`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`
`(51) International Patent Classification 5 :
`A61K 31/33, C12N 9/12
`C12Q 1/42, G0lN 33/48, 33/573
`C07D267/00
`
`(11) International Publication Number:
`
`WO 93/19752
`
`Al
`
`(43) International Publication Date:
`
`14 October 1993 (14.10.93)
`
`\.
`
`J' ,._
`
`(21) International Application Number:
`
`PCT/US93/02459
`
`(22) International Filing Date:
`
`19 March 1993 (19.03.93)
`
`(81) Designated States: AU, CA, JP, European patent (AT, BE,
`CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE).
`
`(30) Priority data:
`07/864,806
`
`7 April 1992 (07.04.92)
`
`US
`
`Published
`With international search report.
`
`(7l)Applicant: DANA-FARBER CANCER
`INSTITUTE,
`INC. [US/US]; 44 Binney Street, Boston, MA 02115
`(US).
`
`(72)1nventors: BIERER, Barbara, E. ; 399 Hammond Street,
`Newton, MA 02167-1225 (US). AVRUCH, Joseph; 277
`St. Paul Street, Brookline, MA 02146 (US).
`
`(74) Agent: FRASER, Janis, K.; Fish & Richardson, 225 Fran(cid:173)
`klin Street, Boston, MA 02110-2804 (US).
`
`(54) Title: INHIBITION OF P70 S6 KINASE
`
`(57) Abstract
`
`A method of screening for an antiproliferative or immunosuppressive agent, which method includes the steps of (1) con(cid:173)
`tacting a eukaryotic 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 antiproliferative or immunosuppres(cid:173)
`sive agent; and methods of treatment using such compositions.
`
`1·
`/ ..
`
`
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to ;ctentify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`CA
`CF
`CG
`CH
`Cl
`CM
`cs
`CZ
`DE
`DK
`ES
`Fl
`
`Au,tria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Ccntml African Republic
`Congo
`Swiu.crland
`Cote d'Ivoire
`Cameroon
`c~.cchoslovakia
`(:...cch Republic·
`Germany
`Denmark
`Spain
`Finland
`
`FR
`GA
`GB
`GN
`GR
`HU
`IE
`IT
`JP
`KP
`
`KR
`KZ
`LI
`LK
`I.U
`MC
`MG
`ML
`MN
`
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Democratic People', Republic
`of Korea
`Republic of Korea
`K.!7.akhstan
`Liechtenstein
`Sri l.anl.a
`Luxembourg
`Monaco
`Mad~gascar
`Mali
`Mongolia
`
`MR
`MW
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SK
`SN
`SU
`TD
`TG
`UA
`us
`VN
`
`Mauritania
`Malawi
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Soviet Union
`Chad
`Togo
`Ukraine
`United States of America
`Viet Nam
`
`-!
`
`r ,._~ ._" ..
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`- 1 -
`
`INHIBITION OF P70 S6 KINASE
`The invention was made in the course of work
`funded in part by a grant from the U.S. government, which
`5 has certain rights in the invention. The field of the
`invention is antiproliferative or immunosuppressive
`agents.
`
`Background of the Invention
`A conserved response of many eukaryotic cell types
`10 to mitogenic signals is the phosphorylation of multiple
`serine residues on the 40S ribosomal subunit protein S6
`(Erikson, J. Biol. Chem. 266:6007-6010, 1991). This
`phosphorylation increases the efficiency of protein
`synthesis, which appears to be required in several steps
`15 of cell cycle progression (Erikson, supra). Recently,
`two families of S6 kinases have been characterized at the
`enzymatic and molecular levels:
`the 85-90 kDa (rsk) S6
`kinase family (Jones et al., Proc. Natl. Acad. Sci. USA
`85:3377-3381,, 1988), referred to herein as p90 S6
`20 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
`25 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
`30 cell.
`In some cases 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
`
`,.
`
`,,.
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`-
`
`2 -
`
`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,
`5 acting to remove the phosphate groups added by the
`kinases, and thereby deactivating (or in some cases,
`activating) the substrate polypeptide. In general, each
`member of such a cascade transmutes signals by
`sequentially phosphorylating (if it is a kinase) or
`10 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.
`In T cells expressing the interleukin-2 (IL-2)
`15 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 IL-2 generated
`response is an increase in S6 phosphorylation and a
`20 concomitant increase in the rate of protein synthesis (J.
`Biol. Chem. 262:4624-4630, 1987). Similarly, the multi(cid:173)
`faceted response of insulin-dependent H4 hepatoma cells
`to stimulation by insulin includes an increase in the
`level of S6 phosphorylation, apparently attributable at
`25 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
`30 cellular proteins (termed FK506-binding proteins, or
`FKBPs) to form a biologically active complex, their
`mechanisms of immunosuppression differ significantly.
`The FK506/FKBP complex inhibits a very early step in
`antigen-induced T cell activation, preventing
`35 proliferation of activated T cells by blocking the
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`-
`
`3
`
`-
`
`induction of cytokine gene transcription. Liu et al.
`(Cell 66:807-815, 1991) have recently shown that this
`occurs through the binding of the FK506/FKBP complex to
`calcineurin, a calcium-dependent phosphatase thought to
`5 play a role in T cell signal transduction, thereby
`blocking the phosphatase activity of this enzyme and
`short-circuiting signal transduction along this pathway.
`In contrast, the rapamycin/FKBP complex apparently does
`not bind to or affect the activity of calcineurin, and
`10 acts by blocking a later step in antigen-induced T cell
`activation, one that occurs subsequent to binding of IL-2
`to its receptor (i.e., at a point after the initial
`induction of cytokine gene transcription). The entity
`directly targeted by the rapamycin/FKBP complex has not
`15 been identified, although it has been shown not to be the
`IL-2 receptor itself.
`
`Summary of the Invention
`As disclosed herein, it has now been shown that
`the addition of rapamycin to an IL-2-stimulated T cell
`20 line results in the inhibition of p70 S6 kinase, which
`kinase may constitute the direct target of the rapamycin
`effector complex, or may be downstream of the actual
`target. Furthermore, experiments are herein disclosed
`which demonstrate that rapamycin will inhibit both basal
`25 and insulin-stimulated proliferation and p70 S6 kinase
`activity in an insulin-dependent hepatoma cell line, an
`observation that has broad implications with respect to
`novel medical applications for rapamycin. 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 antipr9liferative agents which act by the same or
`a related mechanism to that of rapamycin:
`i.e., which
`
`30
`
`~
`
`•
`
`
`
`WO 93/19752
`
`PCT /US93/02459
`
`-
`
`4 -
`
`ultimately result in a decrease in p70 S6 kinase activity
`such an assay will pick up not
`in the treated cell.
`only those compounds or complexes which act on the same
`enzyme as that targeted by the rapamycin/FKBP complex,
`5 but also those which act upstream or downstream of that
`particular enzyme in the p70 S6 cascade. The screened
`compounds or complexes (generally referred to as
`compositions} which result in decreased p70 S6 kinase
`activity may act to inhibit a kinase (e.g., p70 S6 kinase
`10 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). The screen will
`also identify those compositions which activate a kinase
`15 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
`20 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
`25 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. The
`30 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
`35 inactivates p70 S6 kinase in vivo, or a phosphatase which
`
`
`
`WO93/t9752
`
`PCT /US93/02459
`
`-
`
`5 -
`
`In preferred embodiments,
`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
`5 kinases and phosphatases upstream of p70 S6 kinase in the
`p70 S6 kinase cascade.
`In order to maximize the window
`of response in this assay, the cell may optionally be
`exposed to an appropriate mitogen (preferably a growth
`factor) before or during step 1. For example, where the
`10 cell is a hematopoietic cell, 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-
`15 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.
`Alternatively, candidate antiproliferative or
`immunosuppressive agents (including, for example, analogs
`of rapamycin and analogs of the rapamycin/FKBP complex)
`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
`(2) determining whether the candidate composition
`30 inhibits the biological activity of the kinase, such
`inhibition being an indication that the candidate
`composition is an antiproliferative or immunosuppressive
`agent. Alternatively, the in vitro assay may employ p70
`S6 kinase as the substrate, and an enzyme just upstream
`
`25
`
`20
`
`...
`
`.•
`
`
`
`WO93/19752
`
`PCT/US93/02459
`
`-
`
`6
`
`-
`
`of p70 S6 kinase as the effector. This method would
`include the following steps:
`(l) providing a sample containing p70 S6 kinase
`and either (i) a serine/threonine kinase capable of
`5 phosphorylating and activating p70 S6 kinase, or (ii) a
`serine/threonine phosphatase capable of dephosphorylating
`and deactivating p70 S6 kinase;
`(2) contacting the sample with a candidate
`antiproliferative or immunosuppressant composition; and
`(3) determining whether the amount of
`phosphorylation of the p70 S6 kinase in the presence of
`the candidate composition is lower than the amount of
`phosphorylation of p70 S6 kinase in the absence of the
`candidate composition, such a lower amount of
`l5 phosphorylation being an indication that the candidate
`composition is an antiproliferative or immunosuppressive
`agent.
`
`io
`
`The invention also includes methods of inhibiting
`the proliferation or immune response of a cell (e.g., a
`20 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)r by introducing into the animal a
`composition that interrupts the p70 S6 kinase cascade via
`a mechanism such as (i) directly inhibiting the activity
`25 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
`30 threonine residue on p70 S6 kinase. By "directly" is
`meant that the altered activity of the given kinase or
`phosphatase results from the interaction between the
`composition (or a complex containing the composition) and
`the given kinase or phosphatase, and not between the
`35 composition and an enzyme upstream to the given kinase or
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`- 7 -
`
`phosphatase. Although it is not known which, if any, of
`these possible mechanisms is the mechanism of
`rapamycin/FKBP, the experimental results obtained with
`rapamycin indicate that a composition which functioned by
`5 any one of these mechanisms would produce the desired
`result.
`
`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
`10 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
`15 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. This method may be used to treat an animal
`20 having a condition characterized by proliferation of a
`cell in response to the given mitogen. The term 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. Examples of such
`25 mitogens include lectins and growth factors (defined as
`naturally-occurring proteins or glycoproteins which acts
`as endogenous mitogens in vivo) 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),
`30 fibroblast growth factor (FGF), platelet-derived growth
`factor (PDGF), and insulin.
`Also within the invention is an analog of
`rapamycin or of the rapamycin/FKBP complex, which analog,
`when introduced into a cell, modulates the activity of a
`35 serine/threonine kinase or serine/threonine phosphatase
`
`
`
`WO 93/19752
`
`PCT /US93/02459
`
`-
`
`8
`
`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
`5 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
`10 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
`15 in the same manner as rapamycin, the rapid screening of
`large numbers of such analogs is now feasible.
`Other features and advantages of the invention
`will be apparent from the following detailed description,
`and from the claims.
`
`20
`
`Detailed Description
`The drawings are first described.
`Drawings
`Fig. 1 is a set of SDS-PAGE gels illustrating the
`effect of IL-2 and PMA on S6 kinase activity, MAP kinase
`25 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)
`induc~ion of tyrosine
`30 phosphorylation. For the in vitro kinase assays (A-C),
`the positions of the corresponding substrates are
`indicated on the left. For the antiphosphotyrosine
`immunoblot (D), the positions of molecular weight markers
`(in kDa) .are indicated on the left.
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`-
`
`9
`
`-
`
`10
`
`15
`
`Fig. 2 is a graph and a set of immunoblot
`autoradiograms illustrating the chromatographic
`resolution of p70 and p90 S6 kinases.
`IL-2-deprived
`cells were either left untreated or treated with
`5 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 o.o
`to 0.5 M, and fractions were collected and analyzed.
`(A) S6 kinase activity. Open circles: untreated,
`unstimulated.
`·closed triangles: untreated, stimulated
`with IL-2. Closed circles:
`treated with rapamycin,
`stimulated with IL-2.
`(B) Immunoblotting with p70 S6 kinase-specific antiserum.
`(C) Immunoblotting with p90 S6 kinase-specific antiserum.
`Treatments and stimulations are indicated on the left,
`fraction numbers are indicated at the bottom, and the
`positions of molecular weight markers (in kDa) are
`indicated on the right. The positions of p70 and p90 S6
`20 kinases are indicated with arrows on the right. The 90
`kDa proteins detected by the anti-p70 S6 kinase antiserum
`(fractions 14-15) correspond to the recently described
`high molecular weight forms of the p70 S6 kinases (Grove
`et al., Mel. Cell. Biol. 11:5541-5550, 1991). Note that
`25 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 SOS-PAGE gels illustrating the
`30 effects of rapamycin and FK506 on S6 ~inase 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).
`
`35
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`- 10 -
`
`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. For the in vitro kinase assays (A-C),
`5 the positions of the corresponding substrates are
`indicated on the left. For the antiphosphotyrosine
`immunoblot (D), the positions of molecular weight markers
`(in kOa) are indicated on the left.
`Fig. 4A is a set of SOS-PAGE gels illustrating the
`10 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% co2 . Cells were then treated with insulin (10- 6 M,
`30 min) or were left untreated. Cells were harvested
`15 into homogenization buffer (10 mM KPi, pH 6.5/1 mM
`EDTA/SmM EGTA/l0mM MgC12/2 mM DTT/ 1 mM V04/50 mM ~(cid:173)
`glycerophosphate/0.1% Triton X-100/2 mM leupeptin/2mM
`pepstatin/0.2 mM PMSF); balanced for protein; and assayed
`for S6 kinase activity according to Nemenoff (Arch.
`20 Biochem. Biophys. 245: 196-203, 1986).
`Fig. 4B is an SOS-PAGE gel illustrating the in
`vivo incorporation of 32Pi into ribosomal protein S6. H4
`hepatoma cells (24-hr serum-starved) were washed with
`phosphate-free, serum-free DMEM, then incubated with 0.5
`25 mCi 32Pi/plate in DMEM for 1 hr. Rapamycin was then added
`at the indicated final concentrations (in quadruplicate).
`After 1 hr of rapamycin incubation, 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
`30 then harvested in 0.5 ml extraction buffer (10 mM KPi, pH
`6.5/1 mM EDTA/5mM EGTA/10 mM MgC12/2 mM DTT/1 mM V04/
`50 mM ~-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.
`35 Supernatants were then balanced for protein (BioRad), and
`
`.
`
`
`
`WO 93/19752
`
`PCT /US93/02459
`
`- 11 -
`
`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
`5 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
`10 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 ~-glycerophosphate, pH 7.2/lmM DTT/lmM EGTA/0.lmM
`15 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
`20 using 40S ribosomes and SKAIPS peptide. Fractions were
`also assayed for total protein and conductivity.
`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
`25 hepatoma cells were incubated in the presence (right) or
`the absence (left) of 20 nM rapamycin for 1 hr at 37°C,
`5% CO2 , after which cells were harvested and extracts
`prepared. Top panel: Extracts were assayed for total S6
`kinase activity using 40S ribosomes as substrates for
`30 phosphorylation. Middle panel: Extracts were
`imm_µnoprecipitated with an affinity-purified polyclonal
`p70 S6 kinase antibody (1 µg/ tube) in the presence of 10
`µl protein A Sepharose beads. After a 3 hr incubation at
`5°C, immunoprecipitated material was washed 3X in
`35 homogenization buffer and 0.25 M NaCl+ 0.1% Triton x-
`
`'>
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`- 12 -
`
`100, followed by one wash in 20 mM Tris HCL pH7.4/lmM
`EGTA/2mM EDTA/2mM DTT/lOmM ~-glycerophosphate/0.1% Triton
`X-100/10% glycerol.
`Immunoprecipitates were then assayed
`for S6 kinase activity using exogenous 40S ribosomes as
`5 substrate. Lower panel: Extracts were
`immunoprecipitated with an affinity-purified polyclonal
`p90 S6 kinase antibody (1 µg per tube) as in the middle
`panel, followed by a determination of S6 kinase activity.
`
`Fig. 7A is a pair of graphs comparing the effects
`10 of rapamycin on p70 S6 kinase, p90 S6 kinase, and
`erkl/MBP kinase. cos cells were transfected with
`expression constructs encoding the rat p70 S6 kinase
`(Grove et al., Mal. Cell. Biol. 11:5541-5550, 1991), the
`rat p90 S6 kinase or the rat p44 erkl/MBP kinase, all of
`15 which had been tagged at the amino terminus with a
`nonapeptide epitope from influenza hemagglutinin (Field
`et al., Mol. Cell. Biol. 8:2159-2165, 1988}. Two days
`following transfection, the cells were treated with the
`indicated concentrations of rapamycin for 60 min and with
`20 100 nM phorbol 12-myristate 13-acetate (PMA) for the
`final 15 min. Cells were lysed with homogenization
`buffer [as in Fig. 4a, but containing 0.1% (w/v) Triton
`X-100], and the extract was clarified by centrifugation
`at 100,000 x g. Recombinant protein was
`immunoprecipitated with the anti-epitope antibody 12CA5
`(immobilized on Sepharose beads) for 3 hat 4°C; pellets
`were washed (3 x 1 ml) in assay buffer [homogenization
`buffer without the MgC12 and with 10% (w/v) glycerol].
`S6 kinase activity was determined as above and p44
`30 erkl/MBP kinase was assayed similarly, using 0.5 mg/ml
`myelin basic protein (MBP) as substrate. Results are
`expressed as the mean pmol of substrate labeled during
`the 15 min assay; error bars indicate standard deviation
`of replicate determinations (upper panel, n=5; lower
`
`25
`
`
`
`WO 93/19752
`
`PCT /US93/02459
`
`- 13 -
`
`panel, n=6). 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
`5 the immunoprecipitants was equivalent.
`Fig. 7B is a bar graph which presents data
`indicating that rapamycin but not FK506 inhibits
`cos cells were transfected
`recombinant p70 S6 kinase.
`with vector or a p70 S6 kinase expression construct, as
`10 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
`15 visualized by chemiluminescence, demonstrating that
`recovery in each of the immunoprecipitants was
`equivalent.
`Fig. 7C is a graph, an autoradiogram of an SDS(cid:173)
`PAGE gel, and a Western blot illustrating the rapamycin
`20 dose response of p70 S6 kinase with respect to
`autophosphorylation of p70 S6 kinase and phosphorylation
`of 40S ribosomal subunits. COS cells were transfected,
`treated with rapamycin, extracted, and immunoprecipitated
`as in 7A. Upper panel: 40S kinase activity was
`25 determined from replicate sampling of duplicate
`immunoprecipitations (mean and standard deviation, n=6).
`Middle panel: Autophosphorylation was determined from
`kinase assays lacking substrate using aliquots of the
`immunoprecipitations from cells transfected with vector
`30 or recombinant p70 S6 kinase (as indiGated) 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
`35 protein w.as detected with the 12CA5 monoclonal antibody
`
`
`
`WO 93/19752
`
`PCT/US93/02459
`
`- 14 -
`
`and visualized by chemiluminescence, demonstrating that
`recovery in each of the immunoprecipitants was
`equivalent.
`Fig. 8 is a pair of graphs illustrating the
`5 rapamycin-mediated inhibition of basal and insulin(cid:173)
`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
`10 assay. Serum-starved (24 hr) rat H4 hepatoma cells, 4 x
`104 cells/well, were cultured at 37°C, 5% CO2 in the
`absence or presence of 10-6 M insulin in the absence or
`presence of rapamycin at the indicated concentrations.
`Proliferation was assessed by the incorporation of [ 3H]-
`15 thymidine in a 16 hr pulse following a 32 hr incubation.
`This experiment is representative of four similar
`experiments.
`Fig. 9 is an illustration of the chemical and 3-
`dimensional structures of FK506, rapamycin, and 506BD
`(Bierer et al., Science 250:556-559).
`
`20
`
`Inhibition of IL2 stimulation of p70 S6
`
`Example 1:
`kinase
`MATERIALS AND METHODS
`Cell culture and stimulation of cells. The IL-2-
`25 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 IL-2 (12~5-25 units/ml,
`30 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 106 cells/ml
`and incubated for 3 hat 37° c. Cells were left
`untreated or treated with 100 nM rapamycin, 100 nM FK506
`
`
`
`WO 93/19752
`
`PCT /US93/02459
`
`- 15 -
`
`or equivalent ethanol diluent (final concentration 0.1%)
`for the last hour and during the subsequent stimulation
`period. Aliquots of 4-10 x 106 CTLL-20 cells were either
`left untreated or incubated in the presence of human
`5 recombinant IL-2 (25 units/ml) or phorbol-12-myristate-13
`acetate (PMA, 50 ng/ml, Sigma) for the indicated times at
`In vitro
`37°C, and then divided into two aliquots.
`In vitro kinase assays were performed
`kinase assays.
`essentially as
`described (Calvo et al., supra). 1-2 x
`10 106 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 mM EGTA, 10 mM MgCl2, 50 mM ~(cid:173)
`glycerophosphate, 1 mM sodium vanadate, 2 mM DTT, 40
`µg/ml PMSF, 10 µg/ml leupeptin, and 1 µg/ml pepstatin.
`15 After 30 min on ice, nuclei were pelleted by a 5-min
`microfuge centrifugation, and supernatants were used for
`kinase assays.
`For the detection of total S6 kinase activity,
`direct S6 phosphorylation assays were performed with 5 µl
`20 of supernatant in a total volume of 30 µl containing 20
`mM Tris-HCl pH 7.25, 10 mM MgC12 , BSA 10-100 µg/ml, 50 µM
`ATP with 5 µCi [y-32P]ATP, and 40S ribosomal subunits as
`substrate (0.1-0.25 mg/ml) prepared from Xenopus laevis
`as described (Erikson et al., J. Biol. Chem. 261:350-355,
`25 1986). For S6 kinase assays specific for p90 S6 kinase,
`100 µl of supernatant were incubated with 5 µl of the
`specific rabbit antiserum 125 (Sweet et al., Mel. Cell.
`Biol. 10:2787-2792, 1990), and immunocomplexes were
`adsorbed to staphylococcus aureus, washed as previously
`30 described (Vik et al., Proc. Natl. Acad. Sci USA 87: 2685-
`2689, 1990), and subjected to S6 phosphorylation assays
`as described above.
`To assay for MAP kinase activity, 5 µl of
`supernatant were used in a total reaction volume of 30 µl
`35 containing 20 mM Tris-HCl pH 7. 25, 10 mM MgCl2, 100 µM
`
`
`
`WO93/19752
`
`PCT /US93/02459
`
`15
`
`- 16 -
`ATP with 5 ~Ci [y-32PJATP, 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
`5 analyzed as previously described (Calvo et al., supra).
`Antiphosphotyrosine immunoblotting analysis. 3-8
`x 106 cells were washed, lysed, and centrifuged as
`described (Calvo et al., supra). The protein
`concentration in the supernatants was determined by a
`10 colorimetric method (Bradford, Anal. Biochem. 72:248-254,
`1976). Equivalent protein amounts of each supernatant
`were resolved by reducing 8-10% SOS-PAGE and analyzed by
`immunoblotting with the antiphosphotyrosine mAb 4G10 and
`anti-mouse IgG alkaline phosphatase-conjugated antibody
`(Promega) as described (Calvo et al., supra).
`Ion-exchange chromatography and anti-p70/p90 S6
`kinase immunoblotting analysis. 1.4 x 108 cells were
`lysed in homogenization buffer (5 mM EGTA, 1 mM EDTA, 10
`mM MgC12 , 50 mM fi-glycerophosphate, 2 mM DTT, 0.2% Triton
`20 x-100, 10% glycerol, 1 mM PMSF, 10 ~g/ml leupeptin, and
`10 ~g/ml pepstatin) and centrifuged at 100,000 x g for
`30 min at 4°C.
`supernatants, normalized for total
`protein content, were applied to a Mono Q (Pharmacia)
`anion-exchange chromato