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WO2004003222A2 - Procede de recherche systematique d'inhibiteurs de kinases - Google Patents

Procede de recherche systematique d'inhibiteurs de kinases Download PDF

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Publication number
WO2004003222A2
WO2004003222A2 PCT/US2003/020280 US0320280W WO2004003222A2 WO 2004003222 A2 WO2004003222 A2 WO 2004003222A2 US 0320280 W US0320280 W US 0320280W WO 2004003222 A2 WO2004003222 A2 WO 2004003222A2
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WIPO (PCT)
Prior art keywords
protein
phosphorylation
heterotypic
complexes
phospho
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Ceased
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PCT/US2003/020280
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English (en)
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WO2004003222A3 (fr
Inventor
Emanuel Petricoin Iii
Lance Liotta
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Priority to AU2003245722A priority Critical patent/AU2003245722A1/en
Publication of WO2004003222A2 publication Critical patent/WO2004003222A2/fr
Publication of WO2004003222A3 publication Critical patent/WO2004003222A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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

Definitions

  • the present invention is directed to methods for screening for kinase inhibitors that are useful in disease therapy.
  • Protein phosphorylation is a critical chemical reaction that plays a major role in normal cellular physiology. Protein signal events can be mediated by protein complex formation followed by phosphorylation or dephosphorylation of binding partners. Consequently, protein kinases, and phosphorylation of protein substrates are an important category of therapeutic targets. Many successful pharmaceuticals target kinases or phosphoproteins (Moasser MM, Basso A, and Averbuch SD, Rosen N. The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells.
  • Iressa The tyrosine kinase inhibitor ZD1839
  • a current weakness in the field of kinase drug screening is the lack of knowledge concerning the in-vivo cellular context of the inhibited target.
  • Thiesing, J. T., Ohno-Jones, S., Kolibaba, K. S., and Druker, B. J. (2000) Efficacy of STI571 , an abl tyrosine kinase inhibitor, in conjunction with other antiieukemic agents against bcr-abl-positive cells. Blood. 96, 3195-3199.
  • Phosphorylation events are influenced by upstream protein signal events and in turn mediate their effect through downstream interconnections.
  • high throughput screening for kinase inhibitors is most often done using isolated targets in silico. Consequently the pathway or network interconnections important in the physiologic context are not observable.
  • Such contextual dependent events can have a profound influence on harmful toxicity and the suitability of specific combinatorial therapy.
  • Tyrosine kinase inhibitors from rational design to clinical trials. Med Res Rev. 21 , 499-512. Zwick, E., Bange, J., and Ullrich, A. (2002) Receptor tyrosine kinases as targets for anticancer drugs. Trends Mol Med. 8, 17-23.). These assays are limited by the purified substrates available and do not give a broad view of the phosphoproteomic network. Moreover, drugs active against a single kinase may cross-react with related known or unknown kinases. This can have a profound impact on the therapeutic effectiveness.
  • FIGURE 1 is a schematic drawing showing capture of heterotypic protein complex consisting of multiple different proteins A, B, C, D and E having two classes of protein phosphorylation of amino acids. The example shown is serine phosphorylation and tyrosine phosphorylation.
  • An object goal of the invention is to provide a means to obtain a multiplexed readout for the state of many signaling pathway intersections or junctures at once.
  • the invention is based in part on employing phospho-specific antibodies that act as a surrogate detectors for kinase activity acting on a given substrate.
  • Signaling pathways are comprised of coalescing complexes of serine, threonine, and tyrosine phosphorylated proteins.
  • the invention is further based on the concept that complexes comprised of different types of proteins, some of which are phosphorylated on tyrosine residues, and some of which are phosphorylated on the serine residues form the key junctures of cellular signaling networks.
  • the identification of such heterotypic complexes before and after treatment with an agent which perturbs one or more interconnection in the network of cellular signaling proteins will provide direct information about pathway interconnections related to the perturbed target.
  • the invention provides a means of obtaining this information which is not possible to obtain by prior methods such as Chin et al U.S. Patent No. 6,197,599 which a) separate or denature protein binding partners and thereby destroy the information content embodied in the interacting clusters of phosphorylated proteins, or which examine the kinase activity of the lysed cellular enzyme instead of the phosphorylated substrate and the binding partners which make up intersections of networks and circuits. Extracting the kinase from the cell removes the kinase from the context of its neighboring cellular inhibitors and substrates, and destroys information about the past effect of a treatment on networks influenced by the target kinase.
  • the present invention overcomes the disadvantages of the prior technique by examining specifically the native previously formed and intact heterotypic phosphorylated complexes of proteins that reflect actual signal pathways and networks that were actually in-use prior to lysis of the cell.
  • the present invention employs high affinity anti-phosphotyrosine, and/or anti- phosphoserine, and/or anti-phosphothreonine antibodies as a reporter for protein complexes that are captured on a parallel multiplex array or other suitable means ( Figure 1 ).
  • the bait on the capture surface is an antibody against a specific phosphorylation event modifying an amino acid, which is not detected by the reporter antibody.
  • An example bait would be an antibody specifically recognizing the phospho-serine modification of AKT kinase at serine (position 473).
  • the bait antibody can be an immobilized monoclonal or polyclonal antibody against a specific serine phosphorylation site on a specific protein, or it could be any type of ligand with said specificity.
  • a reporter antibody is then used to detect a different class of phosphorylation events on the captured protein complex.
  • Rabbit polyclonal anti- phosphotyrosine 4G10 for example, is a suitable probe for the existence of a bound complex that contains both serine and tyrosine phosphorylated proteins.
  • Table 1 lists those specific substrates (phospho-specific events) that are monitored simultaneously when human peripheral blood lymphocytes are incubated with interferon alfa 2a (1000 u/ml) for 30 minutes with interleukin2 (50 ng/ml) and the array is incubated with a monoclonal 4G10 anti-phosphotyrosine antibody reporter.
  • Each phospho-specific event is monitored on the same array using a polyclonal antibody that recognizes each substrate only when it is specifically phosphorylated at that site and a monoclonal anti-phosphotyrosine antibody that recognizes the heterotypic complex formed when those pathways are activated.
  • a further advantage of the invention is that it provides a method to assess the state of hundreds of signaling pathways simultaneously, even with small number of capture and reporter antibodies. This is because a large plurality of different combinations and permutations of heterotypic protein complexes signal nodes can be identified with a limited set of bait or capture ligands. The change in the number and composition of the heterotypic complexes will be a direct reflection of the pertubations in the proteomic network caused by the treatment effect.
  • signaling complexes are always comprised of coalescing proteins that are serine, tyrosine, and/or threonine phosphorylated.
  • the protein captured by the bait or capture ligand is not necessarily tyrosine phosphorylated, but it can be associated with a protein that has undergone tyrosine phosphorylation. The combination will be detected and read out. This association will only occur if the initial protein is activated and forming a complex with its binding partners. The readout with the anti- phosphotyrosine antibody can be accomplished even if the epitope is masked by the complex itself.
  • the invention will provide utility for mapping cellular protein networks by combining the survey of heterotypic phospho-complexes with the use of tyrosine and serine phosphatase inhibitors, such as a combination of sodium pervanadate (tyrosine phosphatase inhibitor) and calyculin A (serine phosphatase inhibitor).
  • tyrosine and serine phosphatase inhibitors such as a combination of sodium pervanadate (tyrosine phosphatase inhibitor) and calyculin A (serine phosphatase inhibitor).
  • tyrosine phosphatase inhibitors sodium pervanadate
  • calyculin A serine phosphatase inhibitor
  • the invention becomes useful for drug screening when the survey of heterotypic phosphoproteins is combined with the phosphatase inhibitor treatment in the presence or the absence of a specific kinase inhibitor, or drug.
  • a specific kinase inhibitor or drug.
  • By pre-treating cells with, for example, a MEK kinase inhibitor, and then shutting off phosphatases activity using sodium pervanadate and calyculin A all signaling pathways downstream of ERK kinase will be seen as a blank spot on the array. In this way, one can elucidate the entire signaling pathway in the cell that is affected by the drug, and identify those pathways that are not affected and therefore, unlinked to the pathway.
  • the invention will uniquely and simultaneously generate at least two types of critical information:
  • the information obtained about interconnections and pathways is not limited to events in the cellular cytoplasm, but extends in to the nucleus and out to the cell surface. This is because protein phosphorylation events modulate transcription factor binding to DNA or RNA.
  • isolating the nuclear, cytoplasmic, and membrane compartment of the cell it is possible to use the invention method to characterize phospho-networks localized to specific cellular compartments. What following are a series of examples which are present to more particularly describe the invention without limiting the same.
  • a protein planar array is prepared using dozens or hundreds of immobilized antibodies that recognize only the specific phosphorylated forms of a specific protein (e.g. an antibody that only recognizes AKT kinase when it is phosphorylated on the serine at amino acid 473).
  • the array is incubated with a whole cell lysate under native conditions. During the incubation, protein complexes, reflecting the state of cellular signaling and recapitulating the "in use" information archive that occurred within the cell of interest, will bind to specific regions on the array depending on whether or not these complexes contain the phosphorylated, and thus activated, protein of interest.
  • the detection of that heterotypic complex containing proteins that are both serine, threonine, and tyrosine phosphorylated occurs through the use of a labeled second antibody that recognizes any protein that is tyrosine phosphorylated.
  • the label can be detected by colorimetric, fluorescent, or chemilumenescent means.
  • a further embodiment would be to use as a reporter an antibody that recognizes any and all proteins that are serine or threonine phosphorylated.
  • the bait antibody will only bind complexes that contain, for example, only phosphorylated AKT kinase at amino acid serine position 473, the anti-phosphotyrosine readout occurs since protein complexes are heterotypic in nature, containing both serine, threonine and tyrosine phosphorylations. In this way, heterotypic phosphoprotein complexes can be characterized and their constituents identified.
  • the arrays can be queried with lysates of cells that have been perturbed by a treatment agent, for example, a drug that is a putative PI3 kinase inhibitor.
  • the resultant heterotypic complex containing serine 473 phosphorylated AKT that forms under untreated conditions will be disrupted since the complex will not form as the kinase activity is blocked. This will result in a blank spot on the array where the anti-phospho serine 473 antibody is located.
  • the heterotypic complexes act as a sentinel for the state of the protein signal network and identify the perturbation by a treatment agent.
  • hundreds of specific "nodes" in the signaling network can be analyzed concomitantly at a time using one type of reporter molecule such as an anti-phophotyrosine antibody. Treatment agent affects on other targets and other pathways can thus be discovered and identified without any prior knowledge of their involvement.
  • Example 2 In another example, a protein array consisting of dozens or hundreds of immobilized antibodies that recognize only the specific phosphorylated forms of a specific protein (e.g. an antibody that only recognizes AKT kinase when it is phosphorylated on the threonine at amino acid 473) is incubated with a cellular whole cell lysate under native conditions. During the incubation, protein complexes, reflecting the state of cellular signaling that occurred within the cell of interest, will bind to specific regions on the array depending on whether or not these complexes contain the phosphorylated, and thus activated protein of interest.
  • a specific protein e.g. an antibody that only recognizes AKT kinase when it is phosphorylated on the threonine at amino acid 473
  • protein complexes reflecting the state of cellular signaling that occurred within the cell of interest, will bind to specific regions on the array depending on whether or not these complexes contain the phosphorylated, and thus activated protein of interest.
  • the array After washing the array free of non-specific protein interactions using, for example, phosphate buffered saline, the array is subjected to denaturing conditions, such as transient heating at 65 degrees C for 10 minutes. All protein complexes specifically captured at any given site will be unfolded and will adhere to the surface of the array. This unfolding can allow for sites, or epitopes on proteins that may be hidden in the heterotypic complex to now be addressable by a second molecule such as anti-phosphotyrosine.
  • a further extension of the art would be to incubate the now denatured array with a third or additional labeled antibody that recognizes, for example, ERK kinase when it is tyrosine phosphorylated.
  • the experimentalist can specifically identify the proteins that ERK is specifically interacting with in the heterotypic complex.
  • the arrays can be queried with a perturbed system as in example 1 except in this iteration, affect of the treatment agent on the ability of any given molecule (e.g. ERK) to interact in a signaling network can be specifically identified and its interacting partners immediately identified without any prior knowledge that they may occur.

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Abstract

La présente invention concerne un procédé permettant de détecter l'effet d'un agent thérapeutique ou prophylactique sur le réseau de signalisation cellulaire dont la médiation fait intervenir des événements de phosphorylation. Un tel procédé comprend plusieurs opérations. On commence par préparer un lysat cellulaire natif avant et après traitement avec un inhibiteur de phosphatase avec le candidat médicament, auquel cas le lysat considéré retient au moins un complexe protéine-protéine. On réalise ensuite une caractérisation du nombre et de la composition des complexes phospho-protéiques hétérotypiques qui conviennent au moins deux types différents de différents acides phospho-aminés.
PCT/US2003/020280 2002-06-26 2003-06-26 Procede de recherche systematique d'inhibiteurs de kinases Ceased WO2004003222A2 (fr)

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AU2003245722A AU2003245722A1 (en) 2002-06-26 2003-06-26 Method for screening for kinase inhibitors

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US39168202P 2002-06-26 2002-06-26
US60/391,682 2002-06-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005020754B3 (de) * 2005-05-02 2007-01-11 Altana Pharma Ag Zelluläres Assayverfahren zur Identifizierung von PKCtheta-Inhibitoren

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* Cited by examiner, † Cited by third party
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US6630312B2 (en) * 1999-08-10 2003-10-07 Joslin Diabetes Center, Inc. Method for identifying compounds for treatment of insulin resistance
US20010031469A1 (en) * 2000-01-03 2001-10-18 Stefano Volinia Methods for the detection of modified peptides, proteins and other molecules
AU2001233276A1 (en) * 2000-02-03 2001-08-14 Immunomatrix Inc. Method and apparatus for signal transduction pathway profiling

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005020754B3 (de) * 2005-05-02 2007-01-11 Altana Pharma Ag Zelluläres Assayverfahren zur Identifizierung von PKCtheta-Inhibitoren

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AU2003245722A8 (en) 2004-01-19
WO2004003222A3 (fr) 2004-04-22

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