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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • 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
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • 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
  • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
  • G01N33/9493—Immunosupressants
• • 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
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. They have routinely been expressed in E. coli as fusion proteins with glutathione-S- transferase (GST). This usually provides high level expression and straightforward affinity purification on glutathione-Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, precipitating the complex with glutathione-Sepharose, washing the beads, and then counting the beads to determine bound radioactivity [Isakov et al., J. Exp.
• the protocol requires separation of bound complex from free phosphopeptide by washing of the glutathione-Sepharose beads. This is a nonequilibrium procedure that risks dissociation of the bound ligand, particularly when off-rates are fast. Thus, there is the possibility of misleading results. Finally, due to the number of manipulations and centrifugations involved, the protocol is very tedious to conduct manually and is not readily adaptable to robotic automation to increase throughput.
• the instant invention covers a method of screening for compounds capable of binding to a fusion protein which comprises combining a test compound, a tagged ligand, a fusion protein (target protein, peptide linker and FK506-binding protein), a radiolabeled ligand, and coated scintillation proximity assay (SPA) beads, and then measuring the scintillation counts attributable to the binding of the tagged 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 tagged ligand.
• a fusion protein target protein, peptide linker and FK506-binding protein
• SPA scintillation proximity assay
• This invention provides an immediate means of making use of SPA technology for the functional assay of ligand binding to a single or multiple signal transduction domain(s), for example a phosphopeptide binding to an SH2 domain.
• the present invention does not require specialized radiochemical synthesis and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors. BRIEF DESCRIP ⁇ ON OF THE FIGURES Figure 1.
• T e present invention relates to a method of screening for compounds which preferentially bind to a target protein.
• 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 tagged ligand, the fusion protein, a radiolabeled ligand and coated scintillation proximity assay
• SPA beads (SPA) beads; b) incubating the mixture for between about 1 hour and about 24 hours; c) measuring the SPA bead-bound counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound using scintillation counting; and d) determining the binding of the tagged 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.
• fusion protein refers to a "target protein” fused to an "FK506-binding protein” (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 GLPRGS.
• target protein refers to any protein that has a defined ligand.
• target protein includes 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 (1995); Bolen, Curr. Opin. Immunol., 1, 306-31 1 (1995); Kuriyan & Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993); Cohen et al., Cell, 80, 237-248 (1995)].
• SHI Src homology 1
• SH2 Src homology 2
• SH3 Src homology 3
• PH pleckstrin homology
• 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. Examples of 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 ZAP, SYK and LCK. The DNA sequences were obtained from
• tagged ligand refers to a biotinylated or epitope tagged ligand for the target protein.
• radiolabeled ligand refers to a [ 3 H]- or [1 25 I]- labeled ligand which binds to the FKBP.
• An example of a radiolabeled ligand useful in the instant invention is [3H]-dihydroFK506.
• coated scintillation proximity assay beads (SPA beads) refers to streptavi din-coated scintillation proximity assay beads when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated scintillation proximity assay beads when the tagged ligand is epitope-tagged.
• control assay refers to the assay when performed in the presence of the tagged ligand, the fusion protein, the radiolabeled ligand and the coated scintillation proximity assay beads, 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.
• host cells 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 techinques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
• E. coli containing an expression plasmid with the target gene 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 are primarily located there and can be released by a standard freeze/thaw treatment of the cell pellet. Following centrifugation, the resulting supematant 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 thrombin site located between FKBP and the target protein can be used as a means to cleave FKBP from the fusion; such cleaved material may be a suitable negative control for subsequent assays.
• 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 tagged ligand is mixed with the FKBP fusion protein in a suitable buffer in the presence of the radiolabeled ligand in the well of a white micropiate. After a suitable incubation period to allow complex formation to occur, coated SPA beads are added to capture the tagged ligand and any bound fusion protein. The plate is sealed, incubated for a sufficient period to allow the capture to go to completion, then counted in a multiwell scintillation counter.
• Screening for agonists/antagonists/inhibitors is carried out by performing the initial incubation prior to the capture step with SPA beads in the presence of a test compound(s) to determine whether they have an effect upon the binding of the tagged ligand to the fusion protein. This principle is illustrated by Figure 1.
• the PCR reaction contained the following primers:5'- GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' and 5'- TACGAATTCTGGCGTGGATCCACGCGGAACCAGACCTTCCAGT TTTAG-3' 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 BamHI and ligated into Ncol andit ⁇ mHI 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.
• ZAP-70 was prepared by PCR to contain a BamHI 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 inco ⁇ orated a stop codon followed by a BamHI 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 and 5 -GACCTAGGATCCCTAATTAACATTTCCCTGTGTGCCGAT-3 * .
• the expression vector for the SH2 domain of Lck fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers: 5 -ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3' and 5 -ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTGT
• Step A Process for Expression of FK-ZAP
• E. coli BL21(DE3) cells containing the pET9dFKBPt/ ZapSH2 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-ZAP 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-ZAP
• the affinity matrix for purification of FK-ZAP was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ⁇ l ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, and washing out unbound peptide. Frozen cells containing FK-ZAP were thawed in warm water, refrozen on dry ice for about 25 min., then thawed again.
• E. coli BL21(DE3) cells containing the pET9dFKBPt/ 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-
• Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01 % TWEEN-20, pH 7.0. 10 ⁇ l of a DMSO solution of test compound(s) and 120 ⁇ l of biotinyl- phosphopeptide stock solution were dispensed into the wells of a 96-well Packard Optiplate. Next, 20 ⁇ l of a mixture of FK-ZAP protein and 3H-dihydroFK506 were added to each test well. Finally, 50 ⁇ l of a 4 mg/ml suspension of SPA beads were dispensed to each well.
• the plate was sealed and incubated between 1 and 8 hours. Bead-bound radioactivity was then measured in a Packard Topcount micropiate scintillation counter.
• the assays were conducted as set forth in Example 8, except that FK-LCK replaced FK-ZAP and the tagged ligand was 25 nM biotiny 1 -EPQp YEEIPI YL.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• GAGCCCGAAC CCTGGTTCTT CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420
• ATCCGTAATC TGGACAACGG TGGCTTCTAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600

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• 1996
  • 1996-09-11 JP JP9512067A patent/JP2000501171A/ja active Pending
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