+

WO2004027018A2 - Dosage de cellule kinase a adhesion focale inductible - Google Patents

Dosage de cellule kinase a adhesion focale inductible Download PDF

Info

Publication number
WO2004027018A2
WO2004027018A2 PCT/IB2003/003968 IB0303968W WO2004027018A2 WO 2004027018 A2 WO2004027018 A2 WO 2004027018A2 IB 0303968 W IB0303968 W IB 0303968W WO 2004027018 A2 WO2004027018 A2 WO 2004027018A2
Authority
WO
WIPO (PCT)
Prior art keywords
fak
cells
cell
phosphorylation
expression
Prior art date
Application number
PCT/IB2003/003968
Other languages
English (en)
Other versions
WO2004027018A3 (fr
Inventor
Walter Gregory Roberts
Pamela Matthews Whalen
Ethan James Tekly Ung
Original Assignee
Pfizer Products Inc.
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 Pfizer Products Inc. filed Critical Pfizer Products Inc.
Priority to CA002497434A priority Critical patent/CA2497434A1/fr
Priority to EP03797456A priority patent/EP1546372A4/fr
Priority to JP2004537413A priority patent/JP2006500022A/ja
Priority to AU2003259498A priority patent/AU2003259498A1/en
Publication of WO2004027018A2 publication Critical patent/WO2004027018A2/fr
Publication of WO2004027018A3 publication Critical patent/WO2004027018A3/fr
Priority to NO20051869A priority patent/NO20051869L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • This invention relates to methods and compositions for inducing the expression of the focal adhesion kinase (FAK) gene, which encodes a signaling protein involved in growth factor response and cell migration and is also implicated in disease.
  • the invention is also directed to the identification of FAK inhibitors.
  • FAK focal adhesion kinase
  • FAK is a cytoplasmic, non-receptor tyrosine kinase.
  • FAK transduces signaling from a diverse group of stimuli (e.g. integrins, cytokines, chemokines, and growth factors) to control a variety of cellular pathways and processes including cell proliferation, migration, morphology, and cell survival.
  • stimuli e.g. integrins, cytokines, chemokines, and growth factors
  • FAK is found at elevated levels in most human cancers, particularly in highly invasive metastases. It has been shown that expression of the dominant-negative FAK-related nonkinase (FRNK) in human tumor cells results in rounded morphology of the cells, the irreversible loss of focal plaques, and subsequent cell death.
  • FRNK dominant-negative FAK-related nonkinase
  • the controlled expression of FRNK results in decreased tyrosine phosphorylation of FAK, suggesting that inhibition of FAK phosphorylation may yield a therapeutic
  • FAK enzyme activity resulting in phosphorylation at Y397 is the critical step in integrin signal transduction (Guan, JL, Int. J. Biochem.Cell.Biol. 29: 1085-96, 1997).
  • the transmembrane integrin receptors are important for linking the extracellular matrix (ECM) proteins with the cellular actin cytoskeleton and the nucleus to regulate cell morphology, tissue architecture, and attachment-induced gene expression.
  • An objective of the invention is to design and develop a FAK drug discovery pathway that tracks the biochemical mechanism of FAK.
  • a number of exogenous stimuli can lead to FAK phosphorylation such as (1 ) integrin binding to ECM ligands (e.g. Integrin ⁇ 1 to Fibronectin); (2) cytokine or chemokine stimulation (e.g. Endothelin1/2, Bombesin, or PMA); (3) growth factor stimulation of tyrosine kinase receptors (e.g.
  • PDGFBB PDGFBB
  • integrin antibody cross-linking e.g. Anti- ⁇ 1
  • cell-to-cell and cell-to-ECM contact e.g. cell suspension
  • the invention relates to a method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising:
  • An embodiment of the invention provides a method for measuring the cytotoxicity of a test compound comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; adding a cytotoxicity indicator to said cells; and, detecting the cytoxicity of the test compound.
  • the cytoxicity of the test compound is determined by the colorimetric conversion of the cytotoxicity indicator, wherein the amount of converted cytotoxicity indicator is proportional to the number of living cells.
  • An embodiment of the present invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, wherein said recombinant nucleic acid molecule is selected from the group consisting of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID
  • An embodiment of the invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule that encodes a protein comprising a sequence selected from the group consisting of SEQ ID NOS: 1 , 2, 3 and 4, and wherein expression of said protein requires the presence of an inducing agent.
  • Figure 1 shows a schematic representation of the detection of phosphorylated FAK using a horseradish peroxidase-conjugated phosphotyrosine antibody (pY54 HRP ).
  • Figure 2 shows a schematic representation of the detection of phosphorylated FAK using an unconjugated phosphotyrosine antibody (pY54) followed by a secondary mouse horseradish peroxidase antibody.
  • FIG. 3 shows a schematic representation of a FAK inducible cell-based assay of the invention.
  • the invention is directed to an inducible cell-based assay for FAK.
  • the cell-based assay exploits the biology of FAK and an inducible gene expression system to exogenously control FAK expression and FAK phosphorylation at the tyrosine residue at position 397 (Y397).
  • Y397 tyrosine residue at position 397
  • the cell- based assay of the present invention is flexible and can measure FAK phosphorylation at Y397, total FAK phosphorylation, identify mutant FAK proteins and measure a combination of protein and phosphotyrosine.
  • the inducible FAK cell-based assay of the present invention is advantageous in that it provides tight control over ectopic-basal level expression of FAK and rapid de-repression of FAK gene expression via an exogenous stimulant.
  • the cell-based assay is flexible such that the final read-out is mechanistically relevant to FAK biology as measured for phosphotyrosine FAK Y397 , total FAK phosphotyrosine profile, FAK or mutant proteins, or some combination of protein and phosphotyrosine.
  • the present invention has been successivefully used to identify a number of FAK inhibitors.
  • the term "tight control” refers to the controlled expression of the FAK gene that occurs in the presence of an exogenous stimulant.
  • the invention provides an inducible gene expression system for FAK, where the expression of FAK is induced in the presence of an appropriate inducible agent.
  • the present invention provides a method for the inducible expression of FAK, wherein the regulated expression of the FAK gene does not adversely affect cell viability.
  • An embodiment of the invention is directed to a cell-based assay for the screening of FAK inhibitors.
  • the cell-based assay exploits the biology of FAK and an inducible gene expression system to exogenously control FAK expression and FAK phosphorylation at the tyrosine residue at position 397 (Y397).
  • the cell-based assay is mechanistically relevant to FAK biology and measures changes in FAK phosphorylation.
  • the cell-based assay of the present invention is flexible and can measure FAK phosphorylation, total FAK phosphorylation, identify mutant FAK proteins and measure a combination of protein and phosphotyrosine.
  • An embodiment of the invention provides a method for identifying cell-active inhibitors of FAK comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; capturing the expressed FAK using a FAK capture agent; exposing the captured FAK to an anti-phospho- tyrosine antibody; and, detecting the phosphorylation of said FAK.
  • the extent of phosphorylation of the FAK is determined by the binding of the anti-phospho- tyrosine antibody to the captured FAK, wherein the amount of anti-phospho-tyrosine antibody binding to the captured FAK is proportional to the amount of phosphorylation of said FAK.
  • the method for identifying cell-active inhibitors of FAK comprises an optional step of coating the mammalian cells on a first solid phase.
  • the first solid phase is preferably a well of a first microtiter plate.
  • the cells coated on the first solid phase are lysed with a lysis buffer, prior to the capture of the expressed FAK.
  • the lysis buffer optionally comprises a solubilizing detergent.
  • the FAK capture agent is coated on a second solid phase, which is preferably a well of a second microtiter plate.
  • the test compound inhibits the phosphorylation of FAK at
  • An embodiment of the invention provides a method for measuring the cytotoxicity of a test compound comprising, stably transfecting mammalian cells with a gene encoding FAK, wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; adding a cytotoxicity indicator to said cells; and, detecting the cytoxicity of the test compound.
  • the cytoxicity of the test compound is determined by the colorimetric conversion of the cytotoxicity indicator, wherein the amount of converted cytotoxicity indicator is proportional to the number of living cells.
  • the method for measuring the cytotoxicity of a test compound comprises an optional step of coating the mammalian cells on a solid phase.
  • the solid phase is preferably a well of a first microtiter plate.
  • An embodiment of the invention provides a method for identifying cell-active inhibitors of focal adhesion kinase (FAK) comprising, coating a first solid phase with a homogeneous population of mammalian cells so that the cells adhere to the first solid phase, wherein said cells are stably transfected with a gene encoding FAK, and wherein said gene is expressed in the presence of an inducing agent; adding an inducing agent to induce the expression of said gene encoding FAK; adding a test compound; solubilizing the adhering cells to release the cell lysate; coating a second solid phase with a FAK capture agent so that the FAK capture agent adheres to the second solid phase; exposing the cell lysate to the adhered FAK capture agent so that the FAK capture agent captures FAK; exposing the captured FAK to an anti-phospho-tyrosine antibody; and, measuring binding of the anti-phospho-tyrosine antibody to the captured FAK, wherein the amount of anti-phospho-tyrosine
  • Another embodiment of the present invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, wherein said recombinant nucleic acid molecule is selected from the group consisting of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8, and wherein expression of said sequences requires the presence of an inducing agent.
  • An embodiment of the invention provides a mammalian cell stably transfected with a recombinant nucleic acid molecule, that encodes a protein comprising a sequence selected from the group consisting of SEQ ID NOS: 1 , 2, 3 and 4, and wherein expression of said protein requires the presence of an inducing agent.
  • FAK is also known as the Protein-Tyrosine Kinase 2, PTK2. Any active FAK variant can be employed in the above assay. Inactive mutants can also be used in the assay for various control purposes. Additional variants of FAK that can be employed in the above described assay include, wild type (WT) human FAK at 153012 with 1052 amino acids (SEQ ID NO:1); splice variants of FAK such as described in Andre, E. & Becker-Andre, M., Expression of an N-terminally truncated form of human focal adhesion kinase in brain. Biochem. Biophys. Res. Commun.
  • inducing agent is an agent, compound, or chemical that produces a signal to noise ratio of at least 6-fold.
  • inducing agents include but are not limited to Mifepristone (Ru486) and other antiprogestins such as Org31806 and Org31376. See O'Malley et. al., Cell, 69, 703-713 (1992).
  • capture agent is an agent, compound or chemical that is capable of capturing any form of focal adhesion kinase, including FAK tagged with histidine residues, streptavidin or other comparable affinity tags.
  • the capture agent includes, but is not limited to, the phosphotyrosine FAK Y397 specific antibody, a general phosphotyrosine antibody, anti-FAK antibody, anti-histidine tag antibody and molecules containing biotin which would facilitate the capture of streptavid in-modified FAK.
  • the capture agent includes a combination of one or more antibodies, including but not limited to the combination of goat anti-rabbit antibody and the phosphotyrosine FAK Y397 specific antibody.
  • anti-phospho-tyrosine antibody includes but is not limited to phosphotyrosine FAK Y397 specific antibody as well as general phosphotyrosine antibodies, where the latter are capable of recognizing any phosphorylated tyrosine residue, including but not limited to the tyrosine residue at position 397 of FAK.
  • a cytoxicity indicator is an agent, chemical or compound that is used to assess cell viability.
  • cytoxicity indicators include, but are not limited to tetrazolium salts (e.g. MTT, XTT, WST-1 ) that are especially useful for this type of analysis.
  • MTT is a yellow tetrazolium salt that is cleaved to purple formazan crystals by metabolic active cells.
  • the solubilized formazan product may be spectrophotometrically quantified using an ELISA reader or other spectrophotometric device. The number of living directly correlates to the amount of purple formazan crystals formed, as monitored by the absorbance.
  • an embodiment of the present invention is carried out using a inducible FAK gene expression system having the ability to provide consistent and tight regulation of gene expression.
  • an inducible FAK gene expression system which comprises a system for the regulated expression of a transgene, i.e., FAK.
  • FAK expression is "off” in the absence of an inducing agent, but "on” in its presence.
  • the system consists of two genes: one which codes for a regulatory protein, and the other which codes for the inducible transgene of interest. Expression of the regulatory protein is driven by a constitutive promoter.
  • the inducible FAK transgene has a promoter that consists of a minimal promoter linked to multiple copies of a binding site that is capable of binding the regulatory protein.
  • the regulatory protein is a transcription factor that consists of the yeast GAL4 DNA binding domain, a truncated human progesterone receptor ligand binding domain, and the human p65 activation domain from NF- ⁇ B to facilitate tight regulation of the transgene over basal expression.
  • the plasmid encoding the regulatory protein contains the GAL4 promoter which establishes a positive feedback loop to facilitate rapid response upon ligand treatment.
  • the exogenous control of regulatory protein expression is achieved using a small molecule ligand. Tight regulation is achieved by species selective binding of the regulatory protein to the promoter of the transgene, and through ligand-dependent conformational activation of the regulatory protein.
  • the invention has the following advantages: ⁇ Induction of FAK in cells using an inducible system, which allows for tight repression of gene expression when not induced, resulting in viable cell clones.
  • ⁇ Detection of phosphorylated FAK may be used to identify inhibitors of FAK kinase activity. Some of the methods of detection of phosphorylated FAK include Polyacrylamide gel electrophoresis-based assays and ELISA-based assays for identification of FAK inhibitors. Other suitable detection systems may also be used.
  • the assay allows for detection of total FAK protein, total phosphorylated FAK protein, or FAK protein phosphorylated at a given tyrosine (e.g. tyrosine 397).
  • the cell-based system may be used for in vivo screening of FAK inhibitors, since the transfected cells are tumorigenic and FAK can be induced in vivo by feeding animals Mifepristone. The system's utility in vivo has been demonstrated.
  • ECM-matrigel-induced attachment of cells resulted in no stimulation of FAK phosphorylation as measured by pY54 HRP (Horseradish Peroxidase (HRP)- conjugated phosphotyrosine antibody) detection, and control detection using secondary Anti- Mouse HRP (2°M HRP ) alone resulted in nonspecific increases in assay signal in wells incubated with lysate derived from cells stimulated on matrigel.
  • HRP HapY54 HRP
  • S/N signal and noise ratio
  • Clones were generated that harbored FAK cDNA transcripts that encode either a constitutively active protein (CD2 « FAK), a membrane bound tyrosine-dead protein (CD2'FAK Y397F ), or cytoplasmic FAK mutants that lack key downstream tyrosine residues (e.g., FAK Y86 F , FAK Y925F , and FAK Y861F/Y925F ).
  • a constitutively active protein CD2 « FAK
  • CD2'FAK Y397F membrane bound tyrosine-dead protein
  • cytoplasmic FAK mutants that lack key downstream tyrosine residues (e.g., FAK Y86 F , FAK Y925F , and FAK Y861F/Y925F ).
  • constructs are based on the published sequence for human FAK at GenBank accession number L13616, see Whitney.G.S. et. al., DNA Cell Biol. 12 (9), 823-830 (1993), and each construct was sequence confirmed and aligned against the published sequence.
  • the DNA constructs were engineered to include specific 5' and 3' restriction sites that are unique and distinct over the prior art.
  • the 3' ends of certain constructs were also engineered to include tagged epitopes such as V5 and histidine tags.
  • NIH3T3 NIH Swiss Mouse fibroblast NIH3T3 (ATCC Accession No. CCL-92), the human epidermoid carcinoma A431 (ATCC Accession No. CRL1555), and the human glioblastoma astrocytoma U87MG (ATCC Accession No. HTB-14).
  • Each cell line exhibits unique characteristics that makes its application favorable for overexpression of FAK.
  • NIH3T3 cells offer species specific overexpression of FAK and ease of transfectability
  • A431 cells express moderate levels of endogenous FAK and provide a tumor background more representative of a FAK native environment
  • U87MG cells lack the putative FAK negative regulator PTEN (a tumor suppressor phosphatase).
  • PTEN a tumor suppressor phosphatase
  • the pSwitch regulatory vector was co-transfected with either the pGene Vector V5His, or the pGeneFAK WT V5His, or the pGeneFAK mutants (pGeneFAK K 54R V5His, and pGeneFAK Y397F V5His) into A431 cells.
  • pGeneFAK K 54R V5His pGeneFAK K 54R V5His
  • pGeneFAK Y397F V5His pGeneFAK Y397F V5His
  • NIH3T3:FAK Y397F V5His NIH3T3:Vector, U87MG:FAK WT V5His, and U87MG:FAK Y397F V5His were successfully isolated, selected, confirmed and validated.
  • the FAK inducible assay of this embodiment of the invention measured FAK phosphorylation at Y397.
  • A431:FAK WT V5His cells (about 1.0 x 10 4 to 1.0 x 10 7 cells) are seeded on U-bottom 96-well plates and allowed to attach at 37°C, 5% C0 2 for 4 to 6 hoursprior to overnight incubation in the presence of 0.1 nM Mifepristone.
  • A431 :FAK WT V5His induced cells are either left untreated or treated with inhibitory compounds for 30 minutes at 37°C, 5% C0 2 and lysed in RIPA lysis buffer (50 mM Tris-HCI, pH7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na 3 V0 4 , 1 mM NaF, and one CompleteTM EDTA-free protease inhibitor pellet per 50 ml solution).
  • RIPA lysis buffer 50 mM Tris-HCI, pH7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na 3 V0 4 , 1 mM NaF, and one CompleteTM EDTA-free protease inhibitor pellet per 50 ml solution.
  • NIH3T3:FAK T V5His clones and NIH3T3:FAK Y397F V5His clones were seeded to approximately 80% confluency in T25 flasks and were either left untreated or stimulated with 0.1 nM Mifepristone for approximately 16 hoursat 37°C, 5%C0 .
  • Cell lysates were prepared and immunodepleted of total FAK protein using the Anti-FAK (UBITM, Lake Placid, N.Y.) monoclonal antibody.
  • FAK immunocomplexes were then subject to SDS-Polyacrylamide gel electrophoresis and immunoblotted with Anti-FAK (A17) polyclonal antibody.
  • Table 1 shows densitometric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK protein levels as compared with unstimulated cells.
  • Table 2 shows NIH3T3 clones induced to express FAK T or FAK Y39 F proteins.
  • FAK T and FAK Y397F proteins were captured either to measure total FAK protein or phosphotyrosine FAK Y397 induced by mifepristone.
  • Table 2 Mifepristone induced FAK protein levels as measured by optical density 450 measurements
  • NIH3T3 clones yield a robust phosphotyrosine FAK Y397 and FAK protein signal-to-noise ratio of ⁇ 7 and ⁇ 6, respectively. Both the NIH3T3 tyrosine-dead clone and the vector NIH3T3 transfectants confirm assay specificity for phosphoFAKY397.
  • Table 3 shows A431 :FAK clones assayed under optimized conditions.
  • Table 3 Mifepristone induced FAK protein levels as measured by optical density 450 measurements
  • the A431 :FAK clones yielded a robust phosphotyrosine FAK Y397 and FAK protein signai-to-noise ratio of -17 and -11 , respectively.
  • Both the A431 tyrosine-dead clone and the vector A431 transfectants confirmed assay specificity for phosphoFAKY397.
  • Induction of phosphotyrosine Y397 and FAK proteins were controllable by varying assay conditions such as mifepristone time of incubation, mifepristone concentration, cell density, and antibody concentrations.
  • A431:FAK WT cells were either left untreated or stimulated with mifepristone.
  • a washout experiment was performed to demonstrate a time-dependent decrease in FAK protein and phosphotyrosine Y397 levels (i.e. cells stimulated with mifepristone overnight (16hs) were subsequently cultured in fresh growth media in the absence of mifepristone over time).
  • A431:FAK T cells stimulated with mifepristone were detached and suspended in fresh growth media for 15 minutes followed by re-plating for 4, 24, 48, and 72 hourson tissue culture treated petri dishes.
  • Table 4, experiment A shows densimetric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK phosphorylation as compared to a no stimulation control.
  • FAK may be achieved by re-plating suspended cells to allow cell attachment.
  • Table 4 demonstrates that the mechanism of FAK activation is intact under the regulatory system discussed above. In other words, exogenous stimulation with mifepristone leads to the activation of FAK that is mechanistically relevant.
  • the FAK inducible cell-based assay of the invention was successfully employed in the identification of a number of FAK inhibitors, including PP1 and staurosporin.
  • the assay was used to determine the cytoxicity of particular compounds on FAK-expressing cells.
  • the test compound was placed in contact with FAK-induced or uninduced control cells.
  • Table 5 shows changes in FAK phosphorylation in OD 45 o units upon treatment with increasing concentration of a FAK inhibitor.
  • Table 6 shows densimetric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK phosphorylation in the presence of increasing concentration of the FAK inhibitor.
  • the FAK inducible cell-based assay was used as described in Figure 3 to screen a FAK inhibitor.
  • a 10 ⁇ M curve of an FAK inhibitor was performed in 1/2 log dilutions as shown in Table 5. The % inhibition was determined and the inhibition concentration at 50% (IC 50 ) was calculated.
  • A431 :FAK WT cells were seeded in T25 flask to -80% confluency and were either left untreated or stimulated overnight with 0.1 nM mifepristone. Subsequently, stimulated cells were treated with the same FAK inhibitor as in Table 5 at the same 1/2 log concentrations of a 10 ⁇ M curve for 30 minutes at 37°C, 5% C0 2 . Cell lysates were then prepared and total protein concentration determined by protein assay.
  • the calculated Inhibition Concentration at 50% for the FAK inhibitor was -0.93 uM.
  • Table 6 shows densimetric quantitation measured in arbitrary "band light" units of the autoradiographic film and fold changes in FAK phosphorylation in the presence of increasing concentration of the FAK inhibitor.
  • ECM stimulation of FAK was achieved via plating 2.0x10 5 cells/well (e.g. A2058) on commercially available fibronectin (FN) 96-well plates or on FN 96-well plates prepared in- house. Cells were allowed to attach for 15, 30, 60, or 90 minutes at 37°C, 5%C0 2 to induce integrin engagement and subsequent FAK phosphorylation.
  • FN fibronectin
  • Cells lysates were prepare in RIPA lysis buffer (50 mM Tris-HCI, pH7.4, 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na 3 V0 , 1 mM NaF, and one CompleteTM EDTA-free protease inhibitor pellet per 50 ml solution) and transferred to Anti-FAK coated 96-well plates to capture total FAK protein. Phosphotyrosine FAK proteins were measured using the general phosphotyrosine antibody Py54. Similarly, cells were stimulated on commercially available matrigel coated plates or on matrigel plates prepared in-house.
  • cells were allowed to attach to either commercially available ECM coated flask or to ECM coated flask prepared in-house for the indicated times above.
  • Cell lysates were prepared in RIPA lysis buffer and total and phosphotyrosine FAK proteins were either pull-down or immunodetected using Anti-FAK antibodies or Anti-phosphotyrosine antibodies.
  • Cells e.g. A2058 human metastatic meloanomas
  • Starved Cells (e.g. A2058) were pre-chilled at 4°C for 30 minutes prior to incubation with varying concentrations (1 :100, 1 :330, 1 :1000, 1 :3,300, or 1 :10,000 of stock concentration of 200 ⁇ g/ml) of ⁇ 1-lntegrin antibody (4° C, 30 minutes).
  • ⁇ 1-lntegrin clustering was induced with 1 :500 dilution of Goat anti-mouse secondary antibody for 30 minutes at 4°C.
  • FAK WT and FAK mutants Noninducible and Tet-OnTM/Tet-OffTM Systems
  • the full-length FAK WT cDNA was cloned from a T-cell cDNA library using the primers FAK ⁇ 'Bam: GGATCCATGGCAGCTGCTTACCTTGAC (SEQ ID NO.: 9) and FAK3'Bam: GGATCCTCACTCACTCAGTGTGGTCTCGTCTGCCCA (SEQ ID NO.: 10) in RT-PCR and was sequence confirmed against the accession number L13616.
  • Site-directed mutagenesis of the FAK WT template was performed using the Stratagene QuikChangeTM Site-Directed Mutagenesis Kit.
  • the full-length Focal Adhesion Kinase (FAK WT ) cDNA was cloned from a T-cell cDNA library using the above primers in RT-PCR and was sequence confirmed against the accession number L13616.
  • Site-directed mutagensis of the FAK WT template was performed using the Stratagene QuikChangeTM Site-Directed Mutagenesis Kit.
  • the full-length FAK WT , the tyrosine-dead mutant (FAK Y397F ), and the kinase-dead mutant (FAK K454R ) cDNAs were subcloned into the BamHI-Apal sites of the pGeneV5/His-A plasmid.
  • the dominate-negative FAK-related nonkinase (FRNK) cDNA was subcloned as a Kpnl-Apal insert into the cassette of the pGeneV5/His A-vector. These plasmids were confirmed via DNA sequencing and restriction digestion analysis. Subsequently, these constructs were co-transfected with the pSwitch construct encoding the GeneSwitchTM Protein into NIH3T3, A431, or U87MG cells using Stratagene's GeneJammerTM Transfection reagent.
  • Transfectants were grown and selected in DMEM growth media (10%FBS, Pen/Strep/Glu) spiked with 750 ⁇ g/ml of Zeocin and 50 ⁇ g/ml of hygromycin antibiotics. Hygromycin and Zeocin resistant clones were selected and expanded in culture for screening in both Western and RT-PCR format.
  • A431 « FAK T and A431 « FAK Y397F clones were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight ( ⁇ 16hrs).
  • A431 transfectants were then lysed in RIPA lysis buffer (50 mM Tris-HCI, pH7.4, 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na 3 V0 4 , 1 mM NaF, and one CompleteTM EDTA-free protease inhibitor pellet per 50 ml solution) and cell lysates were assayed for total protein concentration.
  • RIPA lysis buffer 50 mM Tris-HCI, pH7.4, 1 % NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mM EDTA, 1 mM Na 3 V0 4
  • Clones were also screened and verified via RT-PCR. Cytoplasmic mRNA transcripts were isolated and purified from A431 « FAK wild-type and mutant clones for cDNA synthesis using random hexamers. Polymerase chain reactions were subsequently performed on cDNA libraries derived from A431 « FAK transfectants using primers specific to the N-terminus, the internal FAK sequences, and the C-terminus of FAK. Primers specific to the antibiotic transcripts Zeocin or Hygromycin and GAPDH were also used to amplify these genes as internal controls for transfection as well as for the quality of cDNA libraries. Optimization of FAK inducible cell-based System
  • A431 « pGene Vector , A431 « FAK WT , A431 « FAK K454R , and A431 « FAK Y397F clones were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1 , 10, or 100 nM of Mifepristone ligand overnight ( ⁇ 16hrs).
  • A431 transfectants were then lysed in RIPA lysis buffer (described above) and cell lysates were assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK, or FAK mutant, proteins and phosphotyrosine FAK Y397 using standard western blotting techniques. To determine that optimization of Mifepristone concentration in western format translates into an ELISA system, Mifepristone concentration and time of incubation were also optimized in ELISA format.
  • A431'pGene Vector , A431 « FAK WT , and A431 « FAK Y397F clones were seeded in 96-well U-bottom plates at a cell density of 1.2 x 10 6 cells/ml. Cells were allowed to sit at 37°C, 5% C0 2 for 6 to 8 hours prior to FAK induction with Mifepristone ligand. A431 clones were subsequently left untreated or treated with 0.1, 10, 100, or 110 nM of Mifepristone for -0.5, 1.0, 2.0, 4.0 or 24.0 hoursat 37°C, 5% C0 2 .
  • Parameters can be optimized for the particular high thoughput screening system employed, including evaluating the types of 96-well plate (e.g., anti-rabbit, protein A, or protein G), capture antibody concentration, detection antibody concentration, secondary horseradish peroxidase (HRP) antibody concentration, cell density, blocking buffer (e.g. SuperBlock Blocking TBS, 3%BSA blocking), and time of compound treatment.
  • types of 96-well plate e.g., anti-rabbit, protein A, or protein G
  • capture antibody concentration e.g., detection antibody concentration
  • secondary horseradish peroxidase (HRP) antibody concentration e.g. SuperBlock Blocking TBS, 3%BSA blocking
  • cell density e.g. SuperBlock Blocking TBS, 3%BSA blocking
  • blocking buffer e.g. SuperBlock Blocking TBS, 3%BSA blocking
  • A431 FAK WT cells were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight ( ⁇ 16hrs).
  • A431 FAK WT uninduced and induced cells were washed with 10 ml of PBS and suspended in 15 ml growth media (DMEM 10%FBS, Pen/Strep/Glu, 750 ⁇ g/ml Zeocin, 50 ug/ml Hygromycin) for 30, 60, 90, or 120 minutes at 37°C, 5% C0 2 .
  • Cells were subsequently lysed in RIPA lysis buffer (described above) and cell lysates were assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK or phosphotyrosine FAK Y397 proteins using standard western blotting techniques.
  • A431 « FAK WT and A431 «FAK Y397F clones were seeded in T25 flasks to near 80% confluency and were either left untreated or treated with 0.1 nM of Mifepristone ligand overnight ( ⁇ 16hrs).
  • A431*FAK WT cells were subsequently treated with increasing concentrations of a FAK inhibitor (half-log dilutions of a 10 ⁇ M-starting solution) identified by using the FAK inducible cell-based assay.
  • Cell lysates were then prepared in RIPA lysis buffer and assayed for total protein concentration. Equivalent total protein concentrations were subject to western analysis immunoblotting for FAK, general phosphotyrosine FAK, or phosphotyrosine FAK Y397 proteins using standard western blotting techniques.

Landscapes

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

Abstract

L'invention concerne un dosage cellulaire qui exploite le biologie de FAK conjointement avec un système d'expression génique inductible afin de contrôler depuis l'extérieur l'expression de FAK et la phosphorylation de FAK du résidu de tyrosine au niveau de la position 397 (Y<397>). Ce dosage de cellule kinase de l'invention est flexible et peut mesurer le phosphorylation de FAK au niveau de Y397, la phosphorylation de FAK totale, identifier les protéines de FAK mutantes et mesurer une combinaison de protéine et de phosphotyrosine.
PCT/IB2003/003968 2002-09-19 2003-09-08 Dosage de cellule kinase a adhesion focale inductible WO2004027018A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002497434A CA2497434A1 (fr) 2002-09-19 2003-09-08 Dosage de cellule kinase a adhesion focale inductible
EP03797456A EP1546372A4 (fr) 2002-09-19 2003-09-08 Dosage de cellule kinase a adhesion focale inductible
JP2004537413A JP2006500022A (ja) 2002-09-19 2003-09-08 誘導可能な局所接着キナーゼ細胞アッセイ
AU2003259498A AU2003259498A1 (en) 2002-09-19 2003-09-08 Inducible focal adhesion kinase cell assay
NO20051869A NO20051869L (no) 2002-09-19 2005-04-18 Celleanalyse av induserbare focal adhesjonskinase

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41207802P 2002-09-19 2002-09-19
US60/412,078 2002-09-19

Publications (2)

Publication Number Publication Date
WO2004027018A2 true WO2004027018A2 (fr) 2004-04-01
WO2004027018A3 WO2004027018A3 (fr) 2004-09-16

Family

ID=32030798

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2003/003968 WO2004027018A2 (fr) 2002-09-19 2003-09-08 Dosage de cellule kinase a adhesion focale inductible

Country Status (9)

Country Link
EP (1) EP1546372A4 (fr)
JP (1) JP2006500022A (fr)
KR (1) KR20050052663A (fr)
CN (1) CN1681939A (fr)
AU (1) AU2003259498A1 (fr)
CA (1) CA2497434A1 (fr)
NO (1) NO20051869L (fr)
RU (1) RU2005107701A (fr)
WO (1) WO2004027018A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009046129A2 (fr) * 2007-10-01 2009-04-09 Columbia University Procédés de traitement du syndrome de détresse respiratoire de l'adulte

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100934706B1 (ko) * 2006-12-07 2009-12-31 재단법인서울대학교산학협력재단 Ｔｍ４ｓｆ５의 기능을 저해하는 항암물질의 스크리닝 방법및 칼콘계 화합물을 함유하는 항암조성물
CN109112213B (zh) * 2017-06-22 2021-01-12 中国科学院动物研究所 检测黏着斑激酶结构变异体的pcr引物及其检测方法与应用
KR102007450B1 (ko) 2018-05-31 2019-08-05 한국과학기술연구원 대장암 치료제 발굴을 위한 신규 표적 치료 작용점의 스크리닝 방법 및 이를 이용하여 선별한 대장암 치료 예후 바이오마커
KR102408538B1 (ko) * 2020-10-29 2022-06-15 한국과학기술연구원 Fak 활성 억제제를 스크리닝하기 위한 조성물, 키트 및 방법

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6395506B1 (en) * 1991-04-18 2002-05-28 Becton, Dickinson And Company Device for monitoring cells
AU5596998A (en) * 1996-12-11 1998-07-03 New York University Medical Center Pyk2 related products and methods
US20010055585A1 (en) * 1997-12-03 2001-12-27 William G. Cance Frnk proteins in the treatment of tumor cells
US6133031A (en) * 1999-08-19 2000-10-17 Isis Pharmaceuticals Inc. Antisense inhibition of focal adhesion kinase expression

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009046129A2 (fr) * 2007-10-01 2009-04-09 Columbia University Procédés de traitement du syndrome de détresse respiratoire de l'adulte
WO2009046129A3 (fr) * 2007-10-01 2009-05-22 Univ Columbia Procédés de traitement du syndrome de détresse respiratoire de l'adulte
US8420080B2 (en) 2007-10-01 2013-04-16 The Trustees Of Columbia University In The City Of New York Methods for treating adult respiratory distress syndrome

Also Published As

Publication number Publication date
EP1546372A2 (fr) 2005-06-29
JP2006500022A (ja) 2006-01-05
CN1681939A (zh) 2005-10-12
EP1546372A4 (fr) 2006-08-23
AU2003259498A1 (en) 2004-04-08
NO20051869D0 (no) 2005-04-18
WO2004027018A3 (fr) 2004-09-16
CA2497434A1 (fr) 2004-04-01
KR20050052663A (ko) 2005-06-03
NO20051869L (no) 2005-06-16
RU2005107701A (ru) 2005-08-27

Similar Documents

Publication Publication Date Title
Renshaw et al. Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase
Michaud et al. 14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3-and Ras-independent manner
Wright et al. The STE20 kinase HGK is broadly expressed in human tumor cells and can modulate cellular transformation, invasion, and adhesion
Lim et al. RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation
Bräuner-Osborne et al. Pharmacology of muscarinic acetylcholine receptor subtypes (m1–m5): high throughput assays in mammalian cells
Aronheim et al. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions
Ambrosino et al. Negative feedback regulation of MKK6 mRNA stability by p38α mitogen-activated protein kinase
Salmon et al. The p38 mitogen-activated protein kinase is activated by ligation of the T or B lymphocyte antigen receptors, Fas or CD40, but suppression of kinase activity does not inhibit apoptosis induced by antigen receptors.
Feldhahn et al. Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells
NO313103B1 (no) Fremgangsmåte for screening for proteininhibitorer og aktiveringsmidler, samt testceller og testkit
WO2017121865A1 (fr) Récepteur de mic-1 et utilisations correspondantes
Krautwald et al. Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway
Musumeci et al. Hck inhibitors as potential therapeutic agents in cancer and HIV infection
US9404080B2 (en) Human taste cells capable of continuous proliferation
Rahimi et al. EGFR variant‐mediated invasion by enhanced CXCR4 expression through transcriptional and post‐translational mechanisms
WO2004027018A2 (fr) Dosage de cellule kinase a adhesion focale inductible
US5874230A (en) Assays using TRAF2-associated protein kinase polypeptides
Thomas et al. Characterisation and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor
AU2018322286B2 (en) Fusion protein of DCTN1 protein with RET protein
JP2022068272A (ja) サイトカイン産生抑制剤
CN114957444B (zh) 一种nmdar的nr1亚基缺失突变体、突变体细胞及构建方法和应用
US7348151B1 (en) Method for the cellular high-throughput-detection of nuclear receptor ligand interactions
EP0711341A1 (fr) Procedes de determination de la fonction d&#39;un recepteur
Boni Successful preservation of BCR-ABL1 protein and direct measure of kinase activity in peripheral blood of CML and Ph+ ALL patients unveil a kinase inhibitory activity present in chronic phase CML cells
Mahlmann et al. Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 512/DELNP/2005

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 166872

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 2003259498

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2497434

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2003797456

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2005107701

Country of ref document: RU

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020057004717

Country of ref document: KR

Ref document number: 2004537413

Country of ref document: JP

Ref document number: 20038221519

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 1020057004717

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 2003797456

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2003797456

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载