WO2000000584A2 - Reactifs immobilises pour dosages de kinase - Google Patents

Reactifs immobilises pour dosages de kinase Download PDF

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Publication number
WO2000000584A2
WO2000000584A2 PCT/US1999/014566 US9914566W WO0000584A2 WO 2000000584 A2 WO2000000584 A2 WO 2000000584A2 US 9914566 W US9914566 W US 9914566W WO 0000584 A2 WO0000584 A2 WO 0000584A2
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phosphatidylinositol
composition
pip
bisphosphate
phosphate
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PCT/US1999/014566
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English (en)
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Glenn D. Prestwich
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University Of Utah Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/653—Five-membered rings
    • C07F9/65306—Five-membered rings containing two nitrogen atoms
    • C07F9/65312—Five-membered rings containing two nitrogen atoms having the two nitrogen atoms in positions 1 and 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
    • 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

Definitions

  • This invention relates to in vitro assays of kinase activity. More particularly, the invention relates to scintillant-tethered phosphoinositide polyphosphates (PIP n s). methods of making thereof, and methods of use thereof.
  • PIP n s scintillant-tethered phosphoinositide polyphosphates
  • Phosphoinositide (PI) metabolism plays an important role in the control of diverse cellular processes such as proliferation and differentiation, apoptosis. and control of cell shape and cell migration.
  • P. DeCamilli et al. 271 Science 1533-1539 (1996); P. Janmey. 2
  • PI3K Inhibitors of PI3K. such as wortmannin, G.D. Prestwich, 7 Chemtracts-Org. Chem. 301-305 (1994); R. Yao & G.M. Cooper. 267 Science 2003-2006 (1995), and LY294002, have further confirmed these distinct cellular functions. C.L. Carpenter & L.C. Cantley, 1288 Biochim. Biophys. Acta Ml 1-M16 (1996). PI3K may also be involved in mediating several insulin-regulated metabolic pathways. P.R. Shepherd et al., 17 J. Mol. Endocrinol. 175-184 (1996), leading to diabetes, including glucose uptake, antilipolysis, glycogen synthesis, and the suppression of hepatic gluconeogenesis. D.J. Withers et al., 391 Nature 900-904 (1998).
  • the PI3K family of lipid kinases includes PI3K ⁇ . a G protein-activated PI3K. and the yeast PI3K encoded by the Vps34 gene. PI3Ks are responsible for the phosphorylation of inositol lipids via transfer of the ⁇ phosphate of ATP to the D-3 hydroxyl position. B.
  • PI3K is a heterodimeric protein consisting of an 85 kDa regulatory subunit (p85) that contains two Src homology-2 (SH2) domains and an SH3 domain, and a 1 10 kDa catalytic subunit (pi 10) in which kinase activity resides.
  • p85 85 kDa regulatory subunit
  • SH2 Src homology-2
  • pi 1 10 kDa catalytic subunit
  • yeast Vps34p kinase has a substrate specificity restricted to PI. M. Susa et al., 267 J. Biol. Chem. 22951- 22956 (1992); B. Vanhaesebroeck et al., 27 Cancer Surv. 249-270 (1996).
  • PI3K As various growth factors and cytokines bind to their cell-surface receptors. intracellular PI3K becomes activated. At the membrane, PI3K converts PI(4,5)P 2 to
  • PI(3,4,5)P 3 as well as generating PI(3,4)P 2 from inositol 5'-polyphosphate (FIG. 2).
  • PI(3,5)P 2 phosphatidylinositol 3,5-bisphosphate
  • J. Peng & G.D. Prestwich, Tetrahedron Lett. (1998); C.C. Whiteford et al., 323 J. Biochem. 597-601 (1997) was identified with unknown biological relevance.
  • Stokoe et al. 277 Science 567-570 (1997), act as second messenger molecules and induce activity of multiple downstream effectors that include SH2 and pleckstrin homology (PH) domains of serine/threonine and tyrosine kinases as well as various cytoskeletal proteins.
  • PH pleckstrin homology
  • BAD promotes cell death by blocking the activity of the cell survival factors, Bax-Bcl-x L by displacing Bax and binding to Bcl-x L .
  • Bcl-x L becomes capable of promoting cell survival by repressing apoptosis pathways involving the activity of Apaf-1 , cytochrome c (cyt.c), and the caspase protease cascade.
  • T.F. Franke & L.C. Cantle 390 Nature 116-117 (1997).
  • compositions and methods that would expedite the processing of samples in kinase assays and would be more amenable to automation would be significant advancements in the art.
  • Such assays would require no addition of scintillation cocktail and would be less problematic and less expensive for radiowaste disposal.
  • providing scintillant-tethered phosphoinositide polyphosphates and methods of making and using thereof would be significant advancements in the art.
  • PIP n is a phosphoinositide polyphosphate, L in a linker moiety, and S is a scintillant.
  • PIP n is a member selected from the group consisting of PI. PI(3)P. PI(4)P, PI(5)P, PI(4,5)P 2 , PI(3,4)P 2 , PI(3,5)P 2 , and PI(3,4,5)P 3 .
  • L is a member selected from the group consisting of succinimide and poly(ethylene glycol) linkers.
  • S is 2-(4-amino- methylphenyl)-5-(4-biphenyl)-1.3.4-oxadiazole (i.e., amino-PBD).
  • Another aspect of the invention comprises a composition represented by the formula
  • PIP n is a phosphoinositide polyphosphate, L in a linker moiety, S is a scintillant, and Matrix is a solid support.
  • PIP n is a member selected from the group consisting of PI, PI(3)P, PI(4)P, PI(5)P. PI(4,5)P,, PI(3,4)P 2 , PI(3,5)P 2 , and PI(3,4,5)P 3 .
  • L is a member selected from the group consisting of succinimide and poly(ethylene glycol) linkers.
  • S is 2- (4-amino-methylphenyl)-5-(4-biphenyl)-l,3,4-oxadiazole (i.e., amino-PBD).
  • the Matrix is preferably a hydrophobic polymer and more preferably polystyrene. In an especially preferred embodiment of the invention the Matrix is in the form of a microtiter plate.
  • a method for assaying a phosphatidylinositol kinase comprises:
  • PIP n -L-S-Matrix wherein PIP n is a phosphoinositide polyphosphate, L in a linker moiety.
  • S is a scintillant, and Matrix is a solid support;
  • reaction mixture effective amounts of a sample to be tested containing the phosphatidylinositol kinase. reaction buffer, and ATP labeled with a low- energy ⁇ -emitter to form a reaction mixture;
  • the low-energy ⁇ -emitter is 33 P.
  • PIP n -L-S wherein PIP n is a phosphoinositide polyphosphate, L in a linker moiety, and S is a scintillant comprises:
  • a method for screening compounds for a drug that interferes with phosphatidylinositol kinase activity comprises: (a) providing a composition represented by the formula
  • PIP -L-S-Matrix wherein PIP n is a phosphoinositide polyphosphate, L in a linker moiety, S is a scintillant, and Matrix is a solid support;
  • reaction mixture (b) contacting the composition with effective amounts of a compound to be tested, reaction buffer, phosphatidylinositol kinase, and ATP labeled with a low-energy ⁇ -emitter to form a reaction mixture;
  • FIG. 1 shows selected phosphoinositide kinase pathways.
  • FIG. 2 shows that phosphorylation of Akt by PIP 3 -activated PDKl blocks caspase activation and prevents apoptosis.
  • FIG. 3 shows a representative SCLNTI-PIP and its graphic representation.
  • FIG. 4 shows the principle of the scintillation proximity assay (SPA).
  • FIG. 5 shows chemical structures, ⁇ abs . and ⁇ em of common scintillants.
  • FIG. 6 shows commercially available phosphoinositide polyphosphates.
  • FIG. 7 shows synthesis of ⁇ -l-( -(6-aminohexanoyl), 2-(9-hexanoyl-PI(4,5)P 2 .
  • FIG. 8 shows modified PIP n s that are substrates for PI3K.
  • FIG. 9 shows chemical synthesis of 2-(4-aminomethylphenyl)-5-(-biphenyl)- 1,3,4- oxadiazole (amino-PBD); reagents: a, MeOH/H 2 S0 4 ; b. H 2 NNH 2 ; c, toluene/heat; d.
  • FIG. 10 shows synthesis of three SC ⁇ NTI-LINKER modules from desymmetrized PEG.
  • FIG. 1 1 shows coupling of SCLNTI-LLNKER modules to four PIP scaffolds to give SCINTI-PIPs; for PEG-6 and PEG-85, X may be either NH or O.
  • FIG. 12 shows the principle of scintillation proximity assay as applied to SCINTI-PIP.
  • an effective amount of a phosphatidylinositol kinase for carrying out an assay thereof is an amount of such enzyme that will phosphorylate a selected amount of substrate in a selected period of time under standard conditions. This amount can be easily determined without undue experimentation by a person skilled in the art.
  • effective amounts of reaction buffer and ATP for carrying out a phosphatidylinositol kinase assay are amounts known in the art or amounts that can be readily determined by a person skilled in the art for obtaining a selected result.
  • PIP ⁇ s are key signaling molecules in cellular communication.
  • PIP n s are biosynthesized by the interplay of kinases and phosphatases.
  • isozymes of PI 3- kinase, PI 4-kinase, and PI 5-kinase have been identified and are important in insulin signaling, apoptosis, cytoskeletal remodeling, and protein trafficking. Few specific inhibitors for these enzymes exist.
  • the development of targeted therapeutic agents is substantially enhanced with rapid assays for lipid kinase activities that can be used in, for example, a 96- well format.
  • Vast libraries of potential inhibitors can be screened with such a high throughput assay.
  • the present assay expedites the processing of samples and can be automated.
  • the assay requires no addition of scintillation cocktail and is less problematic and less expensive for radiowaste disposal.
  • the present invention in which a scintillant is covalently linked to a substrate molecule, can be used for assaying any PI kinase.
  • SCLNTI-PIPS sintillant phosphoinositide polyphosphates
  • SCINTI-PIPS are a class of hybrid fluorophore -phosphoinositide polyphosphates that can be used to immobilize a PIP n substrate on a plastic surface, as shown in FIG. 3. Similar to the scintillation proximity assay (SPA; Amersham) and FLASHPLATE (NEN Life Science Products) technologies, N. Nelson, 165 Anal. Biochem. 287-293 (1986); N. Bosworth & P. Towers, 341 Nature 167-168 (1989); S.
  • SPA scintillation proximity assay
  • FLASHPLATE N. Nelson, 165 Anal. Biochem. 287-293 (1986); N. Bosworth & P. Towers, 341 Nature 167-168 (1989); S.
  • the ⁇ -particle energy excites the scintillant to emit a visible photon.
  • Unbound radioactivity is too distant from the scintillant (and thus quenched by the solvent medium) to cause scintillation.
  • the photon flux produced form the scintillant is proportional to the degree of phosphorylation.
  • the need to coat the plate with scintillation cocktail in the form of fluoromicrospheres or beads is not necessary, since the hydrophobic anchor is also an effective fluorophore and scintillant.
  • 96-well microplates are chemically modified with a thin layer of polystyrene-based scintillant to provide a platform for nonseparation assays using a variety of isotopes without the addition of liquid scintillation cocktail.
  • the SCINTI-PIPS assay greatly increases the efficiency by reducing the distance between scintillant and ⁇ -emitter by several orders of magnitude.
  • Fluorophores consisting of bis-phenyl-substituted oxadiazoles have proven to be superior scintillants compared to those commonly used, such as 2,5-diphenyloxazole (PPO) and l,4-bis([5-phenyl]-l,3-oxazol-2-yl])-benzene (POPOP).
  • PPO 2,5-diphenyloxazole
  • POPOP l,4-bis([5-phenyl]-l,3-oxazol-2-yl])-benzene
  • FOG. 5 2-(4-t-butyl-phenyl-5-biphenyl)-l,3,4-oxadiazole
  • the number of nitrogen atoms in the five-membered ring of a conjugated system causes a hypochromic or blue shift in both the absorption and emission spectra.
  • This type of fluorophore strongly absorbs in an effectively irreversible fashion to polystyrene surfaces as well as to other plastics.
  • a PEG linker extends into the aqueous medium. G.M. Bonora, 1 17 Gazz. Chim. Ital. 379-380 (1987). PEG is used because of its biocompatibility, lack of toxicity and immunogenicity. and nonbiodegradability. S. Zalipsky, 6 Bioconjugate Chem. 150-165 (1995). Attachment of biologically relevant molecules (such as .s' «-l-0-(6-aminohexanoyl)PIP n s) to PEG is achieved either with commercially available bisfunctionalized PEG derivatives (Shearwater Polymers,
  • a modular orientation such as this provides groups for specific coupling as well as creating an immobilized backbone that can be generalized to any receptor-ligand interaction.
  • the PIP n s comprise eight molecular scaffolds based on the number and position of phosphates on the inositol head group (FIG. 6). PIP n s are commercially available (Echelon Research Laboratories, Salt Lake City. Utah), and synthetic routes are known. G.D. Prestwich et al.. in Advances in Phosphoinositides (K. Bruzik & C.S. Chen eds; American Chemical Soc. 1998); Q.-M. Gu & G.D. Prestwich, 61 J. Org. Chem.
  • FIG. 7. J. Chen et al., 61 J. Org. Chem. 6305-6312 (1996). Similar approaches have been developed for PI(3)P, PI(4)P, L. Feng et al., J. Org. Chem. (1998), and PI(5)P, J. Peng & G.D. Prestwich, Tetrahedron Lett. (1998). An alternative synthetic route involves the triester- modified PIP n derivatives. Q.-M. Gu & G.D. Prestwich, 61 J. Org. Chem. 8642-8647 (1996).
  • amino-PBD tetherable scintillant fluorophore 2-(4-amino-methylphenyl)-5-(4-biphenyl)-l,3.4-oxadiazole
  • a hydrophilic linker is used with or without a PEG spacer for coupling to a "tetherable" PIP n to form the immobilized phosphoinositide, SCLNTI-PIPS. as shown in FIG. 3.
  • Three tetherable SCINTI-LLNKER modules are shown in FIG. 10.
  • PEG linker addresses potential problems of water solubility and steric access of the kinase to the head group.
  • PEG linker (“PEG-0").
  • PEG-0 A recently-described, convenient route to desymmetrized PEGs may also be used.
  • This method comprises preparing the symmetrical diazide followed by a biphasic mono-reduction method in which the amino-azide is continuously extracted into a reductant-free aqueous phase.
  • the SCLNTI-LLNKER modules comprise a short-chain hexa(ethyleneglycol) and the commercially available PEG 3,400.
  • the relatively less expensive scintillants are chemically activated to provide a maximally economical convergent synthesis, as shown in FIGS. 10 and 11.
  • the amino-PBD is succinylated and activated to an NHS ester for connection to the amino terminus of a desymmetrized PEG.
  • the SCFNTI-PEG6 and SCLNTI-PEG85 are further converted to active NHS esters for optimal coupling to the aminoacyl-PIP n as the limiting and more costly reagent.
  • SCLNTI-PIPS plates are prepared by coating standard 96-well plates with several concentrations of each of four SCLNTI-PIPS (FIG. 12) to determine which provides the most efficient substrate for kinase activity. These details are presented below. Importantly, the present approach is benchmarked by two comparisons: (a) the FLASHPLATE type of SPA. and (b) coating with common non-covalent scintillators, e.g. PPO/POPOP. and commercial i- Bu-PBD are performed as described below (FIG. 12).
  • the desired di-C 16 PIP n e.g. PI (Echelon Research Laboratories Cat. No. P-0016), PI(4)PP (Echelon
  • PI(5)P Echelon Research Laboratories Cat. No. P- 5016
  • PI(4.5)P2 Echelon Research Laboratories Cat. No. P4516
  • LUVs unilamellar phospholipid vesicles
  • the PEG linker provides water solubility for the SCLNTI-PIPS while preserving the hydrophobic adsorption by the aromatic scintillant.
  • the PEG6 may provide the best balance of water solubility and adsorptive tenacity, but more hydrophilic and more hydrophobic materials can be made since solubilities of the PIP n s change with increasing phosphorylation.
  • Vesicle-type coating is merely illustrative, since other coating methods may be used.
  • coating could proceed by evaporation of organic solvents, and with PEG85 a water solution could be used and evaporated under reduced pressure to allow adhesion of the SCLNTI-PIP. It has been shown that methanol, DMSO, and glacial acetic acid are compatible with the 96-well microtiter plate and may be used as coating solvents. Chloroform or any chloroform-methanol mixture is unsuitable.
  • PI3K will not phosphorylate PI(4)P or PI(4,5)P 2 in the absence of PS carrier, although it will phosphorylate PI.
  • the details for using an insect-cell expressed recombinant PI3K (as a glutathione (GST)- fusion protein) to form lipids is described below.
  • the PI3K assay is conducted as follows. The supernate from one cell pellet is diluted in NP-40 lysis buffer, snap frozen in liquid nitrogen, and stored as aliquots at -70 °C Under these conditions, the enzyme is stable for months.
  • Crude enzyme from a 10.000 x g supernate can be used for PI3K assays.
  • pure enzyme can be obtained from an aliquot by thawing on ice, addition to a 1 : 1 slurry of GSH-beads (Pharmacia) pre-equilibrated in NP-40 lysis buffer, and incubation for 1 hr at 4°C The beads are washed three times, once with PBS + 1% NP-40, then with 100 mM Tris-HCl pH 7.0 + 1 M LiCl. and finally with TNE (20 mM Tris-HCl pH 7 + 100 mM NaCl + 1 mM EDTA).
  • the enzyme is eluted with 20 mM HEPES pH 7.0 + 1 mM EDTA containing 10 mM GSH, and aliquots of the PI3K preparation are added to the 96-well plate.
  • the reaction is initiated by addition of an aliquot (from 1-100 nCi of ⁇ - 3j P-ATP) with a multichannel pipettor. allowed to proceed for 15 minutes at room temperature, and quenched by addition of 1 N HC1.
  • the plate is read in a 96-well plate reader, such as a Wallac MICROBETA unit or a Packard TOP COUNT unit.
  • a typical screening experiment contains (a) a control well (absence of ⁇ - 33 P-ATP and inhibitor but coated with PS carrier), (b) a well with enzyme, ⁇ - 33 P-ATP, and inhibitor, (c) a well with enzyme and ⁇ - 3 P- ATP, and (d) a well with ⁇ - JJ P-ATP and four times the normal enzyme concentration.
  • Loading of the SCINTI-PIP on the plate with the enzyme and ⁇ - 33 P-ATP concentrations can be optimized such that good kinetics can be obtained under conditions in which the phosphoinositide substrate is in excess.
  • the assay can be used to determine an effective IC 50 for the known PI3K inhibitors, wortmannin (irreversible) and LY294002 (reversible), as well as for screening and assaying unknown inhibitors.
  • Type II PI3K isoforms In addition to recombinant PI3K, a complete panel of Type II PI3K isoforms has been obtained by Dr. Andrew J. Morris (State University of New York-Stony Brook), including p85 ⁇ , PI 10a, pi lO ⁇ , and pi lO ⁇ , in which an epitope tag on the p85 ⁇ facilitates rapid purification of the catalytically active heterodimeric complex.
  • the need to coat the microplate with a scintillant is unnecessary with SCLNTI-PIPS, since the tetherable hydrophobic anchor is simultaneously a highly efficient adhesive moiety as well as scintillant.
  • the proximity between the scintillant and ⁇ -emitter is intramolecular (within 40 to 100 A) and several orders of magnitude greater than the micrometer proximity achieved with the coating protocols, greatly increasing efficiency.
  • These assays can be conducted in 96-well polystyrene microtiter plates with the medium-energy isotope 33 P. Use of 32 P would not be appropriate, since the higher energy of this isotope would result in adjacent wells responding to radioisotope in a different well.
  • a phosphorylation reaction takes place in each well in the presence of a cellular extract, reaction buffer, and enzyme (e.g. insect cell expressed PI3K).
  • the reaction is initiated upon the addition of ⁇ - 33 P-ATP. If bound (within a 10 ⁇ m distance of scintillant), the ⁇ -particle is brought in close enough proximity that it stimulates the scintillant to emit light. However, energy from unbound radioactivity is quenched by solvent (FIG. 4).
  • Table 2 shows illustrative substrates, products, and PI kinases that can by assayed according to the present invention.

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Abstract

La présente invention concerne une composition représentée par la formule PIPn-L-S-matrice, dans laquelle PIPn représente un polyphosphate phosphoïnositide, L représente une fraction de liaison, S représente un scintillant et matrice un support solide. Les fractions de liaison préférées comprennent des agents de liaison succinimide et poly(éthylène glycol), le scintillant préféré étant 2-(4-amino-méthylphényl)-5-(4-biphényl)-1,3,4-oxadiazole. Dans un mode de réalisation préféré, la matrice est une plaque à microtitration. La composition précitée peut être utilisée pour le dosage de phosphatidylinositol kinases ainsi pour le criblage de composés en vue d'inhiber l'activité du phosphatidylinositol. Par ailleurs, cette invention concerne des procédés de fabrication et d'utilisation de ce composé.
PCT/US1999/014566 1998-06-26 1999-06-26 Reactifs immobilises pour dosages de kinase WO2000000584A2 (fr)

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WO2001092560A2 (fr) * 2000-05-31 2001-12-06 Promega Corporation Dosage pour kinases et phosphatases
WO2002101084A2 (fr) * 2001-06-11 2002-12-19 Applied Research Systems Ars Holding N.V. Nouveaux dosages de la scintillation par proximite de molecules de liaison aminoglycoside (abm)
EP1481093A2 (fr) * 2002-02-01 2004-12-01 Beth Israel Deaconess Medical Center, Inc. Modulation de la phosphoinositide phosphate kinase de type ii$g(b)
EP1495129A2 (fr) * 2002-03-29 2005-01-12 The University of Utah Research Foundation Compositions de phospholipides-phosphoinositides hybrides et utilisations de ces compositions
EP1604021A2 (fr) * 2003-03-17 2005-12-14 Smithkline Beecham Corporation Procedes pour identifier des inhibiteurs d'enzyme et des proteines kinases
JP2009149903A (ja) * 2006-04-27 2009-07-09 Intezyne Technologies Inc 化学的に異なる末端基を含むポリ（エチレングリコール）
US7820803B2 (en) 2000-08-23 2010-10-26 Andrew Bruce Holmes Immobilized phosphatidic acid probe
CN114096565A (zh) * 2019-04-10 2022-02-25 康奈尔大学 Pip4k通过非催化依赖性机制抑制胰岛素信号传导并增强免疫功能

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AU2001265231B2 (en) * 2000-05-31 2006-07-06 Promega Corporation Assay for kinases and phosphatases
WO2001092560A3 (fr) * 2000-05-31 2002-08-01 Promega Corp Dosage pour kinases et phosphatases
US6720162B2 (en) 2000-05-31 2004-04-13 Promega Corporation Assay for kinases and phosphatases
WO2001092560A2 (fr) * 2000-05-31 2001-12-06 Promega Corporation Dosage pour kinases et phosphatases
US6893834B2 (en) 2000-05-31 2005-05-17 Promega Corporation Assay for kinases and phosphatases
US7820803B2 (en) 2000-08-23 2010-10-26 Andrew Bruce Holmes Immobilized phosphatidic acid probe
WO2002101084A2 (fr) * 2001-06-11 2002-12-19 Applied Research Systems Ars Holding N.V. Nouveaux dosages de la scintillation par proximite de molecules de liaison aminoglycoside (abm)
WO2002101084A3 (fr) * 2001-06-11 2003-02-20 Applied Research Systems Nouveaux dosages de la scintillation par proximite de molecules de liaison aminoglycoside (abm)
US7157239B2 (en) * 2001-06-11 2007-01-02 Applied Research Systems Ars Holding N.V. Method and kit for identifying and/or quantifying radiolabeled aminoglycoside binding molecules
EP1481093A2 (fr) * 2002-02-01 2004-12-01 Beth Israel Deaconess Medical Center, Inc. Modulation de la phosphoinositide phosphate kinase de type ii$g(b)
EP1481093A4 (fr) * 2002-02-01 2008-10-01 Beth Israel Hospital Modulation de la phosphoinositide phosphate kinase de type ii$g(b)
EP1495129A4 (fr) * 2002-03-29 2006-06-14 Univ Utah Res Found Compositions de phospholipides-phosphoinositides hybrides et utilisations de ces compositions
EP1495129A2 (fr) * 2002-03-29 2005-01-12 The University of Utah Research Foundation Compositions de phospholipides-phosphoinositides hybrides et utilisations de ces compositions
JP2006520600A (ja) * 2003-03-17 2006-09-14 スミスクライン・ビーチャム・コーポレイション 酵素阻害剤およびプロテインキナーゼの同定方法
EP1604021A4 (fr) * 2003-03-17 2006-11-08 Smithkline Beecham Corp Procedes pour identifier des inhibiteurs d'enzyme et des proteines kinases
EP1604021A2 (fr) * 2003-03-17 2005-12-14 Smithkline Beecham Corporation Procedes pour identifier des inhibiteurs d'enzyme et des proteines kinases
JP2009149903A (ja) * 2006-04-27 2009-07-09 Intezyne Technologies Inc 化学的に異なる末端基を含むポリ（エチレングリコール）
JP2009535462A (ja) * 2006-04-27 2009-10-01 インテザイン テクノロジーズ， インコーポレイテッド 化学的に異なる末端基を含むポリ（エチレングリコール）
JP2011058001A (ja) * 2006-04-27 2011-03-24 Intezyne Technologies Inc 化学的に異なる末端基を含むポリ（エチレングリコール）
CN114096565A (zh) * 2019-04-10 2022-02-25 康奈尔大学 Pip4k通过非催化依赖性机制抑制胰岛素信号传导并增强免疫功能
EP3953394A4 (fr) * 2019-04-10 2023-11-01 Cornell University Suppression de la signalisation de l'insuline et amélioration de la fonction immunitaire par les pip4k par l'intermédiaire d'un mécanisme indépendant de l'activité catalytique

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