EP0990028A1 - Criblage d'inhibiteurs de la kinase liee au phosphatidilynositol - Google Patents

Criblage d'inhibiteurs de la kinase liee au phosphatidilynositol

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Publication number
EP0990028A1
EP0990028A1 EP98930061A EP98930061A EP0990028A1 EP 0990028 A1 EP0990028 A1 EP 0990028A1 EP 98930061 A EP98930061 A EP 98930061A EP 98930061 A EP98930061 A EP 98930061A EP 0990028 A1 EP0990028 A1 EP 0990028A1
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European Patent Office
Prior art keywords
cells
wortmannin
kinase
mtor
phas
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EP98930061A
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German (de)
English (en)
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EP0990028A4 (fr
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Robert T. Abraham
Jann N. Sarkaria
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Mayo Foundation for Medical Education and Research
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Mayo Foundation for Medical Education and Research
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Publication of EP0990028A1 publication Critical patent/EP0990028A1/fr
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    • 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
    • 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
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • PIKK phosphatidylinositol kinase-related kinase
  • AT clinical syndrome ataxia-telangiectasia
  • ATM ataxia-telangiectasia mutated
  • the gene product of the ATM gene, ATM protein, is a member of the PIKK family.
  • AT patients are hyper-sensitive to ionizing radiation and suffer from progressive cerebellar degeneration, immunodeficiency, and a dramatically increased incidence of various cancers, particularly lymphomas .
  • Cells derived from AT patients exhibit defects in Gl, S and G2 checkpoints following exposure to ionizing radiation.
  • ATR ATaxia and Rad3 related protein
  • DNA-PK CS forms a heterotrimer with Ku70 and Ku80 which is critical for DNA double strand break repair in cells exposed to ionizing radiation and in normal hematopoietic cells undergoing V(D)J gene rearrangements.
  • rapamycin target protein or mammalian target of rapamycin appears to participate in mitogenic signal transduction.
  • Mammalian TOR protein is also named FRAP or RAFT1. Brown, E.J. et al . , Nature 369:756 (1994); Subatini, M. et al., Cell, 78:35 (1994).
  • the invention is based on the ability of phosphoinositide 3 -kinase related kinase (PIKK) polypeptides to phosphorylate PHAS-1 protein. Assays for identifying agents that inhibit the phosphorylation activity of PIKK polypeptides are described. Such inhibitors have therapeutic applications in transplantation, cancer, and other proliferative disorders .
  • the invention relates to a method for identifying a compound inhibiting the phosphorylation activity of a PIKK polypeptide. The method includes incubating isolated PIKK polypeptide and a substrate of the PIKK • polypeptide with the compound to determine if phosphorylation of the substrate is inhibited.
  • the PIKK polypeptide can be, for example, mTOR, ataxia- telangiectasia mutated protein or Ataxia and Rad3 related protein.
  • PHAS-I protein is a particularly useful substrate of the PIKK polypeptides.
  • a "compound” refers to a biological macromolecule such as an oligonucleotide or a peptide, a chemical compound, a mixture of chemical compounds, or an extract isolated from bacterial, plant, fungal or animal matter.
  • suitable compounds induce radioresistant DNA synthesis in irradiated cells containing the compound.
  • the invention also features an antibody or fragment thereof having specific binding affinity for a conjugate including wortmannin or an analog thereof and a polypeptide.
  • the antibody can be polyclonal or monoclonal.
  • the polypeptide can be, for example, mTOR, DNA-PK, ataxia-telangiectasia mutated protein or Ataxia and Rad3 related protein.
  • the invention also relates to a method for identifying a compound that induces radioresistant DNA synthesis within cells. The method includes irradiating the cells, wherein the cells include an effective amount of the compound, and measuring radioresistant DNA synthesis of the cell. The presence or absence of radioresistant DNA synthesis is correlated with activity of the compound.
  • Figure 1 depicts the eIF-4E binding activity of phosphorylated PHAS-I. Radioactivity bound to PHAS-I in each sample lane was quantitated and normalized to the nonphosphorylated control (lane 1) . The concentration of wortmannin used in the pretreatment is given in ⁇ M.
  • Figure 2 depicts the phosphorylation of PHAS-1 by immunoprecipitated ATM.
  • FIGS 3A-3C depict the covalent modification and inhibition of ATM, DNA-PK and ATR, respectively, by wortmannin.
  • Kinase activity was normalized to 1.0 for 0 ⁇ M wortmannin treatment.
  • Figure 5 depicts wortmannin- induced radiosensitization of A549 cells as measured by a clonogenic assay.
  • Log-phase cells were exposed to graded doses of radiation and then incubated with DMS0( «), 2 ⁇ M( ⁇ ), 10 ⁇ M (v) or 20 ⁇ M ( ⁇ ) wortmannin for 14 days prior to fixation and staining.
  • Figure 6 depicts the effect of wortmannin on radiation- induced G 2 -phase delay m A549 cells synchronized in S-phase by treatment with aphidicolin. Histograms of red fluorescence intensity (DNA content) from 20,000 ungated events are shown from a representative experiment. The numbers in each panel indicate the percentage of G 2 -M cells in a test population.
  • Figure 7 depicts the induction of radioresistant DNA synthesis by wortmannin.
  • Dysfunction of certain PIKKs, including ATM, DNA- PK, and ' possibly ATR leads to defects in DNA damage repair and hypersensitivity to ionizing radiation.
  • PIKK polypeptides are novel molecular targets for the development of agents that, in principle, might sensitize cancer cells to conventional chemotherapeutic agents or ionizing radiation.
  • wortmannin is an effective radiosensitizing agent in tumor cells.
  • wortmannin-induced radiosensitization is manifested at drug concentrations similar to those required for inhibition of DNA-PK and ATM, but not ATR, in A549 lung adenocarcinoma cells.
  • the clinically proven immunosuppressive and antiproliferative activities of rapamycin validate mTOR as a target for drug discovery efforts.
  • PHAS-I as an in vi tro substrate for the phosphatidylinositol kinase- related kinase (PIKK) polypeptides offers a suitable starting point for the implementation of screens for novel inhibitors of this group of protein kinases .
  • PIKK phosphatidylinositol kinase- related kinase
  • the invention features a method for identifying a compound that inhibits the phosphorylation activity of a PIKK polypeptide.
  • the method includes incubating isolated PIKK polypeptide and a substrate of the PIKK polypeptide with the compound to determine if phosphorylation of the substrate is inhibited.
  • the PIKK polypeptide can be, for example, mTOR, DNA-PK, ATM or ATR.
  • polypeptide refers to a chain of amino acids of any length.
  • the PIKK polypeptide can be full-length or can be a single domain of the full-length protein, such as a catalytic or kinase domain.
  • the PIKK polypeptide can be wild- type or mutant.
  • Mutant PIKK polypeptides are catalytically active but have decreased binding affinity for regulatory proteins. For example, deletion of amino acid residues 2432 to 2449 of mTOR yields a protein with a 50-100 fold increase in specific activity. Increased specific activity may be desirable for drug screening protocols .
  • mTOR regulates at least two downstream signaling events in mitogen-stimulated cells. As described herein, one pathway culminates in the phosphorylation of the elF- 4E binding protein PHAS-1, at serine and threonine residues involved in the regulation of eIF-4E binding affinity. The second pathway leads to the phosphorylation and activation of p70S6 kinase (Brown, E.J.
  • the rate of progression of mammalian cells through G 1 phase is thought to be governed in part by the ratio of the translational stimulator, eIF-4E, to hypophosphorylated PHAS-I.
  • the functional consequences of an imbalance between these positive and negative regulators of translation are illustrated by the finding that constitutive overexpression of eIF-4E induces malignant transformation of NIH 3T3 fibroblasts.
  • Lazaris-Karatzas, A. et al . Nature 345:544 (1990); Lazaris-Karatzas, A. et al . , Genes Dev. 6:1631 (1992); Rousseau, D. et al . , Proc. Natl. Acad. Sci.
  • hypophosphorylated PHAS-I serves as a negative growth regulator in normal cells, and may function as a tumor suppressor in vivo .
  • PIKK polypeptide substrates include PHAS-1 polypeptide and a peptide substrate from the amino-terminus of p53. All mammalian members of the PIKK family can phosphorylate PHAS-I to varying extents.
  • PHAS-1 can be expressed and isolated as described below or can be obtained from Stratagene (La Jolla, CA) . Although the actual phosphorylation site(s) in PHAS-I are known only for mTOR, this peptide contains the minimal SQ kinase recognition motif which appears to be shared by ATM and DNA-PK.
  • PIKK polypeptides can be isolated using techniques known in the art.
  • the coding sequence of a PIKK polypeptide can be cloned into a vector and expressed in a host cell.
  • Vectors contain suitable regulatory elements to control the expression of the PIKK polypeptide and are typically a plasmid, cosmid, or a viral vector.
  • Various viral vectors that can be utilized include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous Sarcoma Virus.
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Suitable regulatory elements include promoter nucleic acid sequences, enhancer nucleic acid sequences, inducible elements, transcription termination sequences or other control sequences.
  • plasmid vectors contain promoters and control sequences that are derived from species compatible with the host cell . Promoters suitable for use with prokaryotic hosts illustratively include the -lactamase and lactose promoter systems
  • nucleotide sequences are generally known in the art, thereby enabling a skilled worker to ligate them to a polynucleotide encoding the peptide of interest (Siebenlist, et al . , Cell , 20:269 (1980) using linkers or adapters to supply any required restriction sites.
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40, adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and later promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers, et al , Nature, 273:113 (1978).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenaway, et al . , Gene, 18:355-360 (1982). Promoters from the host cell or related species also are useful herein.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman, et al . , J. Biol . Chem. , 255:2073 (1980) or other glycolytic enzymes (Hess, et al . J . Adv . Enzyme Reg.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degraded enzymes associated with nitrogen metabolism, metallothionine, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Yeast enhancers also are advantageously used with yeast promoters .
  • Enhancer elements include the SV40 enhancer on the late side of the replication origin (bp 100-270) , the ⁇ cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers .
  • Expression vectors that contain a gene which operatively encodes a polypeptide and are intended to be introduced into eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription which may affect mRNA expression.
  • selectable markers for mammalian cells include dihydrofolate reductase (DHFR) , thymidine kinase or neomycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin neomycin
  • Plasmid cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • recombinant gene expression vectors may be modified to include genes that operatively encode known reporter polypeptides.
  • the pRSV lac-Z DNA vector described in Norton, . et al., Mol. Cell. Biol . , 5:281 (1985), may produce ⁇ - galactosidase with protein expression.
  • Luciferase and chloramphenicol acetyl transferase (“CAT"; see, e.g., Gorman, et al . , supra , re construction of a pRSV-CAT plasmid) may also be used.
  • Convenient plasmid propagation may be obtained in E. coli (see, e.g., Molecular Cloning: A Laboratory Manual )
  • Expressed polypeptides can be isolated from host cells by conventional chromatographic techniques. For example, gel-filtration, ion-exchange or immunoaffinity chromatography can be used to isolate the proteins. Reverse-phase high performance liquid chromatography (HPLC) , ion-exchange HPLC, size-exclusion HPLC, or hydrophobic- interaction chromatography also can be used. See, for example, "Short Protocols in Molecular Biology", Ed. Ausubel, F.M et al . , Greene Publishing Associates and John Wiley & Sons, 1992, Chapter 10.
  • PIKK polypeptides can be isolated by immunoprecipitation.
  • cultured cells are prepared for lysis by washing in phosphate-buffered saline (PBS) prior to harvesting, then incubating with a lysis buffer in the cold and homogenizing, for example, by briefly sonicating.
  • Lysis buffers can include non- ionic detergents such as Nonidet P-40 (NP-40) , Igepal CA- 630 (Sigma) , chelating agents such as EDTA and EGTA, or high or low salt concentrations.
  • protease inhibitors such as aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride (PMSF) and microcystin.
  • a typical lysis buffer can include, for example, 20 mM HEPES buffer, pH 7.4 , 1.5 mM MgCl 2 , 0.15 M NaCl , 1 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors. After lysis, the lysate is cleared by centrifugation in the cold, i.e.
  • Appropriate assay conditions minimally include a buffer and a phosphate donor such as ATP or GTP .
  • a phosphorylation assay can include approximately 10 mM HEPES, pH 7.4 , 10 mM MgCl 2 , 50 mM NaCl, 10 mM MnC12, 1 mM dithiotheitol (DTT), 10 mM [ 32 P] ⁇ ATP.
  • PIKK polypeptide and about 1 ⁇ g of PHAS-1 can be incubated in the presence of a compound. If a cell extract is the source of PIKK protein, about 100 ⁇ g to about 2000 ⁇ g of total protein is used. Typically, from about 1 nM to about 1 M of a compound can be included in the assay. After an initial screening, the optimal concentration of compound can be refined by using an entire range of concentrations.
  • Kinase assays are typically incubated from about 5 to about 50 minutes in length and are maintained at about 30°C. For example, the incubation can be from about 15 to about 25 minutes. The length of incubation can be adapted to the incubation temperature. For example, if the incubation temperature is about 25°C, incubation time can be increased.
  • Phosphorylation of PHAS-1 polypeptide can be monitored in various ways, for example by electrophoresing the protein mixture through an SDS polyacrylamide gel, which is then dried and exposed to x- ray film. The phosphorylation of PHAS-1 polypeptide is compared with a corresponding assay in the absence of compound. Alternatively, inhibition of phosphorylation can be assessed by measuring the amount of radioactivity incorporated into PHAS-1 polypeptide. In this method, the assay is terminated by addition of an equal volume of 30% acetic acid and then spotted onto P-81 phosphocellulose paper (Whatman LabSales, Hillboro, OR) or other suitable material.
  • P-81 phosphocellulose paper Whatman LabSales, Hillboro, OR
  • the paper is then rinsed for five minutes with 1% phosphoric acid and 10 mM sodium pyrophosphate . After four cycles of rinsing, radioactivity is measured by scintillation counting.
  • the phosphocellulose paper can also be washed with 30% trichloroacetic acid (TCA) for 30 minutes at approximately 65°C, then subsequently washed two to four times with 15% TCA for 15 minutes. After drying, the filters are washed in ethanol , dried and counted in a liquid scintillation counter. Casnellie, J.E., Meth. Enzvmol . , 200:155 (1991).
  • PIKK family members are inhibited by wortmannin in the low nM to the low ⁇ M range. While the mechanism of PIKK inhibition by wortmannin has yet to be defined, it has been extensively studied in PI3K. Wortmannin covalently binds to Lys-802 in the ATP binding domain of PI3K which blocks the binding of ATP in the catalytic cleft. This lysine residue is critical for PI3K kinase activity with a Lys802Arg mutation resulting in a catalytically inactive enzyme.
  • wortmannin may inhibit all PIKK members in a manner similar to PI3K by binding to this crucial lysine residue and thereby acting as a non-competitive inhibitor of ATP binding.
  • Wortmannin inhibition of DNA-PK has been demonstrated.
  • IC 50 s of 250 to 300 nM two other studies have reported IC 50 s of 250 to 300 nM.
  • DNA-PK was immunoprecipitated in a physiologic buffer with mild detergent conditions, which is presumed to preserve the integrity of the DNA-PK heterotrimer . This presumption is supported by the sensitivity of the DNA-PK kinase activity to high salt treatment which has been reported to result in the dissociation of the Ku subunits .
  • Hartley et al used purified DNA-PK CS in their experiments which is more resistant to inhibition by wortmannin than the intact DNA-PK heterotrimer.
  • DNA-PK was not isolated, but assayed directly in cell lysates from SW480 cells treated with wortmannin prior to an in vi tro kinase assay.
  • the DNA-PK 'specific' peptide substrate used in the kinase assay was derived from the amino-terminus of p53 and includes Ser-15.
  • ATM can also phosphorylate Ser-15 of p53.
  • Radiosensitizers achieve synergistic tumor cell killing by either enhancing the level of initial DNA damage caused by radiation or impeding the repair of radiation-induced DNA lesions by inhibiting enzymes involved in DNA metabolism, synthesis and repair.
  • the inhibition of checkpoint PIKKs would not only impair the proximal signaling pathways controlling DNA repair, but also would eliminate crucial cell cycle checkpoints that allow time for DNA repair to occur.
  • the invention also features an antibody or fragment thereof having specific binding affinity for a conjugate including wortmannin or an analog thereof and a polypeptide.
  • the antibody can be polyclonal or monoclonal.
  • the conjugate can include, for example, wortmannin and a PIKK polypeptide, for example, mTOR, DNA-PK, ATM and ATR.
  • wortmannin is conjugated to a polypeptide such as ovalbumin and injected into a host mammal.
  • Various host animals can be immunized by injection of a conjugate including wortmannin and a polypeptide.
  • Host animals include rabbits, chickens, mice, guinea pigs and rats.
  • Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol .
  • Polyclonal antibodies are heterogenous populations of antibody molecules that are contained in the sera of the immunized animals .
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, can be prepared using wortmannin conjugated to a polypeptide such as ovalbumin and standard hybridoma technology.
  • monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler, G. et al . , Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor et al . , Immunology Today, 4:72 (1983); Cole et al . , Proc. Natl. Acad. Sci USA, 80:2026 (1983), and the EBV-hybridoma technique (Cole et al . , "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pp. 77-96 (1983) .
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the monoclonal antibodies of the invention can be cultivated in vi tro and in vivo.
  • Antibody fragments that have specific binding affinity for a conjugate including wortmannin or an analog thereof and a polypeptide can be generated by known techniques.
  • such fragments include but are not limited to F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries can be constructed. See, for example, Huse et al . , Science, 246:1275 (1989).
  • the antibodies or fragments thereof are tested for recognition of wortmannin bound to a polypeptide by Western blotting or immunoprecipitation as described herein.
  • Antibodies of the invention are particularly useful for deconvoluting the methods for identifying inhibitors of PIKK proteins.
  • the antibodies are useful for preventing the re-isolation of wortmannin from the screening assays.
  • the antibodies can be used to identify potential target proteins for wortmannin or analogs thereof in drug- treated cells. 3 . 0 Radioresistant DNA Synthesis
  • the invention also features a method for identifying a compound that induces radioresistant DNA synthesis within cells.
  • the method includes irradiating cells, wherein the cells include an effective amount of the compound, and then measuring radioresistant DNA synthesis of the cell. The presence or absence of radioresistant DNA synthesis is then correlated with the activity of the compound.
  • Radioresistant DNA synthesis is indicative of an abrogation of an S-phase checkpoint and is measured in . the following manner. Cultured cells in exponential growth are harvested and plated in 96 -well plates. After approximately 18 hours, cells are irradiated at room temperature with a sufficient dose rate. The irradiated cells are treated as indicated with a compound for approximately 20 minutes at 37°C prior to pulsing with 3 H- methyl-thymidine for about 40 minutes. Cells are harvested, transferred onto glass filters and lysed in distilled water. Filter-bound radioactivity is then determined by scintillation counting. Compounds identified as inducing radioresistant DNA synthesis can be further assessed to determine if they inhibit the phosphorylation activity of a PIKK polypeptide using the above-described methodology.
  • Example 1 Cell Culture, Antisera and Wortmannin Treatment: Human embryonic kidney 293 cells (HEK 293) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in a 5% C0 2 atmosphere at 37°C. K562 erythroleukemia cells were maintained in RPMI 1640 with 10% FBS. The A549 lung adenocarcinoma cell line was maintained in RPMI 1640 (GibcoBRL) containing 10% FBS. A fibroblast cell line, GM02052, derived from an AT patient, was obtained from the Coriell Institute for Medical Research.
  • FBS fetal bovine serum
  • GM02052 was maintained in minimal essential medium (GibcoBRL) with 15 mM HEPES, 20% FBS, and 2x non-essential and 2x essential amino acids (GibcoBRL) .
  • Wortmannin Sigma was stored at -80°C as a 20 mM stock solution in dimethylsulfoxide (DMSO) and diluted immediately prior to use in either RPMI 1640 for treatment of intact cells or in aqueous buffer. Diluted wortmannin was added directly to the media and incubated at 37°C for one hour prior to lysis for all kinase assays and immunoblotting experiments involving drug treatment of intact cells.
  • Wortmannin-specific monoclonal antibodies were generated in the Mayo Department of Immunology Monoclonal Antibody Facility by immunizing Balb/c mice with wortmannin conjugated to ovalbumin. The resulting hybridoma supernatants were screened for specificity by an enzyme-linked immunosorbent assay with wortmannin conjugated to bovine serum albumin. On the basis of these initial tests, one antibody-producing hybridoma, designed Wm7.1, was selected for further evaluation. Rat brain extracts were treated with drug vehicle or with 1 ⁇ M wortmannin and subjected to SDS-PAGE and immunoblotting with Wm7.1.
  • Wm7.1 specifically reacted with a subset of proteins from wortmannin-treated rat brain extracts, but not with control brain extracts.
  • Wm7.1 used in subsequent experiments was purified from ascites fluid by affinity chromatography over Protein G-Sepharose (Pharmacia) .
  • Polyclonal antibodies were generated against the same immunogen in New Zealand White rabbits. Immunizations, boost and antibody bleeds were performed commercially by Cocalico, Inc. Polyclonal antisera were screened for wortmannin-specific immunoreactivity as described above. Rabbit polyclonal antiserum specific for ATR and antibodies specific for ATM (Ab-3) and DNA-PK (Ab-1) were obtained from Oncogene Research-Calbiochem.
  • mTOR-wt wild type mTOR
  • mTOR-rr rapamycin-resistant mTOR mutant
  • mTOR-kd rapamycin-resistant, kinase-dead mTOR double mutant
  • the cDNAs were tagged at their 5' -termini with nucleotide sequences encoding the six amino acid sequence recognized by monoclonal antibody AU1 (Babco, Richmond, CA) .
  • the PHAS-I expression vector, pCMV4-PHAS-I is described by Lawrence, J. , Adv . Enzyme Regul . , 37:239-267 (1997).
  • Transfections were performed using TransIT polyamino transfection reagent (Pan Vera Corporation, Madison, WI) according to manufacturer's instructions. Approximately 2 ⁇ g pCMV4-PHAS-I and 4 ⁇ g of pcDNA3 or pcDNA3 vectors encoding wild-type or mutant mTOR proteins were used to transfect HEK 293 cells seeded into 60 mm dishes at 5 x 10 5 cells per dish. Alternatively, K562 erythroleukemia cells (10 7 cells per sample) were transfected using a BTX model T820 square- wave electroporator .
  • the cells were mixed with 25 ⁇ g pcDNA3-mTOR or pcDNA3 -mTOR-kd plasmid DNA plus 20 ⁇ g pcDNA3 only as filler DNA. Mock transfections were performed with 45 ⁇ g pcDNA3 only. The cells were electroporated with a single pulse at field settings of 350 V and 10 msec duration. For p38 MAP kinase assays, K562 cells were transfected with FLAG-p38 -encoding plasmid. Control transfections (Co) were performed with pcDNA3 only.
  • lysis buffer 50 mM /3-glycerophosphate, 1.5 mM ethylene glycol-bis (3-aminoethyl ether) N,N,N' ,N' , -tetraacetic acid (EGTA) , pH 7.4, supplemented with 0.5 mM Na 3 V0 4 , 20 nM microcystin-LR, 0.2mM PMSF, 10 ⁇ g/ml leupeptin, 5 ⁇ g/ml aprotinin, 5 ⁇ g/ml pepstatin, and 1% Nonidet P-40 (NP- 40) ) .
  • lysis buffer 50 mM /3-glycerophosphate, 1.5 mM ethylene glycol-bis (3-aminoethyl ether) N,N,N' ,N' , -tetraacetic acid (EGTA) , pH 7.4
  • EGTA ethylene glycol-bis (3-aminoethyl ether) N,
  • cells were osmotically shocked for 10 minutes with 0.4 M sorbitol, then disrupted by sonication in lysis buffer (50 mM Tris HCl, 50 mM ⁇ - glycerophosphate, 100 mM NaCl, pH 7.4, 10% glycerol, 1 mM Na 3 V0 4 , 1 mM DTT, 0.2% Tween-20, and the standard cocktail of phosphatase and protease inhibitors) .
  • Post -nuclear detergent extracts were equalized for protein content and were mixed with reducing SDS-PAGE sample buffer.
  • PHAS-I immunoblots were performed with affinity-purified rabbit polyclonal antibodies generated against a peptide derived from the carboxy-terminus of PHAS-I. Lin, T.A., and Lawrence, J.C., J. Biol. Chem. , 271:30199 (1996).
  • AUl -tagged mTOR polypeptides were blotted with AUl mAb followed by rabbit anti-mouse IgG antibodies (Pierce) .
  • Recombinant p38 was immunoprecipitated with anti-FLAG mAb Ml (Eastman Kodak) .
  • the amount of p38 in the anti-FLAG immunoprecipitates was assessed by immunoblotting with a p38 -specific antibody- (New England Biolabs) . Immunoreactive proteins were detected with horseradish peroxidase coupled to protein A followed by chemiluminescence detection using the ECL reagent (Amersham) .
  • the ATM kinase assay was a modification of a previously described method.
  • A549 cells were lysed for 20 minutes as described above, with the exception that 0.2% Tween-20 was substituted for NP-40 as the detergent and lysates were diluted to 0.5 mg/ml .
  • cells were harvested by trypsinization and lysates were sonicated to maximize the yield of nuclear proteins. Equivalent amounts of protein (0.5 mg) were incubated on ice for 2 hours with ATM-specific antibodies and precipitated with potential sepharose beads.
  • immunoprecipitation Following immunoprecipitation, immune complexes were washed twice in lysis buffer with DTT, phosphatase and protease inhibitors, once in a high salt buffer (0.6 M NaCl/0.1 M Tris, pH 7.4) and once in kinase base buffer. When indicated, immunoprecipitates were incubated with graded concentrations of wortmannin for 30 minutes in the dark at room temperature.
  • the kinase reaction mix was then added to give a final concentration of 10 mM Hepes, 50 mM NaCl, 10 mM MgCl 2 , 10 mM MnCl 2 , 1 mM DTT, 10 mM [ ⁇ - 32 P]ATP (specific activity: 50 Ci/mmol; ICN) and 25 ng/ ⁇ l PHAS-I (Stratagene) in a total volume of 40 ⁇ l .
  • Kinase reactions were incubated at 30°C for 20 minutes. The phosphorylation of appropriate substrates by each kinase followed linear kinetics under the reaction conditions described.
  • the DNA-PK kinase assay was similar to the ATM kinase assay with the exception that the lysates were sonicated prior to clearing, and the protein concentration was adjusted to 0.75 mg/ml prior to immunoprecipitation with anti-DNA-PK antibodies.
  • the immune complexes were washed twice with lysis buffer and twice with kinase base buffer prior to the kinase reaction.
  • the kinase reaction conditions were identical to those described above with the exceptions that the reaction time was 15 minutes at 30°C and the substrate was a 15 amino acid DNA-PK peptide substrate derived from p53 (Promega, catalog #V5811) .
  • the DNA-PK substrate was used at a concentration of 250 ng/ ⁇ l per reaction. Samples spotted onto P-81 paper were rinsed with four 5 minute cycles in 15% acetic acid/10 mM sodium pyrophosphate prior to liquid scintillation counting.
  • ATR kinase assay was identical to the ATM kinase assay, except the kinase reaction was run for 15 minutes at 30°C and was stopped by the addition of an equal volume of 4x SDS-PAGE loading buffer. Samples were separated by SDS-PAGE, and the proteins were transferred onto Immobilon-PVDF membranes. The incorporation of 32 P into PHAS-I was quantitated with an AMBIS 4000 imaging system.
  • the protein kinase activity of native mTOR was assayed by immunoprecipitation of this protein from rat brain extracts. Sabers, C.J. et al . , J. Biol . Chem. ,
  • the extracts were supplemented with 1 mM DTT, 0.2 ⁇ M microcystin LR, and 10 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin.
  • the extracts (1 mg protein per sample) were mixed with affinity-purified rabbit polyclonal antibodies directed against a peptide sequence corresponding to residues 2432-2449 of mTOR.
  • the immune complexes were precipitated with 15 ⁇ l protein A-Sepharose beads and the immunoprecipitates were washed two times in immunoprecipitation buffer (50 mM Tris HCl, 50 mM ( ⁇ -glycerophosphate, 100 mM NaCl, pH 7.4, 10% glycerol, 20 nM microcystin LR, 10 ⁇ g/ml leupeptin, 5 ⁇ g/ml aprotinin, 5 ⁇ g/ml pepstatin A, and 600 ⁇ M PMSF) .
  • the precipitates were washed one time in high-salt buffer (100 mM Tris HCl, pH 7.4, 500 mM LiCl), followed by two washes in kinase buffer.
  • the immunoprecipitates were pretreated with 25 ⁇ l of kinase buffer containing 10 ⁇ g glutathione S- transferase (GST) -FKBP12 fusion protein and, where indicated, 10 ⁇ M rapamycin or 100 ⁇ M FK506.
  • Parallel samples were pretreated in a similar fashion with various concentrations of wortmannin. After 40 minutes at room temperature, the beads were washed two times in kinase buffer.
  • p38 kinase activity toward PHAS-I was assayed as described in Karnitz, L.M. et al . , Mol . Cell . Biol . ,
  • the amount of p38 in the anti-FLAG immunoprecipitates was assessed by immunoblotting with a p38 -specific antibody (New England Biolabs) .
  • the immunoprecipitates were treated with GST-FKBP12 plus rapamycin (F*R) or FK506 (F*»FK) , or with 1 ⁇ M wortmannin (Wm) .
  • Immune complex kinase assays were performed with PHAS-I as the substrate. 32 P-labelled PHAS-I was detected by autoradiography. Expression of recombinant mTOR proteins was detected by immunoblotting with AUl monoclonal antibody.
  • the immunoprecipitated FLAG-p38 was detected by immunoblotting with a p38 -specific antibody.
  • Clonogenic Assay The effect of wortmannin on the radiosensitivity of A549 cells was assessed with a clonogenic assay.
  • A549 cells in log-phase were harvested, resuspended in fresh growth medium and plated in triplicate in 60 mm dishes at cell concentrations estimated to result in 20-100 colonies per dish following treatment. Four hours after plating, cells were irradiated at room temperature with 137 Cs source at a dose rate of 6.4 Gy/min. Wortmannin was added to the indicated samples immediately after irradiation.
  • the final concentration of the drug solvent did not exceed 0.1% (vol/vol) , and this solvent concentration had no effect on either the clonogenicity or radiosensitivity of the A549 cells.
  • Cells were cultured for two weeks prior to fixation and staining with Coomassie Blue. Only colonies with greater than 50 cells were scored. Data shown represent the mean of four independent experiments with error bars representing the standard error of the mean (SEM) .
  • the fixed cells were resuspended in PBS containing 20 ⁇ g/ml propidium iodide and 100 ⁇ g/ml boiled RNaseA, and were incubated from 30 minutes at 37°C for -30 minutes prior to flow cytometric analysis on a Becton- Dickinson FACScan. Twenty-thousand ungated events were collected. Cell cycle distribution was determined with the ModFit software package (Verity) after excluding doublets and clumps by gating on the DNA pulse height versus pulse area displays.
  • Radioresistant DNA Synthesis A549 cells in exponential growth were harvested and plated in 96 -well plates (10,000 cells per well in 0.1 ml) . Each treatment condition was tested in 6 replicate wells. After 18 hours, the cells irradiated at room temperature at a dose rate of 6.4 Gy/min. The irradiated cells were treated as indicated with wortmannin for 20 minutes at 37°C prior to the pulsing with 2 ⁇ Ci per well 3 H-methyl-thymidine (specific activity: 5 Ci/mmol; Amersham) for 40 minutes. Cells were harvested by trypsinization, transferred onto glass filters and lysed in distilled water. Filter-bound radioactivity was determined by scintillation counting. Data shown represent the mean of three independent experiments with error bars representing the standard error of the mean (SEM) .
  • the sensitizer enhancement ratio (SER) for each survival curve was calculated as the ratio of radiation doses, that resulted in 10% survival of the cells in the absence or presence of wortmannin.
  • SER sensitizer enhancement ratio
  • Example 2 Effect of a rapamycin-resistant mTOR mutant on insulin-stimulated PHAS-I phosphorylation: To test whether or not mTOR is an upstream component of the PHAS-I phosphorylation pathway, HEK 293 cells were cotransfected with expression vectors encoding rat PHAS-I and either wild-type (mTOR-wt) or a rapamycin-resistant mTOR (mTOR-rr) mutant that contains a single amino acid substitution (S 2035 ⁇ I 2035 ) within the FKBP12*rapamycin- binding (FRB) domain. Chen, J. et al . , Proc. Natl. Acad. Sci. , 92:4947 (1995).
  • Rapamycin treatment inhibited the insulin-stimulated phosphorylation of PHAS- I in mock-transfected or mTOR-wt -transfected 293 cells, as indicated by the decrease in immunoreactivity of the most highly phosphorylated form of PHAS-I, and by the appearance of hypophosphorylated PHAS-I. In contrast, rapamycin caused no detectable decrease in PHAS-I phosphorylation in mTOR-rr-expressing 293 cells.
  • rapamycin-sensitive activity of mTOR is required for insulin-stimulated PHAS-I phosphorylation in intact cells.
  • FKBP12 "rapamycin to immune complex kinase assays containing mTOR inhibits the autophosphorylating activity of this kinase in vi tro . Brown, E. J. et al . , Nature , 377:442 (1995). Therefore, a functional mTOR catalytic domain may be needed for the phosphorylation of mTOR in intact cells.
  • wortmannin has been widely used as an inhibitor of PI 3 -kinase, this drug also irreversibly inhibits the autophosphorylating activity of mTOR. Brunn, G.J. et al . , EMBO J . , 15:5256 (1996).
  • concentration of wortmannin required to inhibit mTOR autokinase activity by 50% (IC 50 ) was approximately 200 nM, and this activity was maximally inhibited by 1 ⁇ M wortmannin.
  • wortmannin targets the ATP-binding site of mTOR, rather than the FRB domain, the drug can nondiscriminately block the kinase activities of both wild-type mTOR and the rr-mTOR mutant. This prediction was borne out by the finding that 1 ⁇ M wortmannin strongly inhibited the phosphorylation of PHAS-I in insulin-stimulated 293 cells transfected with either mTOR-wt or the mTOR-rr mutant. In contrast, pretreatment of the transfected cells with 0.1 ⁇ M wortmannin, a drug concentration sufficient to fully inhibit endogenous PI 3 -kinase activity (Karnitz, L.M. et al . , Mol . Cell Biol .
  • Example 3 Role of mTOR kinase activity in PHAS-I phosphorylation within intact cells Additional genetic evidence for the involvement of the mTOR kinase domain in PHAS-I phosphorylation was supplied by transfection experiments with a kinase-dead version of the mTOR-rr mutant.
  • the mTOR-rr/kd double mutant contains the rapamycin resistance-conferring S 2035 ⁇ I substitution described above, together with a D 2338 ⁇ A substitution that inactivates the mTOR kinase domain (Brunn, G.J. et al . , EMBO J . , 15:5256 (1996)).
  • the 293 cells were cotransfected with PHAS-I and a mTOR-rr/kd expression vector.
  • Control cell populations were cotransfected with pcDNA3 only, or with the pcDNA3 vector encoding the catalytically-active, mTOR-rr mutant.
  • Rapamycin largely eliminates the contribution of endogenous mTOR to the phosphorylation of the transfected PHAS-I protein, and allows a direct comparison of the capacities of the catalytically-active and -inactive mTOR mutants to support this response in intact cells.
  • Example 4 Phosphorylation of PHAS-I by immunopurified native mTOR
  • mTOR displays serine-specific autokinase activity in immune complex kinase assays. Brunn, G.J. et al . , EMBO J . , 15:5256 (1996); Brown, E.J. et al . , Nature , 377:442 (1995).
  • immune complex kinase reactions were performed with mTOR immunoprecipitates from rat brain extracts and recombinant PHAS-I as the substrate.
  • the recombinant proteins were prepared by transfecting K562 erythroleukemia cells with either the mTOR-wt expression plasmid or a plasmid encoding a kinase-dead mutant mTOR (mTOR-kd) bearing the inactivating D 2338 ⁇ A substitution in the catalytic domain. After transfection, the AUl-tagged mTOR-wt and mTOR-kd proteins were immunoprecipitated from K562 cell extracts with AUl mAb.
  • K562 cells were transfected with an expression vector encoding a FLAG epitope-tagged version of the p38 MAP kinase, which, like ERK1 and ERK 2 (Lin, T.A. et al . , Science, 266:653 (1994); Haystead, T.A.J. et al . , J. Biol . Chem. , 269:23185 (1994)), phosphorylates PHAS-I in vi tro .
  • the FLAG-tagged p38 was immunoprecipitated from cell extracts, and the immunoprecipitates were treated with FKBP12*rapamycin, FKBP12"FK506, or wortmannin prior to the immune complex kinase assay. Although recombinant p38 readily catalyzed the phosphorylation of PHAS-I in vi tro, p38 kinase activity was not inhibited by FKBP12 "rapamycin or 1 ⁇ M wortmannin. These results argue that both FKBP12 "rapamycin and wortmannin inhibit the PHAS-I kinase activity present in mTOR immunoprecipitates by selectively and directly inhibiting the catalytic activity of mTOR itself.
  • Example 5 Phosphorylation of PHAS-I by recombinant, i munopurified mTOR-wt, mTOR-kd protein and FLAG-tagged p38
  • the effect of mTOR-mediated phosphorylation on the eIF-4E binding activity of PHAS-I was examined by Far Western analysis.
  • Recombinant PHAS-I was incubated with control antibody or anti -mTOR antibody immunoprecipitates from rat brain extracts .
  • Immunoblot analysis of the kinase reaction products demonstrated that exposure to mTOR generated 3 electrophoretically distinct forms of phosphorylated PHAS-I.
  • the same sample lanes were probed with 32 P-labeled eIF-4E in a Far Western blot. Phosphorylation of PHAS-I by mTOR strongly inhibited the eIF-4E-binding activity of PHAS-I in this assay.
  • Example 6 ATM Kinase Assay; Before assessing the sensitivity of the various PIKKs to wortmannin, a series of preliminary experiments were performed to document the specificity of the ATM immune complex kinase assay. A549 cell extracts were immunoprecipitated with preimmune serum or with ATM-specific antibodies as described in Example 1. Samples were then pretreated for 30 minutes at room temperature with 1 ⁇ m wortmannin. Immune complex kinase reactions were performed as described in Example 1 and the reaction products were separated by SDS-PAGE. The amount of ⁇ 2 ⁇ > i incorporation into PHAS-1 substrate was measured with a Molecular Dynamics Phosphorimager System and ImageQuant Software. The results of this experiment are depicted in Table 2. ATM immunoprecipitates from A549 cells phosphorylated PHAS-I to an approximately 25-fold higher level than that catalyzed by control antibody immunoprecipitates.
  • Extracts prepared for an ATM-negative fibroblast line were immunoprecipitated with anti-ATM antibodies.
  • the lack of a detectable kinase activity in ATM immunoprecipitates from the AT fibroblast line GM02052 demonstrated that the kinase activity observed was from ATM or a kinase physically associated with ATM ( Figure 2A) .
  • Q ⁇ -ATM immunoprecipitates were immunoblotted for DNA-PK or ATR. No DNA-PK or ATR immunoreactivity was detected in the ATM immunoprecipitates.
  • the DNA-PK and ATR immunoprecipitates were similarly free of contamination by other PIKK family members.
  • Example 7 - Wortmannin Binds PIKK Protein; To determine the relative potency of wortmannin as an inhibitor of the catalytic activities of DNA-PK, ATM and ATR, appropriate PIKK immunoprecipitates were treated with various concentrations of wortmannin prior to the immune complex kinase assays. As seen in Figure 3A-C, the sensitivities of the PIKKs to wortmannin varied over a wide range, with an IC 50 of 150 nM for ATM, 16 nM for DNA-PK and 1.8 ⁇ M for ATR, respectively.
  • wortmannin may also inhibit PIKKs by a similar mechanism.
  • a monoclonal antibody (Wm7.1) that specifically recognizes wortmannin bound to protein was developed. Using this antibody, wortmannin binding to DNA-PK, ATM, and ATR was detected by immunoblotting PIKK immunoprecipitates which had been incubated with various concentrations of wortmannin. Moreover, the concentrations of wortmannin at which binding was detected correspond with those necessary for kinase inhibition.
  • Example 9 Radiosensitization of Cell Lines Wortmannin has been shown to sensitize a number of human tumor cell lines to radiation.
  • radiosensitization by wortmannin follows a steep dose-response relationship (Figure 5) with no radiosensitization seen at 2 ⁇ M and significant radiosensitization at 20 ⁇ M wortmannin.
  • Wortmannin not only increased the sensitivity of A549 cells to radiation, but it also significantly changed the shape of the radiation survival curve.
  • wortmannin Compared to the DMSO control, 20 ⁇ M wortmannin resulted in an eight-fold increase in the initial slope, a , as described by the linear quadratic equation. This increase in a is suggestive of inhibition DNA repair processes that result in the conversion of potentially repairable DNA lesions into non-repairable lesions. Also consistent with inhibition of repair, wortmannin treatment also resulted in a significant flattening of the shoulder of the radiation survival curve which is reflected by an increase in the a/ ⁇ ratio from 2.0 to 19.5 Gy. The / ⁇ ratio is the dose at which the linear ( ⁇ D) and quadratic ( / SD 2 ) contribution to cell killing are equal and describes how quickly a survival curve begins to bend.
  • ATM deficient, but not DNA-PK deficient cells have a defective S-phase checkpoint (s) which results in one of the hallmark characteristics of AT cells: radioresistant DNA synthesis (RDS) .
  • RDS radioresistant DNA synthesis

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Abstract

Cette invention concerne un procédé permettant d'identifier un composé qui inhibe l'activité de phosphorylation d'un polypeptide de kinase apparenté au phosphoinositide 3-kinase; ainsi que des anticorps présentant une affinité de liaison spécifique pour un conjugué comprenant du wortmannin.
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US7049313B2 (en) 2002-02-25 2006-05-23 Kudos Pharmaceuticals Ltd. ATM inhibitors
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US9644037B2 (en) 2013-10-18 2017-05-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibodies that specifically bind ataxia telangiectasia-mutated and RAD3-related kinase phosphorylated at position 1989 and their use

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BRUNN, GREGORY J. ET AL: "Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3- kinase inhibitors, wortmannin and LY294002." EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, (1996) VOL. 15, NO. 19, PP. 5256-5267., XP000891362 *
BRUNN, GREGORY J. ET AL: "Phosphorylation of the translational repressor PHAS -I by the mammalian target of rapamycin." SCIENCE (WASHINGTON D C), (1997) VOL. 277, NO. 5322, PP. 99-101., XP000891365 *
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