out by performing the incubation in the presence of a test compound(s) to determine whether they have an effect upon the cleavage of the fusion protein substrate. This principle is illustrated by Figures 2 and 3.
  • Scheme 1 depicts the treatment of Compound A, an analog of FK506, with 1 equivalent of p-nitrophenylchloroformate in THF in the presence of a tertiary amine, which gives the p- nitrophenylcarbamate, Compound B.
  • the mtrophenylcarbamate is isolated by silica gel chromatography or used without further purification.
  • the mtrophenylcarbamate is an activated acylating group and will undergo attack by a variety of nucleophiles.
  • the cryptate has two primary amino groups (nucleophiles).
  • the cryptate can be linked to Compound A, other FK506 analogs, and related molecules by a variety of linkages, such as carbamates, amides, ureas, amines, etc.
  • linkages such as carbamates, amides, ureas, amines, etc.
  • One of ordinary skill in the art would be familiar with the standard procedures used to prepare the FK506 analog containing the cryptate molecule.
  • the PCR reaction contained the following primers: 5'-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' (SEQ. ID. NO. 9) and 5'-TACGAATTCTGGCGTGGATCCAC GCGGAACCAGACCTTCCAGTTTTAG-3 * (SEQ. ID. NO. 10) and a plasmid containing human FKBP- 12 as the template.
  • the resulting 367 base pair amplification product was ligated into the vector pCRII (Invitrogen) and the ligation mixture transformed into competent Escherichia coli cells. Clones containing an insert were identified using PCR with flanking vector primers. Dideoxy DNA sequencing confirmed the nucleotide sequence of one positive isolate.
  • the altered 338 base pair FKBP fragment was excised from the pCRII plasmid using Ncol and BamHl and ligated into Ncol an ⁇ BamHl digested pET9d (Novagen) plasmid. Competent E. coli were transformed with the ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for flanking vector sequences.
  • the FKBP fusion cloning vector is called pET9dFKBPt.
  • a DNA fragment encoding for the tandem SH2 domains of ZAP70 was prepared by PCR to contain a BamHl site at the 5 '-end such that the reading frame was conserved with that of FKBP in the fusion vector. At the 3 '-end, the fragment also incorporated a stop codon followed by a BamHl site.
  • the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
  • the expression vector for the tandem SH2 domains of Syk fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Raji cell cDNA (Clontech) and the following primers: 5'-CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3'
  • FKBP FKBP was prepared as in Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
  • GCA-3' (SEQ. ID. NO. 16).
  • Step A Process for Expression of FK-ZAP70
  • E. coli BL21(DE3) cells containing the pET9dFKBPt/ ZAP70SH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml kanamycin at 37 degrees C until the optical density measured at 600 nm was 0.5-1.0.
  • Expression of the FK-ZAP70 fusion protein was induced with 0.1 mM isopropyl beta- thiogalactopyranoside and the cells were grown for another 3-5 hr at 30 degrees C.
  • Step B Process for Purification of FK-ZAP70
  • the affinity matrix for purification of FK-ZAP70 was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ⁇ l IT AM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, (SEQ. ID. NO. 8) and washing out unbound peptide.
  • Frozen cells containing FK-ZAP70 were thawed in warm water, refrozen on dry ice for about 25 min., then thawed again.
  • SykSH2 plasmid were grown, induced, and harvested as described in Example 5.
  • FK-Syk was purified using the same affinity matrix and methodology described in Example 5.
  • E. coli BL21(DE3) cells containing the pET9dFKBPt/ LckSH2 plasmid were grown, induced, and harvested as described in Example 5.
  • the affinity matrix for purification of FK-Lck was prepared by combining agarose-immobilized avidin with excess biotinyl-EPQpYEEIPIYL, (SEQ. ID. NO. 17) and washing out unbound peptide. The remaining methodology for purification was the same as Example 5.
  • a DMSO solution of test compound(s) and biotinyl- phosphopeptide stock solution in a suitable buffer are dispensed into the wells of a 96-well black microplate.
  • a mixture of FK-ZAP protein and Eu(K)-labeled FK506 analog are added to each test well.
  • a solution of SA-XL665 is dispensed to each well and the plate is incubated for an appropriate time. The fluorescence ratio is then measured in a Packard Discovery HTRF analyzer. (Packard Instrument Company, 800 Research Parkway, Meridan, CT 06450)
  • a DMSO solution of test compound(s) and a suitable buffer containing a mixture of FK-ZAP protein, Eu(K)-labeled FK506 analog, and biotinyl-phosphopeptide are dispensed into the wells of a 96-well black microplate.
  • thrombin is added to each test well.
  • a solution of SA-XL665 is dispensed to each well. The fluorescence ratio is then measured in a Packard Discovery HTRF analyzer. (Packard Instrument Company, 800 Research Parkway, Meridan, CT 06450)
  • MOLECULE TYPE protein
  • GAGCCCGAAC CCTGGTTCTT CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420
  • ATCCGTAATC TGGACAACGG TGGCTTCTAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600
  • Lys Thr Val Tyr His Tyr Leu lie Ser Gin Asp Lys Ala Gly Lys Tyr
  • Cys lie Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp Gin Leu Val Glu
  • Lys Thr Gly Lys Leu Ser lie Pro Glu Gly Lys Lys Phe Asp Thr Leu
  • Val Leu Thr Val Pro Cys Gin Lys lie Gly Thr Gin Gly Asn Val Asn 370 375 380
  • Lys His Tyr Lys lie Arg Asn Leu Asp Asn Gly Gly Phe Tyr lie Ser
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 13: CAATAGGATC CATGGCCAGC AGCGGCATGG CTGA 34
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA
  • MOLECULE TYPE Genomic DNA

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Abstract

L'invention concerne un dosage à haute capacité pour le criblage de composés capables de se lier à une protéine de fusion qui consiste en une protéine cible et en une protéine fixant FK506. Elle porte aussi sur un dosage pour le criblage de composés inhibant une protéase.
PCT/US1998/004610 1997-03-14 1998-03-10 Dosage a haute capacite dans lequel des proteines de fusion sont utilisees Ceased WO1998041866A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023600A1 (fr) * 1998-10-19 2000-04-27 Ariad Gene Therapeutics, Inc. Materiaux et methodes impliquant des domaines d'agregation conditionnelle
WO2000037448A1 (fr) * 1998-12-21 2000-06-29 Novartis Ag Colorants fluorescents destines au criblage de phase solide et de phase solution
WO2000050635A1 (fr) * 1999-02-25 2000-08-31 Cyclacel Limited Procedes et compositions dans lesquels on utilise des partenaires de liaison
US6117639A (en) * 1998-08-31 2000-09-12 Vertex Pharmaceuticals Incorporated Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
WO2004003509A3 (fr) * 2002-06-28 2004-08-19 Genepharm Inc Procede d'identification de l'interaction specifique entre un ligand et un recepteur
EP1425581A4 (fr) * 2001-08-23 2005-03-09 Qtl Biosystems Llc Plates-formes de bio-detection et d'analyse quantitative de molecules biologiques
ES2249986A1 (es) * 2004-07-02 2006-04-01 Universidad Complutense De Madrid Sintesis de derivados fluorescentes de tacrolimus (fk506) y su uso en la caracterizacion de la interaccion de fk506 con proteinas de union a fk506.

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
US5498597A (en) * 1992-01-17 1996-03-12 Dana-Farber Cancer Institute, Inc. FKBP-13, an FK506-binding immunophilin
WO1997010502A1 (fr) * 1995-09-15 1997-03-20 Merck & Co., Inc. Dosage a rendement eleve utilisant des proteines de fusion

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
US5498597A (en) * 1992-01-17 1996-03-12 Dana-Farber Cancer Institute, Inc. FKBP-13, an FK506-binding immunophilin
WO1997010502A1 (fr) * 1995-09-15 1997-03-20 Merck & Co., Inc. Dosage a rendement eleve utilisant des proteines de fusion

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117639A (en) * 1998-08-31 2000-09-12 Vertex Pharmaceuticals Incorporated Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
US6528276B1 (en) * 1998-08-31 2003-03-04 Vertex Pharmaceuticals Inc. Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
WO2000023600A1 (fr) * 1998-10-19 2000-04-27 Ariad Gene Therapeutics, Inc. Materiaux et methodes impliquant des domaines d'agregation conditionnelle
WO2000037448A1 (fr) * 1998-12-21 2000-06-29 Novartis Ag Colorants fluorescents destines au criblage de phase solide et de phase solution
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