WO2010141062A1 - Inhibiteurs de janus kinases constitutivement actives et leurs utilisations - Google Patents

Inhibiteurs de janus kinases constitutivement actives et leurs utilisations Download PDF

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WO2010141062A1
WO2010141062A1 PCT/US2010/001538 US2010001538W WO2010141062A1 WO 2010141062 A1 WO2010141062 A1 WO 2010141062A1 US 2010001538 W US2010001538 W US 2010001538W WO 2010141062 A1 WO2010141062 A1 WO 2010141062A1
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jak2
activity
agent
mutant
jakl
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Stefan N. Constantinescu
Alexandra Dusa
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Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
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Ludwig Institute for Cancer Research Ltd
Ludwig Cancer Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • JAK 1 and JAK2 are members of the Janus kinase (JAKs) family of non-receptor tyrosine kinases, crucial to blood formation and immune responses. JAK 1 and JAK2 play a role in downstream signaling pathways such as the JAK/STAT pathway, involved in cytokine signaling. Members of the JAK family possess seven defined regions of conserved homology denoted JAK homology (JH) domains 1-7 (1).
  • JH5-7 make up the amino terminus of JAKs and contain a predicted FERM (Band-4.1, ezrin, radixin, and moesin)-like motif (2), which are important in associating JAKs with their receptors and in some cases receptor cell-surface expression (3, 4, 5).
  • FERM Bit-4.1, ezrin, radixin, and moesin
  • JH3-4 domains display some homology to SH2 domains, their function remains ambiguous (6).
  • the carboxyl terminus is composed of JHl and JH2 and contains the kinase and pseudokinase domains, respectively (7).
  • the JAK2 JHl domain includes all the features of a catalytic tyrosine kinase, while the JH2 domain, though highly sequence-homologous to JHl, lacks several elements conferring catalytic activity.
  • JAKl, JAK2, and JAK3 kinase domains in isolation have been solved in complex with specific inhibitors (8, 9, 10).
  • the JAK2 kinase domain exhibits a typical bilobar arrangement, with a secondary structure profile very similar to other solved kinase domains. (11, 12).
  • the N-terminal lobe of JAK2 is composed of ⁇ -strands and includes a single helix, ⁇ C, while the C-terminal lobe is mostly helical (8, 10).
  • ⁇ C helix
  • C-terminal lobe is mostly helical (8, 10).
  • PV polycythemia vera
  • ET essential thrombocytopenia
  • PMF primitive myelofibrosis
  • V617F mutation induces constitutive tyrosine phosphorylation of JAK2 and STAT5 and renders Ba/F3-EpoR cells cytokine-independent.
  • V617F a comprehensive mechanism of activation of this mutation has yet to be proposed.
  • agents that reduce or inhibit the activation of mutant forms of JAK2 may be designed or identified for use in the treatment of myeloproliferative disorders.
  • agents such as small molecules or peptides may be identified or designed that modulate the activity of JAKl or JAK2, for example, mutant JAKl or mutant JAK2.
  • agents may be identified that interfere with the binding of F617 with F595 of JAK2, or the binding of F658 with F636 of JAKl, or masking/interacting with F595 of JAK2 or F636 of JAKl, thereby inactivating or reducing the activity of the constitutively active kinase.
  • agents may be identified that stimulate the binding of the amino acid residue at position 617 with F595 of JAK2, or the binding of the amino acid at position 658 with F636 of JAKl thereby activating or stimulating the activity of the kinase.
  • Agents that interfere with the interaction of F617 with F595 of JAK2 V617F or with the interaction of F658 with F636 of JAKl may be useful in the treatment of myeloproliferative disorders, such as polycythemia vera, essential thrombocythemia, primitive myelofibrosis, and lymphoblastic leukemia.
  • myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, primitive myelofibrosis, and lymphoblastic leukemia.
  • the invention provides methods of identifying agents that modulate, for example, interfere with, the interaction of F617 with F595 of JAK2 V617F.
  • Agents that may be tested for their ability to modulate this interaction include small molecules, proteins, peptides, aptamers, etc.
  • Agents may be identified by contacting a test agent with JAK2 V617F under suitable conditions for the test agent to interfere with the interaction of F617 with F595.
  • Modulation, for example, interference with the F617-F595 interaction may be determined by assaying the activity of the kinase. An agent that interferes with the interaction between these two amino acids should reduce the constitutive activity of the kinase.
  • an agent that stimulates the interaction between these two amino acids should increase the constitutive activity of the kinase.
  • a plurality of agents ⁇ e.g., a library of chemical compounds) may be screened using the inventive methods.
  • the method may be used in high-throughput screening of a large number of test agents.
  • the invention encompasses agents found to modulate, with the interaction of F617 with F595 of JAK2 V617F, for example, agents that interfere with this interaction.
  • agents may be identified using the inventive assay methods described herein.
  • the modulating agent may be designed in silico based on the three-dimensional structure of the binding pocket. Agents designed in silico may then be tested for their ability to inhibit the constitutive activity of JAK2 V617F.
  • Agents found to inhibit the constitutive activity of JAK2 V617F may be used alone or in pharmaceutical compositions to treat proliferative diseases.
  • the proliferative diseases may be myeloproliferative disorders, such as, but not limited to, polycythemia vera, essential thrombocythemia, and primitive myelofibrosis.
  • myeloproliferative disorders such as, but not limited to, polycythemia vera, essential thrombocythemia, and primitive myelofibrosis.
  • some aspects of the invention provide methods for the identification of agents blocking or inhibiting the interaction of the amino acid F595 and an amino acid K539L, T875N, or R683G in a mutated JAK2. Some aspects of this invention provide methods to identify agents inhibiting constitutive or aberrant K539L, T875N, or R683G mutant JAK2 activity, for example, kinase activity. Some aspects of this invention provide methods of inhibiting K539L, T875N, or R683G mutant JAK2 activity in a cell or a subject.
  • Some aspects of this invention provide methods to use agents found to inhibit K539L, T875N, or R683G mutant JAK2 kinase activity to treat a myeloproliferative disease, a deep venous thrombosis or a leukemia.
  • Some aspects of this invention relate to the discovery that the F636 residue of JAKl interacts with mutated residues implicated in constitutive or otherwise aberrant activation of mutant JAKl, for example, with mutated residue V658F and that inhibition of this interaction inhibits or eliminates the constitutive or aberrant activation of the mutant JAKl. Accordingly, some aspects of the invention provide methods for the identification of agents inhibiting the interaction of the amino acid F636 and amino acid V658F in a mutant JAKl . Some aspects of this invention provide methods to identify agents inhibiting constitutive or aberrant V658F mutant JAKl activity, for example, kinase activity.
  • Some aspects of this invention provide methods of inhibiting V658F mutant JAKl activity in a cell or a subject. Some aspects of this invention provide methods to use agents found to inhibit V658F mutant JAKl kinase activity to treat a myeloproliferative disease or a hematological malignancy, for example, T-ALL (T-adult lymphoblastic leukemia).
  • T-ALL T-adult lymphoblastic leukemia
  • Figure 1 Sequence homology and distances between homologous V617 and F595 positions in other kinases and a model of the relative positions of the two residues in JAK2.
  • A Alignment of part of the pseudokinase domain of JAK2 with other kinases containing a GVCV-like motif.
  • B Distances between the nearest atoms of homologous F595 and V617 residues in the other kinases. Distances were estimated based on the PDB coordinates with UCSF Chimera program (See ref. 32).
  • C Predicted relative position of the JAK2 JHl and JH2 ⁇ C helices based on the homology model of Lindauer et al. (20).
  • residue F595 in the JH2 ⁇ C helix is in the proximity of the mutated V617F residue, also located in the pseudokinase domain, and could make a pi-stacking interaction with the mutated V617F residue, contributing to a change in relative conformations of the JHl and JH2 ⁇ C helices.
  • FIG. 1 Mutation of residue F595 to Ala inhibits the constitutive activity of the JAK2 V617F mutant.
  • the F595A single mutant exhibits an activity level and ligand response similar to WT JAK2.
  • FIG. 3 Effect of the substitution of residue F595 in the JH2 ⁇ C helix on the constitutive activity of JAK2 V617F and on the ligand-induced activation of JAK2 wild-type measured by luciferase assays.
  • A Mutation of F595 to some residues induces up to an 86% decrease in the constitutive activity of JAK2 V617F, depending on the particular substitution, while mutation of F595 to aromatic residues rescues the constitutive activity of JAK2 V617F.
  • B The single substitution mutants at position 595 display Epo responses similar to JAK2 wild-type at various concentrations of Epo and do not induce constitutive activation of JAK2 wild-type.
  • C The JAK2 F595X/V617F double mutants also respond to Epo in a manner analogous to JAK2 wild-type.
  • FIG. 4 Role of JH2 ⁇ C helix residue F595 in the proliferation and activation of Ba/F3 cells stably expressing the Epo receptor and individual JAK2 mutants.
  • A Proliferation assay of sorted Ba/F3-EpoR cells stably expressing each mutant and wild-type, in medium without growth factors for 7 days. JAK2 V617F is able to proliferate constitutively in this minimal medium starting from the first day (black triangles). The F595A/V617F double mutant lost most of its proliferative advantage (green stars), while the Ba/F3-EpoR parental cell line, or expressing either wild-type or F595A alone could not proliferate in the absence of growth factors.
  • JAK2 V617F pJAK2, pSTAT5 and pErkl/2 levels of JAK2 V617F, probed with specific antibodies revealed a constitutive signal in the absence of stimulation. This signal was absent in the cells expressing F595A/V617F but partially rescued in cells expressing F595W/V617F.
  • FIG. 5 Selected cytokine-independent murine JAK2 (mJAK2) V617X mutants display high levels of STAT5 transcriptional activity.
  • mJAK2 cytokine-independent murine JAK2
  • FIG. 5 Selected cytokine-independent murine JAK2 (mJAK2) V617X mutants display high levels of STAT5 transcriptional activity.
  • JAK2 -deficient ⁇ -2A cells which are also deficient in STAT5
  • FIG. 6 Two other JAK2 mutants previously described to be constitutively active, V617W and V617M, show a large decrease in the constitutive activity in the presence of the F595A mutation.
  • A Luciferase assay in ⁇ -2A cells shows that JAK2 V617W alone can induce a constitutive transcriptional activation of STAT5 in the absence of cytokines. When coupled with F595A, the F595A/V617W double mutant loses most of this constitutive advantage, similar to the effect of F595A/V617F.
  • FIG. 7 The JAK2 V617F mutation can induce constitutive activation regardless of the dimeric conformation of the receptor it is bound to, while JAK2 wild-type requires a ligand-activated EpoR dimer in order to signal.
  • A The coiled-coil EpoR constructs were made by replacing the extracellular domain of EpoR with the dimeric coiled- coil domain of the yeast transcription factor Put3.
  • B Luciferase assay in ⁇ -2A cells transfected with each JAK2 and with previously engineered EpoR dimers containing coiled- coil replacements of their extracellular domains.
  • JAK2 wild-type signals best from the EpoR dimeric conformations imposed by cc-EpoR-III and cc-EpoR-VI, previously shown to correspond to activated dimers.
  • V617F has acquired the ability to signal constitutively from both active (cc-EpoR-III, cc-EpoR-VI) and inactive (cc-EpoR-II, cc-EpoR-V) dimeric interfaces.
  • JAK2 F595A/V617F and F595V/V617F have lost the ability to signal constitutively from active and inactive dimeric conformations, suggesting that F595 may play a role in the constitutive activity of JAK2 V617F.
  • residues F617 (red circle) and F595 (red circle in pseudokinase domain helix C, depicted as a grey cylinder) are crucial for initiation activation, which is transmitted to the kinase domain of JAK2, in the absence of the rotation and scissors-like movement of the receptors.
  • FIG. 8 JAK2 V617F and V617W can signal comparatively well from all interfaces of an Epo receptor dimer, while WT can only signal from dimeric interfaces corresponding to an activated EpoR dimer. Eliminating the F at position 595 by mutation to Ala, causes V617F to lose its signaling advantage and to also be constrained to the dimeric interfaces characteristic of an activated EpoR.
  • A Luciferase assay in ⁇ -2A cells transfected with JAK2 WT or V617F and with previously engineered EpoR dimers containing coiled-coil replacements of their extracellular domains imposing all seven possible dimeric orientations.
  • JAK2 WT signals best from the EpoR dimeric conformations imposed by cc-EpoR-III and cc-EpoR-VI, previously shown to correspond to activated dimers (See ref. 23).
  • V617F has acquired the ability to signal constitutively regardless of the particular dimeric interface.
  • JAK2 V617W also displays a comparatively high constitutive STAT5 signal constitutively from all conformations of an EpoR dimer, in a luciferase assay in ⁇ -2A cells.
  • FIG. 10 The F595-F617 interaction is specific to activation of JAK2 V617F and does not affect the JAK2 K539L mutation in the same manner. Predicted arrangement of the JHl and JH2 ⁇ C helices in an inactive conformation (model coordinates from ref. 20), showing the predicted arrangement of residues F595, V617, K539, and D620. [0021] Figure 11. Substitution of F595 to Ala has an inhibitory effect on the constitutive activity of other JAK2 oncogenic mutants.
  • JAK2 K539L, T875N and R683G all induce constitutive activation of JAK2, however all three mutants display a marked decrease in STAT5 transcriptional activity in ⁇ -2A cells when the F595 A mutation is also introduced.
  • B Proliferation assay of sorted Ba/F3-EpoR cells stably expressing each constitutive mutant and wild-type, in medium without growth factors for 7 days. Cells expressing JAK2 V617F (black triangles), K539L (blue lines), T875N (green stars), and R683G (yellow circles) can proliferate constitutively in this minimal medium starting from the first day.
  • the kinase domain is depicted in yellow and the pseudokinase domain in blue.
  • the location of F595 is shown as a green sphere.
  • Circular arrows placed between helices C of JH2 and JHl indicate potential conformational changes originating in JH2 and transmitted to JHl.
  • Solid double-headed arrow placed near the R683G mutation suggests increased flexibility induced by this JH2 hinge mutant.
  • Green star (right panel) suggests T875N mutation changes JH1-JH2 linker segment conformation, which is transmitted to JH2 helix C F595.
  • FIG. 12 The F595A homolog in JAKl, F636A, blocks constitutive activity of JAKl V658F.
  • Transient transfection in the JAKl -deficient U4C cell line indicates a decrease in the STAT3 transcriptional activity in the JAKl F636A/V658F, as compared to JAKl V658F.
  • FIG. 13 Effect of mutating residues in the JH2 helix C on the constitutive and ligand induced activity of JAK2 V617F.
  • Transient transfection of the JAK2-deficient g2A cell line was performed with cDNAs coding for the indicated JAK2 mutants, EpoR, STAT5 and luciferase reporters.
  • Measurement of STAT5 transcriptional activity indicated that residues R588, E592 are not required for constitutive activity of JAK2 V617F.
  • JH2 residues M600, M601 and S602, Q603 are required for constitutive but not Epo-induced activation of JAK2 V617F
  • agent refers to any compound or molecule that is to be tested.
  • agents of the present invention include but are not limited to peptides, small molecules, and antibodies.
  • Agents can be randomly selected or rationally selected or designed.
  • an agent is said to be “randomly selected” when the agent is chosen randomly without considering the specific interaction between the agent and the target compound or site.
  • an agent is said to be “rationally selected or designed”, when the agent is chosen on a non-random basis which takes into account the specific interaction between the agent and the target compound or site and/or the conformation in connection with the agent's action.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human, at any stage of development. In some embodiments, “animal” refers to a non-human animal, at any stage of development. In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or clone.
  • mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • antibody refers to an immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term.
  • the term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody may be monoclonal or polyclonal.
  • the antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.
  • aptamer refers to single stranded oligonucleotides and peptides designed to bind a molecular target with high affinity and/or interfere with the function of a molecular target.
  • the molecular target may be a protein, a nucleic acid, or virtually any other molecule.
  • Aptamers may comprise a variable peptide or nucleotide loop attached at both ends to a scaffold, for example, a protein scaffold.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient of a pharmaceutical composition is combined to facilitate the application or administration.
  • the characteristics of the carrier will depend on the route of administration.
  • physiologically and pharmaceutically acceptable carriers include, without being limited to, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the term "contacting" refers to bringing a JAK2 molecule and a test compound together in a manner that the test agent can affect JAK2 activity. Contacting may be accomplished in a cell-free system, for example, by adding a test agent to a solution comprising an active JAK2 molecule under physiological conditions. Contacting may also be accomplished in a cell, for example, by adding a test compound able to permeate a cell membrane to a culture comprising a cell expressing a JAK2 molecule.
  • the term "effective amount" of an agent refers to an amount sufficient to elicit the desired biological response, for example, a reduction of proliferation of cells in a subject.
  • the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient [0032]
  • the term "in vitro” refers to an artificial environment and to reactions or processes that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.
  • the term "in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • JAKl refers to the Janus Kinase 1 protein.
  • the sequences of the JAKl protein of several species, including human, are well known in the art and exemplary sequences are provided herein. If not further qualified, the term JAKl, is meant to include both wild type and mutant forms, for example, JAKl V658F, of the JAKl protein.
  • JAK2 refers to the Janus Kinase 2 protein. The sequences of the JAK2 protein of several species, including human, are well known in the art and exemplary sequences are provided herein. If not further qualified, the term JAK2, is meant to include both wild type and mutant forms, for example, JAK2 V617F, of the JAK2 protein.
  • JAK2 activity-dependent transcription factor refers to any transcription factor that is affected by JAK2 signaling.
  • the term encompasses transcription factors that are direct phosphorylation substrates of JAK2, for example, some members of the STAT family of transcription factors.
  • the term further encompasses transcription factors activated or inhibited as a result of JAK2 activity, for example, transcription factors that are encoded by target genes of STAT family transcription factors.
  • JAK2 activity-dependent transcription factors are well known in the art and include STAT family transcription factors, for example, STATS.
  • minimal promoter refers to a promoter comprising the binding sites for the base transcriptional machinery, but lacks binding sites for transcriptional transactivators and, therefore, does not exhibit significant transcriptional activity when not bound by a transactivating transcription factor, for example, phosphorylated STAT5.
  • modulating refers to a measurable increase or a decrease in the interaction or activity.
  • an agent identified to modulate mutant JAK2 activity may be either an agent that inhibits ⁇ e.g., decreases) the activity of JAK2 or an agent that stimulates ⁇ e.g., increases) the activity of JAK2.
  • an agent identified to modulate the activity of JAKl may be an agent that inhibits the activity of JAKl or an agent that stimulates the activity of JAKl.
  • the term "pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • a "protein” comprises a polymer of amino acid residues linked together by peptide bonds.
  • the term, as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins.
  • Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, www.cco.caltech.edu/ ⁇ dadg ⁇ /Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed.
  • non-natural amino acids i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, www.cco.caltech.edu/ ⁇ dadg ⁇ /Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels
  • amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in an inventive protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be just a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
  • proliferative disease refers to any disease associated with an undesired and/or abnormal proliferation of cells.
  • the cells may be any type of cell found in the subject.
  • the proliferation may be due to any cause (e.g., any genetic mutation, any signal).
  • proliferative diseases include cancer, neoplasms, inflammatory diseases, autoimmune diseases, graft-vs.-host disease, diabetic retinopathy, and benign tumors.
  • small molecule is used to refer to molecules, whether naturally-occurring or artificially created (e.g. , via chemical synthesis) that have a relatively low molecular weight.
  • a small molecule is an organic compound (i.e., it contains carbon).
  • the small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, heterocyclic rings, etc.).
  • small molecules are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol.
  • Preferred small molecules are biologically active in that they produce a biological effect, for example, a kinase inhibitor produces inhibition of a kinase, in animals, preferably mammals, more preferably humans.
  • the small molecule is a drug.
  • the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
  • drugs approved for human use are listed by the FDA under 21 C.F.R. ⁇ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. ⁇ 500 through 589, incorporated herein by reference.
  • suitable conditions refers to conditions that allow and/or support JAK2 enzymatic activity. Suitable conditions, therefore, allow for a JAK2 molecule to exist in non-denatured form, allow a JAK2 molecule to bind a substrate, and allow a JAK2 molecule to carry out a phosphorylation reaction involving a substrate molecule.
  • a treatment refers to, but are not limited to, one or more clinical intervention with an intent to prevent, ameliorate, or cure a condition or symptoms of the condition in a subject.
  • a treatment is aimed to reduce a level of hematopoietic cell proliferation in a subject having a myeloproliferative disorder.
  • JAKl and JAK2 belong to the Janus kinases (JAKs) family of non-receptor tyrosine kinases, crucial to blood formation and immune responses. JAKl and JAK2 play a role in downstream signaling pathways such as the JAK/STAT pathway, involved in cytokine signaling.
  • JAK homology (JH) domains 1-7 possess seven defined regions of conserved homology denoted JAK homology (JH) domains 1-7 (1).
  • JH5-7 make up the amino terminus of JAKs and contain a predicted FERM (Band-4.1, ezrin, radixin and moesin)-like motif (2), important in association of JAKs to their receptors and in some cases in receptor cell-surface expression (3, 4, 5).
  • JH3-4 domains display some homology to SH2 domains, their function remains ambiguous (6).
  • the carboxyl terminus is composed of JHl and JH2 and contains the kinase and pseudokinase domains, respectively (7).
  • the JAK2 JHl domain includes all the features of a catalytic tyrosine kinase, while the JH2 domain, though highly sequence-homologous to JHl, lacks several elements conferring catalytic activity.
  • JAK2 JHl domain includes all the features of a catalytic tyrosine kinase, while the JH2 domain, though highly sequence-homologous to JHl, lacks several elements conferring catalytic activity.
  • the JAK2 kinase domain exhibits a typical bilobar arrangement, with a secondary structure profile very similar to other solved kinase domains (11, 12).
  • the N-terminal lobe of JAK2 is composed of ⁇ -strands and includes a single helix, ⁇ C, while the C-terminal lobe is mostly helical (8, 10).
  • V617F A single acquired somatic mutation in the pseudokinase domain of JAK2, in the form of a substitution of VaI for Phe at position 617 (V617F), is at the base of >95% Polycythemia Vera (PV) patients and 50-60% of patients with Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF) (13, 14, 15, 16).
  • the V617F mutation induces constitutive tyrosine phosphorylation of JAK2 and STAT5 and renders Ba/F3 cells that express the erythropoietin receptor (EpoR) cytokine-independent.
  • V658F VaI for Phe at position 658 in JAKl, a position homologous to V617 of JAK2, is implicated to cause lymphoblastic leukemia and other myeloproliferative diseases.
  • the present invention is based, at least in part, on the elucidation that the molecular event rendering JAK2 V617F and JAKl V658Fconstitutively active is a pi- stacking interaction of the phenylalanine residue at position 617 of the mutant protein with the phenylalanine residue at position 595 in JAK2, or an interaction of the phenylalanine residue at position 658 of the mutant protein with the phenylalanine residue at position 636 in JAKl . This interaction induces a conformational change resulting in ligand-independent activation of JAK2 V617F or JAKl V658F.
  • Ligand-independent, constitutive activation of JAKl or JAK2 is associated with a number of myeloproliferative disorders, for example, polycythemia vera, essential thrombocythemia, primitive myelofibrosis, and lymphoblastic leukemia.
  • Some aspects of the current invention provide methods to identify an agent that inhibits JAK2 V617F activity and/or JAKl V658F activity.
  • Some aspects of the present invention provide methods to identify an agent that inhibits JAK2 V617F activity and/or JAKl V658F activity but not wild type JAK2 activity and/or wild type JAKl activity, respectively.
  • Agents found to inhibit JAK2 V617F and/or JAKl V658F activity may be useful in the inhibition of aberrant JAKl and/or JAK2 activity in subjects diagnosed with a myeloproliferative disorder.
  • methods to identify an agent that modulates JAK2 V617F or JAKl V658F activity are described herein.
  • methods to identify an agent that inhibits JAK2 V617F or JAKl V658F activity are described herein.
  • methods to identify an agent that inhibits JAK2 V617F or JAKl V658F activity by interfering with the interaction of F595 and F617 in JAK2 V617F or with the interaction of F636 and F658 in JAKl V658F, respectively are described herein. Also described herein are methods to identify an agent that modulates JAK2 V617F activity but does not modulate wild type (wt) JAK2 activity.
  • Methods of administering a JAK2 V617F inhibitor or a JAKl V658F inhibitor to a subject with a myeloproliferative disorder for example, polycythemia vera, essential thrombocythemia, primitive myelofibrosis, or lymphoblastic leukemia are also provided herein.
  • a myeloproliferative disorder for example, polycythemia vera, essential thrombocythemia, primitive myelofibrosis, or lymphoblastic leukemia.
  • Such inhibitors are particularly useful for subjects expressing a mutated form of JAKl or JAK2, for example, JAKl V658F or JAK2 V617F.
  • Janus kinases are a family of intracellular non-receptor tyrosine kinases that transduce cytokine mediated signals via the JAK-STAT pathway.
  • JAK family member proteins range from 120-140 kDa in size and have seven defined regions of homology called Janus homology domain 1-7 (JHl -7).
  • JHl is the kinase domain mediating the enzymatic activity, containing typical features of tyrosine kinases, for example, conserved tyrosines that are essential for JAK activation (e.g.
  • JH2 is a pseudokinase domain structurally similar to a tyrosine kinase and essential for normal kinase activity, but lacking enzymatic activity. This domain may be involved in regulating the activity of JHl.
  • JH3 and JH4 share homology with Src-homology-2 (SH2) domains.
  • JH4-JH7 are called FERM domain (short for band 4.1 ezrin, radixin and moesin) and are involved in association of JAKs with cytokine receptors and/or other kinases.
  • Janus kinase 2 has been implicated, for example, in signal transduction of type II cytokine receptor family members (for example, interferon receptors), GM-CSF receptor family members (for example, IL-3R, IL-5R, and GM-CSF-R), the gpl30 receptor family (for example, IL-6R), and the single chain receptors (for example, Epo-R, Tpo-R, GH- R, PRL-R). JAK2 further transduces signals downstream of the prolactin receptor.
  • type II cytokine receptor family members for example, interferon receptors
  • GM-CSF receptor family members for example, IL-3R, IL-5R, and GM-CSF-R
  • the gpl30 receptor family for example, IL-6R
  • single chain receptors for example, Epo-R, Tpo-R, GH- R, PRL-R
  • JAK2 V617F Mutations in the JAK2 gene leading to aberrant expression or activity patterns of JAK2 are associated with proliferative diseases, for example, leukemia, polycythemia vera, essential thrombocythemia, and other myeloproliferative disorders.
  • a mutation of valine to phenylalanine at the 617 position in JAK2 V617F has been associated with hypersensitivity of hematopoietic cells to growth factors such as erythropoietin and thrombopoietin.
  • the discovery of the elusive molecular mechanism underlying the constitutive activation of JAK2 V617F, as described herein, translates into methods of identifying a JAK2 V617F inhibitor provided by some aspects of this invention.
  • the invention provides methods to reduce the proliferation of hematopoietic cells in a subject having a myeloproliferative disease. In some embodiments, the invention provides methods for treating a proliferative disorder in a subject expressing a mutated form of JAK2, for example, JAK2 V617F.
  • accession numberNP_004963 (www.ncbi.orgwww.ncbi.nlm.nih.gov/), for example, under accession numberNP_004963. [0054] >gi
  • accession number NP_002218 accession number NP_002218. [0059] >gi[102469034
  • Residues F595 of JAK2 and F636 of JAKl are homologous, as are residues V617 of JAK2 and V658 of JAKl .
  • residues V617 of JAK2 and V658 of JAKl are residues V617 of JAK2 and V658 of JAKl .
  • Those of skill in the art will be able to identify additional homologous amino acid residues and sequences of JAKl and JAK2 by methods well known in the art, for example, by sequence alignment. Accordingly, it will be apparent to those of skill in the art that the concepts, methods, and compositions disclosed herein in relation to mutant JAK2 are also applicable in relation to JAKl having the same or similar mutations in homologous residues and vice versa.
  • homologous sequences exist, which, while overall similar, exhibit mutations of one or more amino acid residues.
  • homologous sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical with the JAK2 or JAKl sequences provided herein and are encompassed within the scope of this invention.
  • test agents for example, small organic molecules, peptides, or proteins
  • a test agent can be synthesized or isolated from a natural source.
  • the test agent is purified.
  • a single test agent is characterized by the methods provided herein.
  • a plurality of test agents are characterized, or screened, for example, in parallel, or sequentially.
  • a test agents may be an agent selected, randomly or rationally, from a list of known agents, for example, a list of agents previously synthesized, a list of agents previously administered to a subject, for example, a human subject or a mammalian subject, a list of FDA approved agents, a historical list of agents, for example, a historical list of a pharmaceutical company, etc.
  • Suitable lists of known agents are well known to those of skill in the art and include, but are not limited to, the Merck Index and the FDA Orange Book.
  • Small molecules and libraries of small molecules can be obtained from commercial and academic sources, for example, from Sigma- Aldrich (www.sigmaaldrich.com), ChemDiv (www.chemdiv.com), Evotec (www.evotec.com ), or ICCB (iccb.med.harvard.edu/screening/compound_libraries/index.htm). Combinatorial libraries of small molecules are also well known to those of skill in the art and may be screened for agent that inhibit JAK2 V617F by methods provided herein. [0066] In some embodiments, a library of randomly selected and/or randomly synthesized test agents is screened. In some embodiments, a library of rationally selected or designed test agents is screened. In some embodiments, a library of kinase inhibitors is screened. In some embodiments, JAK inhibitors and closely structurally related test agents are screened.
  • test agents are chosen based on 3D modeling of interactions between the F617 and/or the F595 residue of JAK2 V617F and a test agent.
  • test agents are chosen based on 3D modeling of interactions between the F658 and/or the F636 residue of JAKl V658F and a test agent
  • a test agent is designed in silico based on structural data or interaction data of the target protein, for example, JAK2 V617F or wild type JAK2. Methods of rational design of test agents are well known to those of skill in the related art.
  • test agents are rationally designed de novo.
  • binding affinity and/or selectivity of a test agent is scored in silico, for example, based on an algorithm well known to those of skill in the art, for example, as published in Boehm, 1994, J. Comput Aided MoI Des, 8(3):243-256.
  • Software suitable for various aspects of rational design of test agents is available from commercial and academic sources and is well known to those in the art.
  • Non- limiting examples of software for rational design of test agents are AutoDock (autodock.scripps.edu), Flexidock, SYBYL (by Tripos, tripos.com) SITUS, DOCK, 3D- Dock-Suite (www.bmm.icnet.uk/docking/), ClusPro (structure.bu.edu/Projects/PPDocking/cluspro.html), DOT, SPROUT (www.simbiosys.ca/sprout/index.html), and GrowMol (Ripka et al., Org Lett 2001 26, 3:2309-2312).
  • test agents are chosen based on 3D modeling of interactions between the 617, 539, 875, or 683 residue and/or the F595 residue of JAK2 or mutated JAK2 and a test agent. In some embodiments, test agents are chosen based on 3D modeling of interactions between the 658 residue and/or the F636 residue of JAKl or mutated JAKl and a test agent. Methods of synthesizing rationally designed small molecules are well known to those in the relevant chemical arts.
  • the present invention provides a method for the design and identification of a potential binding compound for V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl, comprising the steps of: (a) using a three-dimensional structure of V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl ; (b) employing the three-dimensional structure to design and/or select the potential binding compound; and (c) synthesizing and/or choosing the potential binding compound.
  • Suitable computer programs which may be used in the design and selection of potential binding compounds include, but are not limited to, GRID (Goodford (1985) J. Med. Chem. 28:849 857); MCSS (Miranker, A. and M. Karplus, (1991) Proteins: Structure. Function and Genetics, 1 1:29-34); AUTODOCK (Goodsell, D. S, and A. J. Olsen (1990) Proteins: Structure. Function, and Genetics 8:195 202); and DOCK (Kuntz, I. D. et al. (1982) J. MoI. Biol. 161:269-288), the entirety of each of which is incorporated herein by reference.
  • Suitable computer programs which may be used in connecting the individual chemical entities or fragments include, but are not limited to, CAVEAT (Bartlett, (1989) Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78:182-196); and 3D Database systems such as MACCS-3D by MDL Information Systems, San Leandro, Calif.), HOOK (Molecular Simulations, Burlington, Mass.) and as reviewed in Martin, Y. C, (1992) J. Med. Chem. 35:2145 2154), the entirety of each of which is hereby incorporated herein by reference.
  • CAVEAT Bartlett, (1989) Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78:182-196
  • 3D Database systems such as MACCS-3D by MDL Information Systems, San Leandro, Calif.), HOOK (Molecular Simulations, Burlington, Mass.) and as reviewed in Martin, Y. C, (1992) J. Med. Chem
  • potential binding compounds may be designed as a whole or "de nov ⁇ " using either an empty active site or, optionally, including some portion(s) of a known inhibitor(s), activator(s) or stabilizer(s).
  • Suitable computer programs include, but are not limited to, LUDI (Bohm, (1992) J Comp. Aid. Molec. Design 6:61-78); LEGEND (Nishibata, Y. and A. Itai, (1991) Tetrahedron 47:8985); and LEAPFROG (Tripos Associates, St. Louis, Mo.).
  • a potential binding compound has been designed, selected, identified, synthesized, or chosen by the methods described herein, the affinity with which that compound binds to V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl may be tested and optimized by computational evaluation.
  • a compound designed, or selected, or synthesized, or chosen as potential binding compound or may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the potential binding compound and the site at which it is bound to V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl make a neutral or favorable contribution to the enthalpy of binding.
  • Suitable computer software which may be used to evaluate compound deformation energy and electrostatic interactions, includes, but is not limited to, Gaussian 92, revision C by M. J. Frisch, Gaussian, Inc., (1992) Pittsburgh, Pa.; AMBER, version 4.0 by P. A.
  • binding compounds may be specifically designed and/or selected and/or synthesized and/or chosen by the above methods to induce non- complementary (e.g., electrostatic) interactions, such as repulsive charge-charge, dipole- dipole and charge-dipole interactions.
  • non- complementary interactions e.g., electrostatic
  • the sum of all electrostatic interactions between the potential binding compound and the site at which it is bound to V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl make a contribution to the enthalpy of binding that is not neutral.
  • the above method comprises using a suitable computer program in designing and/or selecting a potential binding compound. [0075] Additionally, in certain embodiments, the above method comprises using a suitable computer program in conjunction with synthesizing and/or choosing the potential binding compound.
  • the above method further comprises the steps of using a suitable assay, as described herein, to characterize the potential binding compound's influence on V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl activity, stability, folding, and/or intracellular localization.
  • the above method further comprises: (d) contacting the potential binding compound with V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl in the presence of a substrate; and (e) determining the amount of substrate conversion of the V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl compared to a wild-type JAK2 or JAKl, respectively, to determine the effect of the potential binding compound on mutant JAK2 or mutant JAKl enzymatic activity.
  • the above method further comprises the steps of: (d) contacting the potential binding compound with a cell that expresses V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl , for example a cancer cell; and (e) determining the effect of the binding compound on V617F, K539L, T875N, or R683G mutant JAK2, or V658F mutant JAKl activity in the cell.
  • step (e) may comprise determining the effect of the binding compound on the proliferation rate or cell survival.
  • Some aspects of this invention relate to methods for identifying an agent that inhibits JAK2 V617F or JAKl V658F activity. Accordingly, methods are provided herein to determine JAK2 V617F of JAKl V658F activity in the presence of a test agent.
  • Kinase activity assays both cell-based and in cell-free systems, are well known to those of skill in the art. The type of assay is dependent on the specific kinase to be assayed. In some embodiments, a kinase activity assay is used to determine the activity of JAK2 V617F or JAKl V658F in the presence of a test agent.
  • JAKl and JAK2 kinase assays are well known to those of skill in the art (see, for some non-limiting examples, Robers et al., Assay Drug Dev Technol 2008, 6:519-529; Lee et al., J Biomed Sci 2006, 13:773-786; EL-Adawi et al. ; Cardiovasc Res 2003, 57:129-138).
  • JAKl or JAK2 kinase activity may be determined by measuring JAKl or JAK2-mediated phosphorylation of a JAKl or JAK2 substrate, typically a protein or peptide, over time.
  • the kinase activity assay comprises contacting a JAKl or JAK2 molecule with a suitable substrate, for example, a synthetic substrate peptide, and a labeled phosphate group source, for example, a radiolabeled nucleotide, for example, 33 P-ATP or 32 P-ATP, under conditions suitable for JAKl or JAK2 to phosphorylate the substrate, and measuring the transfer of labeled phosphate groups to the substrate over a given time interval.
  • a suitable substrate for example, a synthetic substrate peptide
  • a labeled phosphate group source for example, a radiolabeled nucleotide, for example, 33 P-ATP or 32 P-ATP
  • Substrate peptides and proteins as well as suitable sources of phosphate groups and suitable labels are well known in the art and JAKl and JAK2 kinase activity assays can be purchased from commercial sources for single assays or for use in high- throughput screening methods as provided by some embodiments of this invention (for example, HTScan JAK2 kinase assay, Cell Signaling Technology; JAK2 total ELISA kit, Invitrogen; JAK2 Kinase assay, Perkin Elmer).
  • the substrate may be an enzyme the activity of which changes after phosphorylation by JAKl or JAK2 and may be measured in vitro.
  • JAK2 knockout cells for example, ⁇ -2A cells, are used and JAK2 activity is measured in these cells after artificially expressing JAK2 V617F or wild type JAK2 in these cells.
  • JAKl or JAK2 activity is assayed by measuring the activity of a JAKl or JAK2 substrate, for example, a STAT transcription factor, for example, STAT5, in a cell.
  • the activity of a JAKl or JAK2 transcription factor phosphorylated by JAKl or JAK 2 is measured with a reporter construct comprising a promoter with a binding site for the JAKl or JAK2 phosphorylated transcription factor and a reporter gene, for example, encoding a luciferase, a fluorescent protein, or an antibiotic resistance marker.
  • the reporter construct comprises an artificial promoter, for example, comprising a minimal promoter, for example, a minimal CMV promoter, and a binding site for the JAKl or JAK2 activity-dependent transcription factor, for example, a STAT transcription factor, for example, STAT5.
  • the reporter construct comprises a fragment of a naturally occurring promoter, for example, a promoter bound by a JAK2 activity-dependent transcription factor, operably linked to a reporter gene.
  • a target gene of a JAKl or JAK2 activity-dependent transcription factor for example, a STAT transcription factor target gene, is assayed by methods known in the art to measure JAKl or JAK2 activity. Suitable reporter constructs other than those provided herein will be apparent to those of skill in the art.
  • methods are provided to identify an agent that inhibits JAK2 V617F activity and/or JAKl V658F activity.
  • methods are provided to identify an agent that inhibits JAK2 V617F activity, but does not inhibit wild type JAK2 activity. For example, an agent that interferes with the pi-stacking interaction of F595 and F617 in JAK2 V617F, but does not bind to, or interfere with the induction of the active conformation of wild type JAK2.
  • methods are provided to identify an agent that inhibits JAKl V658F activity, but does not inhibit wild type JAKl activity. For example, an agent that interferes with the pi-stacking interaction of F636 and F658 in JAKl V658F, but does not bind to, or interfere with the induction of the active conformation of wild type JAKl.
  • wild type JAKl or JAK2 activity is determined in the presence of agents identified to inhibit JAK2 V617F activity and/or JAKl V658F activity.
  • JAKl or JAK2 activity is determined with a JAKl or JAK2 kinase assay.
  • JAKl kinase assays are well known in the art and the same assay useful for determining JAKl V658F activity may be used to determine wild type JAKl activity.
  • JAK2 kinase assays are well known in the art and the same assay useful for determining JAK2 V617F activity may be used to determine wild type JAK2 activity.
  • JAKl or JAK2 molecules are contacted with a test agent under suitable conditions or the activity of JAKl or JAK2 is determined under suitable conditions.
  • suitable conditions allow a test agent to interfere with the interaction of F595 and F617 in JAK2 V617F, or with the interaction of F636 and F658 in JAKl V658F.
  • suitable conditions allow a test agent to interfere with the interaction, if any, of amino acid residue 595 and amino acid residue 617 in wild type JAK2, or with the interaction, if any, of amino residue 636 and amino acid residue 658 in wild type JAKl.
  • suitable conditions are physiological conditions.
  • suitable conditions are conditions found in a cell in which JAKl or JAK2 is naturally expressed as well as in cells of the same type in which the JAKl or JAK2 gene has been knocked out.
  • suitable conditions are generated in cell- free assays by providing the necessary salts, co-factors, phosphate source, pH, temperature, reaction partners, and/or adjuvants (for example protease inhibitors, stabilizers etc.), for example, in aqueous solution.
  • Suitable conditions for JAKl and JAK2 kinase activity assays are well known in the art and kits containing buffers and descriptions on how to carry out various in vitro kinase assays can be obtained from commercial sources.
  • Suitable salts and reagents will be apparent to those of skill in the art.
  • a non-limiting example of a commercially available suitable buffer is 50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl 2 , 2 mM DTT, and 0.01% Tween 20.
  • Another non-limiting example of a commercially available suitable buffer is 6.25 mM MOPS, pH 7.2, 3.125 mM ⁇ -glycerol phosphate, 6.25 mM MgCl 2 , 1.25 mM EGTA, 0.5 mM EDTA.
  • Suitable phosphate sources are nucleoside triphosphates, for example, ATP and analogs thereof.
  • a suitable pH is a pH within the range of 7.2-7.5, 5.5-7.2, 7.5-8, 4-5.5, 0-4, or 8-12.
  • Suitable temperatures typically are temperatures allowing for the protein to be active without denaturing the protein. Suitable temperatures typically range from 0 °C-4 0 C, 4 °C-16 °C, 16 °C-25 0 C, 25 °C-37 °C, 37 °C- 39 0 C, 39 °C-50 0 C, or 50 °C-70 °C.
  • Suitable osmolarities are typically within the range of 0.1-10 mM, 10-20 mM, 20-50 mM, 50-100 mM, 100-200 mM, 200-500 mM.
  • the activity of JAKl and/or JAK2 (JAK 1/2) in the presence of a test agent is compared to the activity of JAK 1/2 in the absence of the test agent.
  • the activity of JAK1/2 in the presence of a test agent is compared to the activity of JAK 1/2 in the absence of a test agent.
  • the activity of JAK 1/2 in the presence of a test agent is compared to a reference or control activity. Comparing JAK1/2 activities typically includes a quantification step. The methods used for the quantification of JAK 1/2 activity assay results depend on the readout of the specific assay and may include, for example, quantification of radiation, fluorescence, luminescence, RNA expression, or protein expression.
  • a reference or control activity may be the activity of JAK1/2 in a control sample.
  • a control sample may be a sample comprising the same conditions and reagents as a sample used to determine JAK 1/2 activity in the presence of a test agent, but not containing the test agent, or not containing a test agent.
  • a reference or control activity may be the average activity of JAK 1/2 in multiple control samples, for example, in multiple samples comprising the same conditions and reagents as the samples containing test agents, but without added test agents or containing test agents of known effect on JAK 1/2 activity. Accordingly, a reference or control activity may be a positive or negative reference or control activity.
  • a negative control activity may be a JAK2 activity, for example wild type JAK2 or JAK2 V617F activity, measured under physiological conditions in the absence of a test agent.
  • a test agent may be identified as an agent that inhibits JAK2 activity if the activity measured in the presence of the agent is at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, or 100% less than JAK1/2 activity observed in the absence of the agent, for example, as a negative reference or control activity.
  • a positive reference or control activity may be the activity measured in the presence of a strong JAK 1/2 inhibitor.
  • a test agent may be identified as an agent that inhibits JAK 1/2 activity, if the activity measured in the presence of the test agent is similar or less than the positive control activity.
  • a test agent may be identified as an agent that inhibits JAK1/2 activity, if the JAK1/2 activity in the presence of the test agent is about 300%, about 250%, about 200%, about 150%, about 100%, about 75%, about 50%, about 30%, about 25%, about 10%, or less than 10% the activity observed in a positive control sample, for example, a sample comprising a JAK 1/2 activity inhibitor (for example US Patent Application 2006/0106020, SB1518 (S*Bio), TG101348 (TargeGen), WP1066 (Callisto Pharmaceuticals), AG490 (Tocris)).
  • a JAK 1/2 activity inhibitor for example US Patent Application 2006/0106020, SB1518 (S*Bio), TG101348 (TargeGen), WP1066 (Callisto Pharmaceuticals), AG490 (Tocris)
  • a reference or control activity may be the average JAK1/2 activity in a plurality of samples comprising different test agents, for example, during a screen of a plurality of compounds.
  • a reference or control activity can further be a theoretical or historical activity.
  • the effect on JAK 1/2 activity of a plurality of test agents is screened.
  • at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000 , at least 20000, at least 50000, or at least 100000 test agents are screened. Screening of a plurality of test agents may be performed in parallel or sequentially.
  • methods are provided to identify an agent that inhibits JAK2 V617F and/or JAKl V658F and does not inhibit wild type JAK2 and/or wild type JAKl , respectively.
  • wild type JAK2 is contacted with an agent identified to inhibit JAK2 V617F under conditions allowing the agent to inhibit JAK2 V617F activity and wild type JAK2 activity is measured. If the agent does not significantly affect wild type JAK2 activity, then it is identified as an agent that inhibits JAK2 V617F and does not inhibit wild type JAK2.
  • wild type JAKl is contacted with an agent identified to inhibit JAKl V658F under conditions allowing the agent to inhibit JAKl V658F activity and wild type JAKl activity is measured. If the agent does not significantly affect wild type JAKl activity, then it is identified as an agent that inhibits JAKl V658F and does not inhibit wild type JAKl .
  • An agent that inhibits the activity of the mutant but not the wild type JAK may be useful in decreasing the proliferation of hematopoietic cells in subjects having a myeloproliferative disorder.
  • Methods for the use of an agent that inhibits mutant JAK activity are provided by some aspects of this invention.
  • methods of administering a JAK2 V617F inhibitor or a JAKl V658F inhibitor to a subject having a myeloproliferative disorder or a hematological malignancy are provided.
  • the subject is identified as expressing JAK2 V617F or carrying a gene coding for JAK2 V617F, or as expressing JAKl V658F or carrying a gene coding for JAKl V658F.
  • a treatment according to some aspects of this invention can be a monotherapy, for example, treating a subject only by using one or more methods described herein, for example, to reduce hematopoietic cell proliferation.
  • the treatment also can be an adjunct therapy to one or more other therapies, for example, immune therapies, radiation therapies, and/or chemotherapies.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary agents affecting JAK2 signaling and/or JAKl signaling, such as cytokine inhibitors, and optionally other therapeutic agents.
  • compositions also are capable of being co- mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Therapeutics according to some embodiments of the invention can be administered by any conventional route, for example, injection or infusion over time.
  • the administration may, for example, be oral, intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • an agent inhibiting JAK 1/2 activity may be administered by pulmonary aerosol.
  • an agent is suitable for aerosolic delivery can be readily determined by those of skill in the art. Techniques for preparing aerosol delivery systems containing therapeutic agents are well known to those of skill in the art.
  • compositions of some embodiments of the invention are administered in effective amounts.
  • the desired response may be inhibiting the progression of the disorder. This may involve slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
  • the desired response is a permanent reduction of hematopoietic cell proliferation to levels comparable to those found in healthy individuals.
  • the desired response can be delaying or preventing the manifestation of clinical symptoms characteristic for the disease or condition.
  • the effective amount will depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health care professional treating the subject. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be used for medical reasons, psychological reasons, or virtually any other reasons.
  • Typical dosages for agents identified by methods of this invention are well known to those of skill in the art or can be determined empirically. Typical dosage ranges are (given as weight of agent/body weight of the subject), for example, 1-100 ng/kg, 0.1-1 ⁇ g/kg, 1-100 ⁇ g/kg, 0.1-1 mg/kg, 1-10 mg/kg, 10-100 mg/kg, or 0.1-1 g/kg. It will be apparent to those of skill in the art that the dosage will depend on the structure and characteristics of the agent to be administered, the administration route, and other factors well known to those of skill in the pharmaceutical and medical arts. Administration may be as a single dose, or as multiple, subsequent doses.
  • compositions according to some embodiments of this invention preferably are sterile and contain an effective amount of one or more therapeutic agents as described herein for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by determining the proliferation of hematopoietic cells in a subject after treatment by, for example, cell counting, flow cytometry, FACS, and other methods well known in the art to be suitable to determine cell proliferation.
  • the doses of one or more therapeutic agents as described herein (for example, small molecule, peptide, protein, antibody, or aptamer) administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • compositions may contain suitable buffering agents, for example, acetic acid in a salt form, citric acid in a salt form, boric acid in a salt form, and/or phosphoric acid in a salt form.
  • suitable buffering agents for example, acetic acid in a salt form, citric acid in a salt form, boric acid in a salt form, and/or phosphoric acid in a salt form.
  • compositions also may contain, optionally, suitable preservatives, such as: ascorbic acid, benzalkonium chloride, chlorobutanol, parabens, EDTA, EGTA, and/or thimerosal.
  • suitable preservatives such as: ascorbic acid, benzalkonium chloride, chlorobutanol, parabens, EDTA, EGTA, and/or thimerosal.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. [00101] All methods may include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other examples of compositions include suspensions in aqueous liquids or nonaqueous liquids, such as a syrup, elixir, or an emulsion.
  • compositions for parenteral administration include, without being limited to, sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • aqueous carriers are water, alcoholic/aqueous solutions, emulsions or suspensions, for example, saline and buffered media.
  • parenteral vehicles are sodium chloride solution, Ringers dextrose, dextrose and sodium chloride, and lactated Ringers or fixed oils.
  • intravenous vehicles are fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringers dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases, and the like.
  • the pharmaceutical agents of some embodiments of the invention may be administered alone, in combination with each other, and/or in combination with other drug therapies and/or treatments.
  • therapies and/or treatments may include, but are not limited to, surgical intervention, chemotherapy, radiotherapy, and adjuvant systemic therapies.
  • JAK2 V617F Constitutive Activation Requires JH2 Residue F595: A Pseudokinase Domain Target for Specific Inhibitors
  • This model places residue V617 in a loop connecting ⁇ -strands 4 and 5 in the N-terminal lobe of JH2 and in close proximity to the JH2 ⁇ C helix.
  • the ⁇ 4/ ⁇ 5 loop as well as the ⁇ C- ⁇ 4 loop that precedes ⁇ 4, were previously shown to play regulatory roles in the mechanisms of Src and AbI tyrosine kinases through interactions with the ⁇ C helix in the N-terminal lobe (33, 34).
  • a specific conformation of the ⁇ C helix is essential for kinase activation (18) and members of the kinase family have evolved diverse ways to influence the position of their ⁇ C helices as a means to affect activity (11, 12, 19).
  • V617 to Ala does not support constitutive activation of JAK2 (22).
  • Epo erythropoietin
  • JAK2 WT and JAK2 F595A were not able to support constitutive proliferation in this minimal medium, but as expected they could grow in the presence of Epo, while JAK2 V617F proliferated at a very high rate in the absence of any growth factors (Figure 4a).
  • the cells expressing F595A/V617F lost most of their proliferative advantage, consistent with a specific interaction between F595 and F617 being responsible for the preservation of constitutive activity ( Figure 4a).
  • the sorted Ba/F3-EpoR cells expressing each mutant were stimulated or not with Epo, lysed and immunoblotted in the presence of phosphospecific antibodies (Figure 4c).
  • 293T-derived BOSC cells were used to produce retroviral supernatants of the JAK2 mutants cloned into the bicistronic vector pMX-IRES- GFP.
  • Ba/F3 cells expressing the murine EpoR were infected with the supernatants and sorted for similar GFP levels 72 hours later.
  • the sorted cells stably expressing each JAK2 mutant were washed and their proliferation in growth factor-free medium was followed for 7 days.
  • F595A also decreases activity of JAK2 V617W and V617M
  • Orientation of the dimeric Epo receptor is essential for activation of JAK2 WT, but not for V617F or V617W
  • JAK2 V617F can overcome the requirement for a ligand-induced conformational change and effectively be capable of signaling from all possible relative conformations of a dimeric cytokine receptor, while in the wild-type scenario the proper conformation of JHl ⁇ C-helix can only be achieved by a ligand-induced conformational change in the receptor transmembrane and cytoplasmic domains leading to the proper active conformation and triggering catalytic activity.
  • F595-F617 interaction affects the relative position of the ⁇ C-helices of JHl and JH2
  • the homology model of the combined structure of the JHl and JH2 domains in an inactive conformation proposes an interaction interface between the ⁇ C-helices of both domains defined by two pairs of salt bridges between R588.E890 and E592.R893 (20).
  • both available crystal structures of the active JHl domain of JAK2 (8, 10) describe a E890.R893 salt bridge within the JHl ⁇ C-helix itself.
  • the R588.E890 and E592.R893 salt bridges could be part of a network of interactions responsible for keeping the JHl ⁇ C-helix in an inactive conformation in the absence of ligand.
  • Pseudokinase domain F595 is required for activation of JAK2 K539L
  • Pseudokinase domain ⁇ C helix residue F595 is also required for constitutive activation of the JAK2 mutants T875N and R683G
  • JAK2 K539L is the prototype for all class of exon 12 JAK2 mutations.
  • Another point mutation first identified in a megakaryoblastic cell line derived from an acute megakaryoblastic leukemia (AMKL) patient, and subsequently detected in a screen of human AMKL cell lines for STAT5 activation, presented a T875N substitution in the kinase domain of JAK2 (38).
  • the JAK2 R683G mutation located in the hinge region between N-terminal and C-terminal lobes of the pseudokinase domain was identified in a screen of pediatric acute lymphoblastic leukemia (ALL) patient samples (39).
  • JAK2 K539L, T875N and R683G were shown to exhibit constitutive STAT5 activation and to transform Ba/F3 cells expressing the EpoR to cytokine-independence (37, 38, 39).
  • JAKl helix C residue F636 is also required for constitutive activity of JAKl V658F
  • JAK2 V617F homologous mutation in JAKl , V658F induces constitutive activation of JAKl (30).
  • JAKl V658F mutation was also identified from a screen of acute leukemia patients (40).
  • F595 homologous residue in JAKl, F636 also plays a role in the constitutive activation of this kinase, in a similar manner to JAK2.
  • JAKl V658F mutation was identified (a mutation reported in Staerk J, Kallin A, Royer Y, Diaconu CC, Dusa A, Demoulin JB, Vainchenker W, and Constantinescu SN, JAK2, the JAK2 V617F mutant and cytokine receptors. Pathol Biol (Paris). 2007 Mar;55(2):88-91.) or several other mutations in the pseudokinase domain which are likely to behave similarly and depend on F636.
  • JAK2 V617F Despite an overabundance of data underlining the role of JAK2 V617F in various myeloproliferative syndromes, the mechanism of its constitutive activation has remained elusive.
  • Residue F595 residing in the middle of the pseudokinase domain ⁇ C helix, plays a key role in the constitutive activation of JAK2 V617F and of several JAK2 mutants located in different locations of the protein, JAK2 K539L, JAK2 T875N and JAK2 R683G.
  • Residue F595 is not crucial for the mechanism of ligand-induced JAK2 activation, as shown by the signaling abilities of the F595 single and double mutants in the presence of various concentrations of Epo ligand. Also, mutation of F595 to several different residues did not induce constitutive signaling from wild-type JAK2.
  • JAK2 V617F constitutive activity of a JAKl mutant homologous to JAK2 V617F (JAKl V658F) was also inhibited by the homologous JAKl F595 mutation (F636A), suggesting that our results in JAK2 might be relevant for activation of JAKl mutants recently identified in T-ALL (40, 41).
  • F595A was able to decrease the constitutive activity of JAK2 V617W, a mutant which we previously showed is able to induce a myeloproliferative phenotype in mice, similar to the one induced by V617F 22.
  • Both Phe and Trp are large aromatic amino acids, pointing to the mechanism of constitutive activation being based on the ability of these two amino acids to form strong pi-stacking hydrophobic interactions with F595, when substituted for VaI at position 617.
  • the strict requirement of hydrophobicity is emphasized by the inability of Tyr, which only differs from Phe by an OH group, to induce constitutive activity of JAK2 (22).
  • hydrophobic interaction being pi-stacking
  • JAK2 V617F and V617W mutations in comparison to the other hydrophobic residues such as He, Met, and Leu which can also induce constitutive activity (22).
  • the double mutant F595A/V617F lost its ability to signal constitutively from all conformations of the EpoR dimer, and maintained an activity level similar to WT, pointing to the specific interaction between F595 and F617 being responsible for conferring the constitutive activity.
  • Our results are in line with those of previous publications (28, 29) that showed that the mere presence of a dimeric cytokine receptor promotes JAK2 V617F signaling.
  • JAK2 V617F can induce signaling in the absence of co-expressed EpoR (30, 31), probably via endogenous receptors, such as IL3 receptor beta (29), also pointing towards an intrinsic driving mechanism for activation of the kinase domain.
  • JAK2 V617F When co-expressed with both active and inactive EpoR dimers, JAK2 V617F was able to induce a constitutive signal, regardless of the relative dimeric conformation ( Figure 7B). JAK2 mutants V617I, V617L or V617M could signal from both inactive and active cc-EpoR dimer conformations, but they signaled stronger from active cc-EpoR dimers. JAK2 wild- type, on the other hand, was activated only in the presence of the particular conformations corresponding to an activated EpoR dimer.
  • F617: F595 pair is optimal for constitutive activation, irrespective of receptor conformation, but that other bulky aliphatic residues at 617 can induce constitutive activation of JAK2 via F595, with the caveat that such mutants might additionally require a conformational change of the scaffold receptor, in this case EpoR.
  • F595 is pivotal for initiating autoactivation of JAK2, while it is not crucial for cytokine-induced JAK2 activation.
  • a large conformational change of the receptor that involves rotation (23) and a scissors-like movement (43), brings about kinase domain activation (model, Figure 7C).
  • the F617-F595 interaction might be a unique hydrophobic pocket in the pseudokinase domain of JAK2 V617F that could be targeted by small molecules and facilitate isolation of those that would specifically inhibit JAK2 V617F and spare wild type JAK2.
  • JAK2 kinase domain inhibitors that are ATP-binding competitors are in clinical trials for primary or secondary myelofibrosis (46, 47). These inhibitors do not discriminate between wild-type and mutant JAK2, and can induce unwanted effects, such as anemia and thrombocytopenia.
  • An ideal inhibitor for patients harboring JAK mutants would have to preferentially target the mutant JAK and spare signaling by the wild-type JAK.
  • the region in the middle of the pseudokinase domain helix C around residue F595 could be the target of such a specific inhibitor.
  • Gamma-2A and U4C cells are JAK2-def ⁇ cient and JAKl -deficient human fibrosarcoma cells (36, 48).
  • Ba/F3-EpoR cells are murine IL3-dependent cells that are expressing the murine erythropoietin receptor. Wild-type and mutant JAKs were transfected into BOSC packaging cells to produce retroviruses which were subsequently used to infect Ba/F3-EpoR cells as described (23).
  • GFP positive cells were sorted by FACS 72 hours after infection, washed, and cultured in the absence of cytokines in RPMI medium + 10% FBS. Cell numbers were recorded over a period of seven days with a Coulter cell counter.
  • the V617F mutant which acquired the ability to proliferate in the absence of cytokines was further cultured in RPMI medium + 10% FBS.
  • the sorted Ba/F3-EpoR cells expressing the JAK2 mutants or wild-type (WT) were maintained in RPMI medium + 10% fetal bovine serum (FBS) and WeHI cell supernatant, as a source of IL-3.
  • the STAT5 transcriptional activity of the various mutants was measured in ⁇ -2A cells (fibrosarcoma cells deficient in JAK2) by dual luciferase assays with the STAT reporter, pGRR5-Luc (31). Cells were seeded in 24-well plates overnight and transfected using lipofectamine, with pGRR5, STAT5, EpoR, the cDNA coding for each individual JAK mutant and pRLTK-Luc as an internal control.
  • the STAT3 transcriptional activity of JAKl mutants was measured in JAKl -deficient U4C cells (48) by transiently transfecting each JAKl construct, the pGRR5-Luc and pRLTK-Luc reporters and the interleukin-9 receptor (IL-9R).
  • Medium was changed 4 hours after transfection and stimulation with Epo was performed when stated..
  • the cells were lysed 24 hours after transfection, and luminescence was recorded on a TD-20/20 or Glomax 96-well plate luminometer.
  • Membranes were blocked in 5% milk/TBS-Tween, immunoblotting was performed overnight at 4°C with rabbit anti-phospho-JAK2 (Yl 007/1008) or rabbit anti phospho-STAT5 A/B (Y694) (both from Cell Signaling Technology) in a solution of 5% BSA in TBS-T ween with a 1:1000 antibody dilution. Secondary anti-rabbit-horseradish peroxidase antibodies (GE Healthcare, UK) were used in a 1 : 10,000 dilution in 5% milk/TBS-Tween. The membranes were stripped and reprobed with anti-JAK2 and anti- STAT5, respectively (both from Cell Signaling Technology), using the same dilutions and secondary antibodies as for the phospho antibodies.
  • Western blotting antibodies were directed against: phospho- JAK2 (Millipore), JAK2 (Santa Cruz), beta-actin (Sigma), and phospho-STAT5 A/B (Tyr694), phospho-Erkl/2 (Tyr202/Tyr204), Erkl/2 (Cell Signaling Technology).
  • Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. MoI. Cell 8, 1327-38 (2001).
  • JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.
  • JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model.
  • JAK2V617F-induced polycythemia vera Cancer Cell 13: 311-320.

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Abstract

Certaines mutations dans JAK1 et JAK2 entraînent l'activation constitutive de la kinase respective. Par exemple, la mutation JAK1 V658F, qui rend la kinase JAK1 constitutivement active, est impliquée dans la leucémie lymphoblastique. De manière similaire, la mutation JAK2 V617F, qui rend la kinase JAK2 constitutivement active, est présente chez plus de 95 % des patients atteints de la maladie de Vaquez et chez 50-60 % des patients atteints de thrombopénie essentielle et de myélofibrose primitive. Jusqu'à présent, le mécanisme de l'activation constitutive de JAK1 V658F et JAK2 V617F était resté obscur. La présente invention découle de l'identification d'une interaction hydrophobe entre les résidus F658 et F636 dans JAK1 ou entre les résidus homologues F617 et F595 dans JAK2, toutes les deux étant situées dans le domaine de pseudokinase. L'interaction intramoléculaire des deux résidus phénylalanine est responsable de l'activation constitutive de JAK1 V658F ou JAK2 V617F. Des agents comme des petites molécules, des peptides ou des aptamères peuvent être criblés pour déterminer leur capacité à interférer avec cette interaction, en réduisant ainsi l'activité de la kinase. Une fois identifiés, ces composés peuvent être utiles dans le traitement de troubles myéloprolifératifs, comme la maladie de Vaquez, la thrombopénie essentielle, la myélofibrose primitive et la leucémie lymphoblastique, en particulier chez les sujets exprimant JAK1 V658F et JAK2 V617F.
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WO2015109275A1 (fr) * 2014-01-20 2015-07-23 New York University Modèle atomique pour la janus kinase -2 (jak2) et utilisations associées
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US11661422B2 (en) 2020-08-27 2023-05-30 Incyte Corporation Tricyclic urea compounds as JAK2 V617F inhibitors
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