WO1993019752A1 - Inhibition de kinase s6 p70 - Google Patents

Inhibition de kinase s6 p70 Download PDF

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WO1993019752A1
WO1993019752A1 PCT/US1993/002459 US9302459W WO9319752A1 WO 1993019752 A1 WO1993019752 A1 WO 1993019752A1 US 9302459 W US9302459 W US 9302459W WO 9319752 A1 WO9319752 A1 WO 9319752A1
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kinase
cell
activity
serine
composition
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PCT/US1993/002459
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Barbara E. Bierer
Joseph Avruch
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Dana-Farber Cancer Institute, Inc.
The General Hospital Corporation
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2

Definitions

  • p90 S6 kinase and the 70 kDa S6 kinase family (Banerjee et al. , Proc. Natl. Acad. Sci. USA 87:8550-8554, 1990; Kozma et al., Proc. Natl. Acad. Sci. USA 87:7365-7369, 1990), referred to herein as p70 S6 kinase.
  • the activity of both types of kinases is regulated by serine/threonine phosphorylation (Erikson, supra) , and both are serine/threonine kinases themselves.
  • Eukaryotic cells contain many different kinases as part of various cascades that transmute signals from cell-surface receptors to effector molecules within the cell.
  • the cell-surface receptor is itself a kinase that is activated upon binding its ligand; in other cases, the receptor is associated with a separate protein that acquires kinase activity when the receptor binds its ligand.
  • the newly-activated kinase then phosphorylates the next member of the relevant cascade, thereby activating (or in some cases, deactivating) it.
  • Phosphatases also form an integral part of the cascade, acting to remove the phosphate groups added by the kinases, and thereby deactivating (or in some cases, activating) the substrate polypeptide.
  • each member of such a cascade transmutes signals by sequentially phosphorylating (if it is a kinase) or dephosphorylating (if it is a phosphatase) certain critical residues in the next member of the cascade, thereby activating or deactivating such next member, as the case may be.
  • the interleukin-2 (IL-2) receptor In T cells expressing the interleukin-2 (IL-2) receptor, binding of IL-2 to this receptor triggers a response culminating in proliferation of the T cell (Smith, Ann. Rev. Immunol. 2:319-333, 1984). Evans et al. observed that one aspect of the I -2 generated response is an increase in S6 phosphorylation and a concomitant increase in the rate of protein synthesis (J. Biol. Chem. 262:4624-4630, 1987). Similarly, the multi- faceted response of insulin-dependent H4 hepatoma cells to stimulation by insulin includes an increase in the level of S6 phosphorylation, apparently attributable at least in part to increased activity of the S6 kinases.
  • Rapamycin and FK506 are macrolide antibiotics which are potent immunosuppressive agents. Although the two compounds share certain structural features (see Fig. 9) and are capable of binding to the same family of cellular proteins (termed FK506-binding proteins, or FKBPs) to form a biologically active complex, their mechanisms of immunosuppression differ significantly.
  • FK506/FKBP complex inhibits a very early step in antigen-induced T cell activation, preventing proliferation of activated T cells by blocking the induction of cytokine gene transcription. Liu et al.
  • the information linking p70 S6 kinase inhibition to the immunosuppressive and newly discovered general antiproliferative effects of rapamycin may be utilized to design an assay for screening for immunosuppressive and/or antiproliferative agents which act by the same or a related mechanism to that of rapamycin: i.e., which ultimately result in a decrease in p70 S6 kinase activity in the treated cell.
  • Such an assay will pick up not only those compounds or complexes which act on the same enzyme as that targeted by the rapamycin/FKBP complex, but also those which act upstream or downstream of that particular enzyme in the p70 S6 cascade.
  • compositions which result in decreased p70 S6 kinase activity may act to inhibit a kinase (e.g., p70 S6 kinase itself, or a kinase upstream of p70 S6 kinase) , or may activate a phosphatase (e.g., one which specifically dephosphorylates and thus inactivates p70 S6 kinase, or an enzyme upstream of p70 S6 kinase) .
  • a kinase e.g., p70 S6 kinase itself, or a kinase upstream of p70 S6 kinase
  • a phosphatase e.g., one which specifically dephosphorylates and thus inactivates p70 S6 kinase, or an enzyme upstream of p70 S6 kinase
  • the screen will also identify those compositions which activate a kinase or inhibit a phosphatase upstream of p70 S6 kinase, provided that this activation or inhibition ultimately results in a decrease in p70 S6 kinase activity.
  • the screening method of the invention includes the steps of (1) contacting a eukaryotic cell (e.g., a mammalian cell such as a human cell) with a candidate antiproliferative or immunosuppressive composition; and (2) determining the level of activity of a serine/threonine kinase or a serine/threonine phosphatase in the p70 S6 kinase cascade of said cell in the presence of the candidate composition, wherein a level of said activity that results in a lower p70 S6 kinase activity in the presence of the composition than in the absence of the composition is an indication that the composition is an antiproliferative or immunosuppressive agent.
  • a eukaryotic cell e.g., a mammalian cell such as a human cell
  • a candidate antiproliferative or immunosuppressive composition e.g., a mammalian cell such as a human cell
  • the serine/threonine kinase may be p70 S6-kinase itself, or a kinase which phophorylates and thereby activates p70 S6 kinase in vivo, or a kinase which acts upstream of that point.
  • the serine/threonine phosphatase may be a phosphatase which dephosphorylates and thereby inactivates p70 S6 kinase in vivo, or a phosphatase which acts upstream of that point.
  • step 2 may be readily accomplished by simply measuring the level of p70 S6 kinase activity, which is directly or inversely related to the activities of each of the kinases and phosphatases upstream of p70 S6 kinase in the p70 S6 kinase cascade.
  • the cell may optionally be exposed to an appropriate mitogen (preferably a growth factor) before or during step l.
  • a cytokine such as interleukin 1 (IL-1) , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, erythropoietin (EPO) , Steel factor (stem cell factor) ; granulocyte colony stimulating factor (G-CSF) , macrophage colony stimulating factor (M- CSF) , or granulocyte/macrophage colony stimulating factor (GM-CSF) may be used to trigger proliferation signals; preferably, the cell is a lymphocyte such as a T cell or B cell, and the mitogen of choice is a lymphokine such as one of the interleukins.
  • IL-1 interleukin 1
  • IL-2 interleukin-2
  • candidate antiproliferative or immunosuppressive agents may be screened in vitro in a method including the following steps: (1) combining (a) a sample containing p70 S6 kinase, (b) a substrate for the kinase, and (c) a candidate antiproliferative or immunosuppressive composition; and
  • the in vitro assay may employ p70 S6 kinase as the substrate, and an enzyme just upstream of p70 S6 kinase as the effector. This method would include the following steps:
  • the invention also includes methods of inhibiting the proliferation or immune response of a cell (e.g., a hematopoietic cell such as a T cell, stimulated by a cytokine such as IL-2) of an animal (preferably a mammal such as a human) , by introducing into the animal a composition that interrupts the p70 S6 kinase cascade via a mechanism such as (i) directly inhibiting the activity of the p70 S6 kinase of the cell, or (ii) directly inhibiting the activity of a kinase which phosphorylates in vivo a serine or threonine residue on p70 S6 kinase, or (iii) directly increasing the activity of a phosphatase which dephosphorylates in vivo a serine or threonine residue on p70 S6 kinase.
  • a cell e.g., a hematopoietic cell such as a T
  • Also within the invention is a method of inhibiting cellular proliferation in response to a mitogen other than IL-2, which method includes the steps of (1) providing a cell which proliferates in response to the mitogen; and (2) treating the cell with a composition (which may include rapamycin or an analog of rapamycin) that modulates the activity of a serine/threonine kinase (such a p70 S6 kinase or a kinase which activates p70 S6 kinase) or a serine/threonine phosphatase (such one which dephosphorylates, and thereby deactivates, p70 S6 kinase) in the p70 S6 kinase cascade of the cell, thereby resulting in a decrease in the activity of p70 S6 kinase in the cell.
  • a composition which may include rapamycin or an analog of rapamycin
  • This method may be used to treat an animal having a condition characterized by proliferation of a cell in response to the given mitogen.
  • mitogen is meant to include any entity which, when contacted with a cell, stimulates the cell to proliferate at a rate higher than the rate in its absence.
  • lectins and growth factors defined as naturally-occurring proteins or glycoproteins which acts as endogenous mitogens in vivo
  • growth factors such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, Steel factor, G-CSF, M-CSF, GM-CSF, EPO, epidermal growth factor (EGF) , fibroblast growth factor (FGF) , platel-et-derived growth factor (PDGF) , and insulin.
  • an analog of rapamycin or of the rapamycin/FKBP complex which analog, when introduced into a cell, modulates the activity of a serine/threonine kinase or serine/threonine phosphatase of the p70 S6 kinase cascade, such that the activity of p70 S6 kinase in the cell is lower in the presence of the analog than in its absence.
  • Such analogs may be designed to mimic the molecular structure of rapamycin by using the information known about rapamycin, FK506, and the FKBP-binding analog known as 506BD (shown in Fig. 9) , which does not function as an immunosuppressant.
  • Such analogs would retain the common FKBP-binding site of all three molecules, and would have changes to some portion of the remainder of the molecule.
  • the design and preparation of such analogs is well within the abilities of one of ordinary skill in the art of synthetic organic chemistry. Given the simple and rapid assays provided herein for determining whether a given analog functions in the same manner as rapamycin, the rapid screening of large numbers of such analogs is now feasible.
  • Fig. 1 is a set of SDS-PAGE gels illustrating the effect of IL-2 and PMA on S6 kinase activity, MAP kinase activity, and tyrosine phosphorylation in CTLL-20 cells.
  • Cells were treated with IL-2 or PMA for the indicated times. Lysates were analyzed for (A) total S6 kinase activity, (B) S6 kinase activity due to p90 S6 kinase, (C) MAP kinase activity, or (D) induction of tyrosine phosphorylation.
  • A-C the positions of the corresponding substrates are indicated on the left.
  • Fig. 2 is a graph and a set of im unoblot autoradiograms illustrating the chromatographic resolution of p70 and p90 S6 kinases.
  • IL-2-deprived cells were either left untreated or treated with rapamycin for 60 min, and either left unstimulated or stimulated with IL-2 for an additional period of 60 min.
  • Cell lysates were applied to a Mono Q anion-exchange column and eluted with a linear gradient of NaCl from 0.0 to 0.5 M, and fractions were collected and analyzed.
  • A S6 kinase activity.
  • the 90 kDa proteins detected by the anti-p70 S6 kinase antiserum correspond to the recently described high molecular weight forms of the p70 S6 kinases (Grove et al., Mol. Cell. Biol. 11:5541-5550, 1991). Note that the protein bands migrating at approximately 100-110 kDa which are recognized by the p70 S6 kinase-specific antiserum (fractions 9-10 and 19-21) elute in fractions devoid of detectable S6 kinase activity.
  • Fig. 3 is a set of SDS-PAGE gels illustrating the effects of rapamycin and FK506 on S6 kinase activity, MAP kinase activity, and tyrosine phosphorylation in CTLL-20 cells.
  • Cells were preincubated with rapamycin or FK506 as indicated and either left untreated for 5 min (lanes 1-3) , treated with IL-2 for 5 min (lanes 4-6) or 60 min (lanes 7-9) , or treated with PMA for 5 min (lanes 10-12) .
  • Lysates were analyzed for (A) total S6 kinase activity, (B) S6 kinase activity due to p90 S6 kinase, (C) MAP kinase activity, or (D) induction of tyrosine phosphorylation.
  • A-C the positions of the corresponding substrates are indicated on the left.
  • D antiphosphotyrosine i munoblot
  • the positions of molecular weight markers in kDa
  • Fig. 4A is a set of SDS-PAGE gels illustrating the rapamycin dose-response of S6 kinase activity in H4 cells. Serum-starved H4 cells were treated with the indicated concentrations of rapamycin for 1 hr at 37°C, 5% C0 2 . Cells were then treated with insulin (10 ⁇ 6 M, 30 min) or were left untreated.
  • Fig. 4B is an SDS-PAGE gel illustrating the in vivo incorporation of 32 P i into ribosomal protein S6.
  • H4 hepatoma cells 24-hr serum-starved
  • DMEM phosphate-free, serum-free DMEM
  • Rapamycin was then added at the indicated final concentrations (in quadruplicate) .
  • one half of the plates were incubated for 30 min with 150 mU/ml insulin (10 ⁇ 6 M) , and the other half left untreated.
  • Cells were then harvested in 0.5 ml extraction buffer (10 mM KP , pH 6.5/1 mM EDTA/5mM EGTA/10 mM MgCl 2 /2 mM DTT/1 mM V0 4 / 50 mM 3-glycerophosphate/2 mM leupeptin/2 mM pepstatin/ 10 U/ml aprotinin/0.2 mM PMSF) .
  • Cells were broken in a dounce homogenizer and centrifuged 10 min at 1000 x g. Supematants were then balanced for protein (BioRad) , and a an aliquot was centrifuged. The pellet was resuspended in 200 ml of extraction buffer including 0.1% Triton X- 100, and then treated with SDS sample buffer and run on a 15% polyacrylamide gel (sample balanced for BioRad protein) .
  • Fig. 5 is a set of graphs depicting the results of Mono Q chromatography of H4 hepatoma extracts.
  • H4 hepatoma cells, serum-starved as in Fig. 4 were treated with 10 "6 M insulin for 30 min (left panels) , or 10 -6 M insulin (30 min) preceded by a 1 hr incubation with 20 nM rapamycin (right panels) .
  • Cells were extracted in homogenization buffer and centrifuged as in Fig. 4.
  • Extracts were diluted 3-fold in chromatography buffer (50 mM 0-glycerophosphate, pH 7.2/lmM DTT/lmM EGTA/O.lmM vanadate) and applied to a Mono Q HR (5/5) column equilibrated with chromatography buffer. The column was eluted with a 90 ml gradient (0-0.4 M NaCl) in chromatography buffer with collection of 1 ml fractions. Individual fractions were assayed for kinase activities using 4OS ribosomes and SKAIPS peptide. Fractions were also assayed for total protein and conductivity.
  • chromatography buffer 50 mM 0-glycerophosphate, pH 7.2/lmM DTT/lmM EGTA/O.lmM vanadate
  • Fig. 6 is a set of SDS-PAGE gels illustrating the time course of insulin-stimulated S6 kinase activity in the presence and absence of rapamycin.
  • Serum-starved H4 hepatoma cells were incubated in the presence (right) or the absence (left) of 20 nM rapamycin for 1 hr at 37°C, 5% C0 2 , after which cells were harvested and extracts prepared.
  • Top panel Extracts were assayed for total S6 kinase activity using 40S ribosomes as substrates for phosphorylation.
  • Middle panel Extracts were imm ⁇ noprecipitated with an affinity-purified polyclonal p70 S6 kinase antibody (1 ⁇ g/ tube) in the presence of 10 ⁇ l protein A Sepharose beads. After a 3 hr incubation at 5°C, immunoprecipitated material was washed 3X in homogenization buffer and 0.25 M NaCl + 0.1% Triton X- 100, followed by one wash in 20 mM Tris HCL pH7.4/lmM EGTA/2mM EDTA/2mM DTT/lOmM 3-glycerophosphate/0.1% Triton X-100/10% glycerol.
  • Fig. 7A is a pair of graphs comparing the effects of rapamycin on p70 S6 kinase, p90 S6 kinase, and erfcl/MBP kinase.
  • COS cells were transfected with expression constructs encoding the rat p70 S6 kinase (Grove et al. , Mol. Cell. Biol. 11:5541-5550, 1991), the rat p90 S6 kinase or the rat p44 erfcl/MBP kinase, all of which had been tagged at the amino terminus with a nonapeptide epitope from influenza hemagglutinin (Field et al. , Mol. Cell.
  • Recombinant protein was immunoprecipitated with the anti-epitope antibody 12CA5 (immobilized on Sepharose beads) for 3 h at 4°C; pellets were washed (3 x 1 ml) in assay buffer [homogenization buffer without the MgCl 2 and with 10% (w/v) glycerol] .
  • S6 kinase activity was determined as above and p4 erJcl/MBP kinase was assayed similarly, using 0.5 mg/ml myelin basic protein (MBP) as substrate.
  • the insert shows a Western blot of the recombinant p70 protein detected with the 12CA5 monoclonal antibody and visualized by chemiluminescence (ECL, Amersham) , demonstrating that recovery in each of the immunoprecipitants was equivalent.
  • Fig. 7B is a bar graph which presents data indicating that rapamycin but not FK506 inhibits recombinant p70 S6 kinase.
  • COS cells were transfected with vector or a p70 S6 kinase expression construct, as indicated; treated with 100 nM PMA and with 2 nM rapamycin or 200 nM FK506 (as indicated) ; and extracted, immunoprecipitated, and assayed as in 7A.
  • the insert shows a Western blot of the recombinant p70 protein detected with the 12CA5 monoclonal antibody and visualized by chemiluminescence, demonstrating that recovery in each of the immunoprecipitants was equivalent.
  • Fig. 7C is a graph, an autoradiogra of an SDS- PAGE gel, and a Western blot illustrating the rapamycin dose response of p70 S6 kinase with respect to autophosphorylation of p70 S6 kinase and phosphorylation of 4OS ribosomal subunits.
  • COS cells were transfected, treated with rapamycin, extracted, and immunoprecipitated as in 7A.
  • Middle panel Autophosphorylation was determined from kinase assays lacking substrate using aliquots of the immunoprecipitations from cells transfected with vector or recombinant p70 S6 kinase (as indicated) and treated as indicated with no addition or with rapamycin at the following concentrations: 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, or 3 nM.
  • Lower panel Western blot of samples in the same order as in the middle panel. Recombinant p70 protein was detected with the 12CA5 monoclonal antibody and visualized by chemiluminescence, demonstrating that recovery in each of the immunoprecipitants was equivalent.
  • Fig. 8 is a pair of graphs illustrating the rapamycin-mediated inhibition of basal and insulin- stimulated proliferation of H4 cells.
  • H4 hepatoma cells were grown to confluence in Swims S77 medium (Sigma) with 15% horse serum and 5% fetal calf serum. Confluent cells were maintained on serum-free medium 18 hours prior to assay.
  • Fig. 9 is an illustration of the chemical and 3- dimensional structures of FK506, rapamycin, and 506BD (Bierer et al. , Science 250:556-559).
  • Example 1 Inhibition of IL2 stimulation of p70 S6 kinase
  • the IL-2-dependent murine cell line CTLL-20 (American Type Culture Collection, Rockville, MD) was cultured in RPMI-10%FCS medium as described (Calvo et al., Eur. J. Immunol. 22:457-462, 1992, herein incorporated by reference), containing human recombinant L-2 (12.5-25 units/ml, kindly donated by Hoffmann-LaRoche, Inc.). Cells were recovered by centrifugation, washed 3 times with RPMI 1640, resuspended in RPMI-10%FCS at 1-5 x 10 6 cells/ml and incubated for 3 h at 37° C.
  • 1-2 x 10 6 cells were spun after stimulation and lysed in 0.2 ml of 10 mM potassium phosphate, pH 7.05, 1 mM EDTA, 0.5% Triton X-100, 5 M EGTA, 10 mM MgCl 2 , 50 mM ⁇ - glycerophosphate, 1 mM sodium vanadate, 2 mM DTT, 40 ⁇ g/ml PMSF, 10 ⁇ g/ml leupeptin, and 1 ⁇ g/ml pepstatin. After 30 min on ice, nuclei were pelleted by a 5-min microfuge centrifugation, and supematants were used for kinase assays.
  • S6 kinase assays specific for p90 S6 kinase 100 ⁇ l of supernatant were incubated with 5 ⁇ l of the specific rabbit antiserum 125 (Sweet et al., Mol. Cell. Biol. 10:2787-2792, 1990), and immunocomplexes were adsorbed to Staphylococcus aureus, washed as previously described (Vik et al., Proc. Natl. Acad. Sci USA 87:2685- 2689, 1990), and subjected to S6 phosphorylation assays as described above.
  • MAP kinase activity 5 ⁇ l of supernatant were used in a total reaction volume of 30 ⁇ l containing 20 mM Tris-HCl pH 7.25, 10 mM MgCl 2 , 100 ⁇ M ATP with 5 ⁇ Ci [ ⁇ - 32 P]ATP, and -1 ⁇ g of unactivated Xenopus p90 S6 kinase (referred to as rsk) obtained as described (Vik et al. , supra) . In all cases kinase reactions were carried out at 30°C for 15 min and analyzed as previously described (Calvo et al. , supra) . Antipho ⁇ photyrosine immunoblotting- analysis .
  • Supematants normalized for total protein content, were applied to a Mono Q (Pharmacia) anion-exchange chromatography column and bound proteins were eluted in a 18-ml gradient of 0-500 mM NaCl (Grove et al., Mol. Cell. Biol. 11:5541-5550, 1991). Aliquots (5 ⁇ l) of each 0.75 ml fraction were assayed for S6 kinase activity as described above. Aliquots (16 ⁇ l) of each fraction were resolved by reducing 8% SDS-PAGE and analyzed by immunoblotting with the p ⁇ O-specific rabbit antiserum 125 as described (Vik et al.
  • IL-2 stimulates S6 kinases of the p70 family in CTLL-20 cells.
  • CTLL-20 proliferates in response to IL-2 stimulation.
  • CTLL-20 cells were allowed to reach plateau phase of growth, extensively washed, and starved of IL-2 for 3h. IL-2 deprivation for this period of time did not affect cell viability or subsequent IL-2-sustained proliferation (data not shown) .
  • IL-2-deprived CTLL-20 cells were stimulated with either recombinant IL-2 or PMA, and total S6 kinase activity was measured in cell lysates by phosphorylation of 4OS ribosomal subunits in vitro (Fig. 1A) .
  • IL-2 binding resulted in a time-dependent stimulation of S6 kinase activity, which was detectable at 5 min after addition of IL-2 and increased cumulatively until at least 120 min after IL-2 addition.
  • Incubation with PMA also resulted in a marked stimulation of S6 kinase activity which increased over time compared to untreated cells.
  • MAP kinase has been reported to activate the p90 S6 kinase in vitro (Sturgill et al. , Nature 334:715-718, 1988) .
  • MAP kinase assays were performed using p90 S6 kinase as substrate with lysates from either untreated cells or cells incubated with IL-2 or PMA.
  • MAP kinase activity from lysates of IL-2-stimulated cells was similar to or slightly increased compared to untreated cells (Fig. IC and data not shown) .
  • Tyrosine-phosphorylated MAP kinase can be detected by antiphosphotyrosine immunoblotting in cells stimulated by a variety of external signals, including T cell receptor- CD3 (TCR-CD3) stimulation of T cells (-Calvo et al. , supra ; Hanekom et al., Biochem. J. 262:449-455, 1989).
  • TCR-CD3 T cell receptor- CD3
  • IL-2-stimulated cell lysates contained an increased S6 kinase activity peak which eluted in a position corresponding to p70 kinases (Fig. 2A) .
  • Fig. 2A S6 kinase activity peak which eluted in a position corresponding to p70 kinases
  • Anti-p90 S6 kinase immunoreactive proteins concentrate in fractions 5-11 (Fig. 2B)
  • anti-p70 S6 kinase immunoreactive proteins concentrate in fractions 14-17 (Fig. 2C) , which correspond to the peak of IL-2-stimulated S6 kinase activity.
  • IL-2 stimulates an increase in S6 kinase activity of the p70 family.
  • Rapamycin is a macrolide antibiotic with immunosuppressive activity. Structurally homologous to FK506, rapamycin has been shown to bind to a family of intracellular proteins termed FK506 binding proteins, or FKBPs. While the FK506/FKBP complex has been shown to bind to and inhibit the activity of the serine/threonine phosphatase known as calcineurin (Liu et al. , Cell, 66:807-815, 1991), the target of action of rapamycin is unknown. Rapamycin has been shown to inhibit IL-2- dependent T cell proliferation (Dumont et al., J. Immunol., 144:251-258, 1990; Bierer et al., Proc.
  • Rapamycin did not inhibit the IL-2-induced tyrosine phosphorylation of a number of substrates (e.g., the -100 kDa band, Fig. 3D, lanes 6 and 9 vs 4 and 7) .
  • the target of action of rapamycin thus appears to lie between the early activation of tyrosine kinases and the activation of members of the p70 S6 kinase family, which require serine/threonine phosphorylation for their activation.
  • Example 2 Inhibition of Insulin/Mitogen-Activated p70 S6 Kinase Insulin treatment of serum-starved H4 rat hepatoma cells results in the activation of S6 protein kinases; assays of whole extracts show a progressive increase in total S6 kinase to a plateau at 10 min that is sustained thereafter for at least l hr (Fig. 4) . Incubation of H4 cells with rapamycin for 1 hr prior to insulin addition leads to a dose-dependent (Fig. 4A) and parallel inhibition of basal and insulin-stimulated S6 kinase activities that is essentially complete at 10 nM rapamycin.
  • This inhibition of total extract S6 kinase reflects an inhibition of the p70 S6 kinase, as illustrated by the loss, following treatment of the cells with rapamycin, of (i) p70 S6 kinase activity in immunoprecipitates formed with an anti-p70 peptide antibody (Fig.
  • the p90 S6 kinase contributes less than 5% of the total extract 4OS S6 kinase activity (Fig. 5) . Nevertheless, when examined selectively by immunoprecipitation with an anti-p90 peptide antibody, the p90 S6 kinase can be seen to undergo activation in response to insulin, with a peak of activity at 10 min that begins to fall by 30 min in (Fig. 4B) ; neither the time course nor activity is affected by concentrations of rapamycin that completely abolish p70 S6 kinase activity (data not shown) .
  • Intrinsic ligand-associated tyrosine kinase activity of the insulin receptor results in the characteristic tyrosine phosphorylation of a 180 kDa polypeptide substrate termed IRS-1.
  • Antiphosphotyrosine immunoblots of extracts from rapamycin-treated H4 cells demonstrate that insulin-stimulated tyrosine phosphorylations of the 180 kDa substrate IRS-l is unaltered by concentrations of rapamycin that abolish p70 S6 kinase (data not shown) .
  • Insulin activates an array of proline-directed protein kinases in H4 hepatoma cells that include erkl/erk2 and a form of cdc2. These enzymes are capable of phosphorylating a putative regulatory domain at the carboxy terminus of intact p70 S6 kinase. The activation of these enzymes can be monitored using an serine- threonine-rich synthetic peptide (SKAIPS) corresponding to these p70 regulatory sequences (Mukhopadhyay et al. , J. Biol. Chem. 267:3325-3335, 1992), as well as with myelin basic protein (MBP) as substrate.
  • SKAIPS serine- threonine-rich synthetic peptide
  • rapamycin does not alter the insulin induced phosphorylation of SKAIPS (Fig. 5) or MBP (data not shown) .
  • This result is entirely consistent with the persistence of insulin-activated p90 S6 kinase in the presence of rapamycin, inasmuch as erk/MAP kinases are the likely proximate mediator of insulin-induced p90 S6 kinase activation.
  • the total and Suc-1 precipitable HI kinase activity in H4 cells is unaffected by rapamycin (data not shown) .
  • the differential sensitivity of p70 and p90 S6 kinases to rapamycin was confirmed by examining directly the activity of the recombinant S6 kinases, expressed transiently in COS cells.
  • the kinase activity of an epitope-tagged recombinant p70 S6 kinase was abolished completely by pretreatment of COS cells with rapamycin prior to harvest, while the activity of an epitope-tagged p90 S6 kinase and an epitope-tagged MAP kinase expressed in parallel was entirely unaffected even by 10-fold higher concentrations of rapamycin (Fig. 7A) .
  • Transient transfection of recombinant p70 S6 kinase into COS cells results in an expression of a multiply phosphorylated polypeptide. Only a small proportion of the synthesized protein, the most highly phosphorylated (and thus electrophoretically slower) material is able to stimulate 4OS phosphorylation. Furthermore, this recombinant p70 S6 kinase in COS cells is not regulated: neither treatment with insulin nor with phorbol ester increases its activity. Incubation of COS cells with rapamycin, however, is nevertheless able to inhibit p70 S6 kinase's ability to autophosphorylate with a concentration-dependence that precisely parallels its ability to phosphorylate 40S ribosomes (Fig.
  • Fig. 7C The presence of the electrophoretically retarded band in Fig. 7C indicates that a proportion of the p70 S6 kinase polypeptide is more highly phosphorylated, and while this form of p70 S6 kinase is the most active, it is also the most susceptible to inhibition by rapamycin. The disappearance of this band at 1 nM rapamycin may correlate with decreased phosphorylation of the protein itself. At higher concentrations of rapamycin, a diminution in the autophosphorylation of the lower band is also observed, suggesting that residual activity of p70 S6 kinase may be further decreased by the drug.
  • Concentrations of rapamycin that inhibit p70 S6 kinase activity are associated with an inhibition of the incorporation of 3 H-thymidine into serum-deprived H4 hepatoma cells in the presence or absence of insulin (Fig. 8) .
  • the complex of rapamycin bound to an FKBP has previously been shown to inhibit proliferation of cells by a lymphokine receptor signal, such as IL-2 binding to its receptor (Dumont et al., J. Immunol. 144:251-258, 1990; Bierer et al., Proc. Natl. Acad. Sci. USA 87:9231- 9235, 1990) .
  • the inhibition of insulin-stimulated proliferation observed here suggests that rapamycin may have a more general effect on mitogenesis.
  • IC 50 5nM
  • a component (25-50%) of proliferation remains resistant to the effects of rapamycin.
  • the lack of a direct correlation between p70 S6 kinase activity and proliferation suggests that other biochemical signalling processes impact upon the complex function of mitogenesis.
  • the target of rapamycin action may be upstream of both p70 S6 kinase activation and proliferation, displaying different IC 50 s for each pathway.
  • p70 S6 kinase may itself exert an effect on proliferation if activation of the 40S ribosomal subunit by p70 S6 kinase is a necessary step in achieving a maximal irate of proliferation.
  • the target for rapamycin appears to be situated downstream of insulin receptor kinase and the proximal IRS-1 tyrosine phosphorylation reaction, and on a limb of signal transmission processes distinct from that mediating erk/MAP kinase activation.
  • the rapamycin target appears to be a crucial element linking growth factor receptors to the mitogenic apparatus. This element is also likely to be a proximal upstream activator of the p70 S6 kinase, i.e., an activating p70 kinase-kinase or a regulator of such an enzyme.
  • An assay based upon the above observations may be utilized as a means for screening for an antiproliferative or immunosuppressive agent that inhibits proliferation or immune function via a mechanism that involves the same kinase or phosphatase as that targeted by the rapamycin-FKBP complex- (which may be the p70 S6 kinase, or an enzyme in the p70 S6 kinase cascade which is upstream of the p70 S6 kinase) .
  • the assay used to screen candidate compounds could use one of the cell types employed in the above-described experiments, or another cell type such as any one of the many known hematopoietic cell lines that are growth factor dependent, or a cell line rendered growth factor dependent by transfection with a recombinant cytokine receptor.
  • the assay could even use yeast cells, which have been shown to contain FKBPs and to be sensitive to inhibition by rapamycin. As described in detail above, rapamycin-FKBP can inhibit basal proliferation of cells, so the screening assay could utilize cells which naturally exhibit a significant basal level of proliferation in medium without the addition of any growth factor, cytokine, or other stimulus.
  • the window of response can be maximized by using a cell line that exhibits an increased rate of proliferation in the presence of a particular mitogenic stimulus.
  • cells and appropriate mitogens include: T cell lines, B cell lines, or hematopoietic cell lines and any cytokine to which the cells are responsive, such as an interleukin (IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, or IL-11) or EPO, G-CSF, M-CSF, GM-CSF or Steel factor (stem cell factor) ; H4 rat hepatoma cells and insulin; fibroblast cells and FGF; estrogen-responsive breast cancer cells and estrogen; or other cells which proliferate in the presence of a particular hormone or growth factor.
  • IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, or IL-11 EPO
  • the screen could be carried out as follows: a preparation of cells is mitogen- and/or serum-starved to arrest growth temporarily, and half of the cells are exposed to the candidate antiproliferative/immunosuppressant agent, while the other half serves as control. Serum and/or an appropriate mitogen are added to both-cultures. Each culture is sampled at given time points, and the activity of p70 S6 kinase is measured as described above. A candidate drug which results in a significantly lower p70 S6 kinase activity relative to the control (e.g., 60% or less of the control value, and preferably 20% or less) is classified as showing antiproliferative or immunosuppressant activity in this assay.
  • the candidate drug may be screened in a cell-free assay.
  • the ability of the drug to affect a particular kinase in the p70 S6 kinase cascade is tested as follows: a sample of the kinase to be tested (whether derived from natural cells or produced by recombinant or synthetic means) is mixed with a peptide or protein substrate for the enzyme, 32 P-ATP, and the drug to be screened.
  • the substrate may be, for example, the 4OS ribosomal subunit, purified S6, or an appropriate fragment of S6.
  • the candidate drug is an analog of rapamycin which is active only when complexed with an FKBP, an appropriate FKBP must be included.
  • a parallel incubation is performed without the drug, as a control. After a given period of incubation at 37°C, aliquots from each are taken, and the amount of 32 P associated with the substrate in each aliquot is determined, as a measure of the activity of the kinase in that particular aliquot.
  • An amount of radioactivity in the treated sample higher than that in the control sample is an indication that the drug activates the kinase tested, while an amount lower than in the control sample indicates that the drug inhibits the kinase tested.
  • a sample of the phosphatase of interest is incubated with the candidate drug and a peptide or protein substrate which has serine and/or threonine residues already phosphorylated with 32 P-phosphate.
  • the drug is absent in the control.
  • the amount of radioactivity remaining in the substrate after incubation is a measure of the activity of the phosphatase in this assay, and thus is a measure of the ability of the candidate drug to activate or inhibit the phosphatase.
  • Antiproliferative/immunosuppressant compositions which modulate the activity of some serine/threonine kinase or serine/threonine phosphatase in the p70 S6 kinase cascade, ultimately resulting in a decrease in p70 S6 kinase activity in a cell, are useful for treating conditions characterized by unwanted proliferation of cells or unwanted immune responses.
  • neoplasms of any type e.g., carcinomas, sarcomas, and leukemias
  • non-neoplastic conditions such as unwanted proliferation of capillaries (as in diabetic retinopathy) , of endothelial and/or smooth muscle cells in vascular walls (as in athrosclerosis) , of connective tissue (as in rheumatoid arthritis) , and of skin cells (as in psoriasis) ; autoimmune diseases; allergies; and host-vs-graft or graft-vs-host disease resulting from transplanted cells or organs.
  • compositions useful in such applications include rapamycin or an analog of rapamycin, complexed with an FKBP or an analog of an FKBP; rapamycin alone or a rapamycin analog alone, if an approprite FKBP is present in the cells to be treated; an analog of the rapamycin/FKBP complex (made, for example, by designing a molecule which mimics the molecular structure of the critical portions of the complex) ; or any other composition which, when introduced into the cell to be treated, ultimately results in a decrease in the activity of the p70 S6 kinase, by modulating the activity either of p70 S6 kinase itself, or of a kinase or phosphatase upstream of p70 S6 kinase in the p70 S6 kinase cascade.

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Abstract

Procédé de dépistage d'un agent anti-prolifératif ou immunodépresseur. Il consiste (1) à mettre une cellule eucaryotique en contact avec une composition candidate anti-proliférative ou d'immunodépression; et (2) à déterminer le niveau d'activité d'une sérine/thréonine-kinase ou d'une sérine/thréonine-phosphatase dans la cascade kinase S6 p70 de ladite cellule en présence de la composition candidate. Un niveau d'activité entraînant une activité de kinase S6 p70 moins élevée en présence de la composition qu'en l'absence de celle-ci indique que la composition est un agent anti-prolifératif ou immunodépresseur. On a également prévu des procédés de traitement utilisant ces compositions.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025072A1 (fr) * 1993-04-23 1994-11-10 American Home Products Corporation Conjugues de rapamycine et anticorps contre la rapamycine
WO1998018935A2 (fr) * 1996-10-31 1998-05-07 Novartis Ag P70-s6k ou kinase apparentee, a activite constitutive
WO1998056910A1 (fr) * 1997-06-11 1998-12-17 Chiron Corporation DETECTION DE LA PERTE DU GENE huBUB1 DE TYPE SAUVAGE
WO2000008173A1 (fr) * 1998-08-04 2000-02-17 Ludwig Institute For Cancer Research Identification et caracterisation fonctionnelle d'une nouvelle proteine kinase s6 ribosomique
EP1181938A2 (fr) * 1993-04-23 2002-02-27 American Home Products Corporation Conjugués de Rapamycine et anticorps
US6410312B1 (en) 1997-12-19 2002-06-25 Chiron Corporation huBUB3 gene involved in human cancers
WO2004014222A2 (fr) * 2002-08-12 2004-02-19 The Regents Of The University Of Michigan Diagnostic et traitement de maladies engendrees par des anomalies propres au trajet de la sclerose tubereuse (de bourneville)
US6830909B1 (en) 1998-08-04 2004-12-14 Ludwig Institute For Cancer Research Identification and functional characterization of a novel ribosomal S6 protein kinase
US7122360B1 (en) 1998-06-24 2006-10-17 University Of Dundee Polypeptides, polynucleotides and uses thereof
US7279562B2 (en) 1993-04-23 2007-10-09 Wyeth Rapamycin conjugates

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5093338A (en) * 1991-04-23 1992-03-03 Merck & Co., Inc. Lipophilic macrolide useful as an immunosuppressant
US5102876A (en) * 1991-05-07 1992-04-07 American Home Products Corporation Reduction products of rapamycin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5093338A (en) * 1991-04-23 1992-03-03 Merck & Co., Inc. Lipophilic macrolide useful as an immunosuppressant
US5102876A (en) * 1991-05-07 1992-04-07 American Home Products Corporation Reduction products of rapamycin

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 186, No. 3, issued 14 August 1992, TERADA et al., "Rapamycin Inhibits the Phosphorylation of p70 S6 Kinase in IL-2 and Mitogen-Activated Human T Cells", pages 1315-1321. *
CELL, Vol. 66, issued 06 September 1991, MCKEON, "When Worlds Collide: Immunosuppressants Meet Protein Phosphatases", pages 823-826. *
CELL, Vol. 66, issued 23 August 1991, LIU et al., "Calcineurin is a Common Target of Cyclophilin-Cyclosporin A and FKBP-FK506 Complexes", pages 807-815. *
IMMUNOLOGY, Vol. 72, issued 1991, KAY et al., "Inhibition of T and B Lymphocyte Proliferation by Rapamycin", pages 544-549. *
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol. 89, No. 1, Part. 2, issued January 1992, HULTSCH et al., "Rapamycin Inhibits the Proliferation of IL-3 Dependent PT18 Mast Cells; FK506 Prevents the Effect of Rapamycin", page 238, Abstract No. 376. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 10, issued 05 April 1987, EVANS et al., "Interleukin 2 and Diacylglycerol Stimulate Phosphorylation of 40 S Ribosomal S6 Protein", pages 4624-4630. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 266, No. 10, issued 05 April 1991, ERIKSON, "Structure, Expression, and Regulation of Protein Kinase Involved in the Phosphorylation of Ribosomal Protein S6", pages 6007-6010. *
JOURNAL OF IMMUNOLOGY, Vol. 144, No. 1, issued 01 January 1990, DUMONT et al., "Distinct Mechanisms of Suppression of Murine T Cell Activation by the Related Macrolides FK-506 and Rapamycin", pages 251-258. *
SCIENCE, Vol. 257, issued 14 August 1992, PRICE et al., "Rapamycin-Induced Inhibition of the 70-kilodalton S6 Protein Kinase", pages 973-977. *

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US6541612B2 (en) 1993-04-23 2003-04-01 Wyeth Monoclonal antibodies obtained using rapamycin position 27 conjugates as an immunogen
US7279562B2 (en) 1993-04-23 2007-10-09 Wyeth Rapamycin conjugates
EP1181938A2 (fr) * 1993-04-23 2002-02-27 American Home Products Corporation Conjugués de Rapamycine et anticorps
EP1181938A3 (fr) * 1993-04-23 2002-03-20 American Home Products Corporation Conjugués de Rapamycine et anticorps
WO1994025072A1 (fr) * 1993-04-23 1994-11-10 American Home Products Corporation Conjugues de rapamycine et anticorps contre la rapamycine
US7279561B1 (en) 1993-04-23 2007-10-09 Wyeth Anti-rapamycin monoclonal antibodies
US7897733B2 (en) 1993-04-23 2011-03-01 Pfizer, Inc. Rapamycin conjugates and antibodies
WO1998018935A2 (fr) * 1996-10-31 1998-05-07 Novartis Ag P70-s6k ou kinase apparentee, a activite constitutive
WO1998018935A3 (fr) * 1996-10-31 1998-07-30 Novartis Ag P70-s6k ou kinase apparentee, a activite constitutive
WO1998056910A1 (fr) * 1997-06-11 1998-12-17 Chiron Corporation DETECTION DE LA PERTE DU GENE huBUB1 DE TYPE SAUVAGE
US6489137B2 (en) 1997-06-11 2002-12-03 Chiron Corporation Detection of loss of the wild-type huBUB1 gene
US6410312B1 (en) 1997-12-19 2002-06-25 Chiron Corporation huBUB3 gene involved in human cancers
US7122360B1 (en) 1998-06-24 2006-10-17 University Of Dundee Polypeptides, polynucleotides and uses thereof
WO2000008173A1 (fr) * 1998-08-04 2000-02-17 Ludwig Institute For Cancer Research Identification et caracterisation fonctionnelle d'une nouvelle proteine kinase s6 ribosomique
US6830909B1 (en) 1998-08-04 2004-12-14 Ludwig Institute For Cancer Research Identification and functional characterization of a novel ribosomal S6 protein kinase
US7998718B2 (en) 1998-08-04 2011-08-16 Ludwig Institute For Cancer Research Ltd. Identification and functional characterization of a novel ribosomal S6 protein kinase
AU771307B2 (en) * 1998-08-04 2004-03-18 Ludwig Institute For Cancer Research Identification and functional characterization of a novel ribosomal S6 protein kinase
WO2004014222A2 (fr) * 2002-08-12 2004-02-19 The Regents Of The University Of Michigan Diagnostic et traitement de maladies engendrees par des anomalies propres au trajet de la sclerose tubereuse (de bourneville)
AU2003259803B2 (en) * 2002-08-12 2007-08-02 The Regents Of The University Of Michigan Diagnosis and treatment of tuberous sclerosis
CN100374158C (zh) * 2002-08-12 2008-03-12 密执安州立大学董事会 降低细胞atp水平的药剂在制备药物中的应用
US7169594B2 (en) 2002-08-12 2007-01-30 Regents Of The University Of Michigan Diagnosis and treatment of diseases arising from defects in the tuberous sclerosis pathway
WO2004014222A3 (fr) * 2002-08-12 2004-05-21 Univ Michigan Diagnostic et traitement de maladies engendrees par des anomalies propres au trajet de la sclerose tubereuse (de bourneville)

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