WO2008118626A9 - Inhibitors of jnk and methods for identifying inhibitors of jnk - Google Patents

Inhibitors of jnk and methods for identifying inhibitors of jnk

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Publication number
WO2008118626A9
WO2008118626A9 PCT/US2008/056341 US2008056341W WO2008118626A9 WO 2008118626 A9 WO2008118626 A9 WO 2008118626A9 US 2008056341 W US2008056341 W US 2008056341W WO 2008118626 A9 WO2008118626 A9 WO 2008118626A9
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WO
WIPO (PCT)
Prior art keywords
jnk
jip
compound
polypeptide
fragment
Prior art date
Application number
PCT/US2008/056341
Other languages
French (fr)
Other versions
WO2008118626A3 (en
WO2008118626A2 (en
Inventor
Maurizio Pellecchia
Original Assignee
Burnham Inst Medical Research
Maurizio Pellecchia
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Application filed by Burnham Inst Medical Research, Maurizio Pellecchia filed Critical Burnham Inst Medical Research
Publication of WO2008118626A2 publication Critical patent/WO2008118626A2/en
Publication of WO2008118626A9 publication Critical patent/WO2008118626A9/en
Publication of WO2008118626A3 publication Critical patent/WO2008118626A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • Obesity and type 2 diabetes are the most prevalent and serious of the metabolic diseases, affecting more than 50% of adults in the USA (Must, 1999).
  • Obesity activates the kinase JNK in liver, skeletal muscle, and adipose tissue.
  • Recent in vivo data are in line with previous reports showing that activation of JNK by pro-inflammatory cytokines inhibits insulin signaling in mouse embryonic fibroblasts, 3T3-L1 and 32Dir cells, through phosphorylation of Ser307 of IRSl (Uysal et al., 1997).
  • Insulin activates JNK in L6 myotubes, rat adipocytes and Rat-1 fibroblasts, indicating that JNK could act as an important negative feedback regulator of insulin signaling (Standaert et al., 1999).
  • JNK binds to scaffold proteins and substrates containing a D-domain having the consensus sequence R/KXXXXLXL (Kallunki et al., 1996; Yang et al., 1998).
  • JNK-interacting protein- 1 (JIPl) is a scaffolding protein which enhances JNK signaling by creating a proximity effect between JNK and upstream kinases (Whitmarsh et al., 1998).
  • the JNK-JIPl interaction is mediated by a specific, high affinity D-domain on JIPl.
  • the mechanism of JNKl inhibition by pepJIPl is mainly due to the competition of pepJIPl with other D-domains of substrates or upstream kinases (Bonny et al, 2001; Ho et al, 2003). However, this is not the only mechanism through which pepJIPl inhibits the kinase.
  • the x-ray structure of the catalytic cleft in the complex shows that the ATP- binding site is distorted on binding of pepJIPl to JNKl, resulting in a reduced affinity of ATP to JNKl.
  • pepJIPl inhibits JNKl phosphorylaton of myelin basic protein (MBP), which is a substrate without a docking site.
  • MBP myelin basic protein
  • assays were developed which allow for rapid screening of a number of test compounds to identify modulators of the JIP/JNK interaction.
  • the modulators are JIPl mimmetics.
  • a number of compounds that bind to the JIPl binding pocket of JNK and thereby inhibit its function were identified.
  • the invention also provides pharmaceutical compositions, kits comprising reagents for the disclosed methods, kits comprising compounds and/or pharmaceutical compositions of the invention and methods of treating patients having a metabolic disorder.
  • the invention provides methods to identify modulators of the JIP/JNK interaction, by providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent, washing the solid support to remove molecules not associated with the solid support, separating the label from the ligand; and determining the amount of separated label.
  • the polypeptide comprising JIPl or a fragment thereof is coupled to biotin.
  • the solid support comprises streptavidin.
  • the polypeptide comprising JNK further comprises glutathione S transferase (GST) or a His tag.
  • the ligand is an antibody, e.g., an anti GST antibody.
  • the label is a lanthanide, e.g., europium.
  • the solid support is a multi-well plate.
  • the JNK is JNKl .
  • the JNK is JNK2.
  • the methods further include determining the amount of separated label in the associated molecules. In yet another embodiment, the methods further include determining the amount of separated label in the unassociated molecules.
  • the methods further comprise the step of pre-selecting test agents using virtual docking.
  • the invention provides methods to identify modulators of the JIP/JNK interaction, by providing a sample comprising a test agent, and a polypeptide comprising labeled JIPl or a fragment thereof, wherein the label is a fluorophore; and determining fluorescence polarization in the sample relative to a control sample that lacks the test agent.
  • the JNK is JNKl . In another embodiment, the JNK is JNK2. In another embodiment, the fluorophore is fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • the methods further comprise the step of pre-selecting test agents using virtual docking
  • the invention provides methods to identify a modulator of the JIP/JNK interaction, by contacting a solid support comprising a polypeptide comprising JIP or a fragment thereof with a sample comprising a fusion protein comprising JNK or a fragment thereof, a labeled ligand for JIP, and a test agent, separating molecules not associated with the solid support comprising JIPl or the fragment thereof from molecules associated with the solid support, separating the label from the ligand, and determining the amount of separated label.
  • the polypeptide comprising JIP or the peptide thereof is coupled to biotin.
  • the solid support comprises streptavidin.
  • the JNK fusion protein further comprises GST or a His tag.
  • the ligand is an antibody.
  • the label is a lanthanide.
  • the solid support is a multi-well plate.
  • the JNK is JNKl . In another embodiment, the JNK is JNK2.
  • the methods further include determining the amount of separated label in the associated molecules. In yet another embodiment, the methods further include determining the amount of separated label in the unassociated molecules. In one embodiment, the methods further comprise the step of pre-selecting test agents using virtual docking.
  • the invention provides assays which do not use a solid support, e.g., soluble assays.
  • the invention provides methods for identifying a modulator of JIP/JNK interaction, by contacting a complex comprising polypeptides comprising JIP and JNK, or fragments thereof, each of which contains one of a donor and an acceptor fluorophore, with a test agent, and measuring the FRET between the donor and acceptor fluorophore, thereby identifying a modulator of the JIP/JNK interaction of JIP and JNK.
  • a decrease in the FRET indicates that the test compound modulates the interaction.
  • the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex. In a specific embodiment, the fluorescent acceptor is attached to JIP.
  • the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK.
  • the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
  • the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively.
  • the FRET is time resolved FRET.
  • the methods of the invention also provide isolating and identifying the test compound.
  • the methods further comprise the step of pre-selecting test agents using virtual docking.
  • the invention provides methods for identifying modulators of JIP/JNK interaction by contacting a solution comprising a polypeptide comprising JIP, or a fragment thereof, wherein the polypeptide comprising JIP, or a fragment thereof, comprises a fluorescent donor or acceptor, with a test agent; adding to the solution a polypeptide comprising JNK, or a fragment thereof, comprising a donor or acceptor, wherein the JNK polypeptide comprises either a fluorescent donor or acceptor suitable to form a fluorescent donor/acceptor pair with the JIP fluorophore; and measuring the FRET between the donor and acceptor fluorophore, thereby identifying a modulator of the JIP/JNK interaction.
  • a decrease in the FRET compared to a solution lacking the test compound indicates that the test compound modulates the interaction of JIP and JNK.
  • the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex.
  • the fluorescent acceptor is attached to JIP.
  • the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK.
  • the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
  • the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively.
  • the methods of the invention further provide for isolating and identifying the test compound.
  • the invention provides methods for inhibiting JIP/JNK interaction in cells comprising contacting cells with an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides methods for treating a metabolic disorder comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides methods for treating diabetes, comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides methods for treating obesity comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides compounds for the treatment of a metabolic disorder, wherein the compound has the structure set forth as formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides compounds for the treatment of a metabolic disorder, wherein the compound has a structure set forth in Table I, Ia, II, Ha, III, IV, V, or VI set forth herein below.
  • the metabolic disease is obesity or diabetes.
  • the invention provides compounds having the structure set forth as formula (II), (III), (IV) or (V) further described herein below.
  • the invention provides pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier therefor.
  • the pharmaceutical compositions are for the treatment of a metabolic disorder, e.g., obesity or diabetes.
  • the invention provides a kit comprising one or more reagents useful in the methods of the invention and instructions for use.
  • kits for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent donor and a JIP polypeptide, or fragment thereof, comprising a fluorescent acceptor and instructions for use.
  • kits for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent acceptor and a JIP polypeptide, or fragment thereof, comprising a fluorescent donor and instructions for use.
  • the invention provides kits comprising one or more compounds or pharmaceutical compositions of the invention and instructions for use.
  • the invention provides a compound identified by any one of the methods described herein.
  • the methods of the invention may comprise an additional step of screening a potential modulator of JNK/JIP interaction against a JNK2 mutant to determine active site binding location.
  • the additional step may comprise the step of screening a potential modulator of JNK/JIP interaction against R127A and C167S mutants of JNK2 to determine if the compound is binding in the JIP pocket.
  • Figure 1 is a schematic representation of the use of Delfia ® (Dissociation enhanced lanthanide fluorimmuno assay) to identify JIPl mimics.
  • Delfia ® Dissociation enhanced lanthanide fluorimmuno assay
  • Figures 2A-B set forth a (A) graph showing titration of a JIP peptide with Delfia ® and JNK and (B) dinase assay with GST-cJun and JIPl.
  • Figure 3 sets forth exemplary compounds (5328208 and 5245976) identified with Delfia ® and JNK as modulators of the JIP1/JNK interaction.
  • FIGS 4A-B depicts NMR binding data for (A) 5328208 and (B) 5245976 in the presence of GST-JNK2.
  • Figure 5 depicts the results of a displacement assay for 5245976.
  • Figure 6 sets forth analogs of 5245976 (6115184, 7246555 and 7261715) and their percent displacement.
  • Figure 7 depicts the results of a competition assay for 11E5, 11E6, 11E8 and 11E9.
  • Figure 8 depicts the results of a kinase assay for 11E9.
  • the IC 50 of 11E9 was determined to be approximately 25 ⁇ M.
  • Figure 9 sets forth analogs of 5245976.
  • Figure 10 sets forth the structures of compounds identified after molecular docking,
  • Figure 10 also sets forth the percent inhibition for these compounds at 25 ⁇ M. The percent inhibition is 80%, 83%, 80%, and 100% for 17A5, 17C6, 19E2 and 24Dl, respectively.
  • Figures HA-C set forth IC 50 data for 17C6, 19E2 and 24Dl.
  • Figure 12 sets forth compounds identified by NMR (7E5 and 7El 1).
  • Figure 13 sets fort TIr, STD, and WaterLogsy NMR data for 7E5 and 7El lat 400 ⁇ M in the presence of 5 ⁇ M JNK2.
  • Figures 14A-B sets forth the kinase assay results for 5245976.
  • Upper panel lanes 1-11 Hek293T cells transfected with a plasmid encoding HA-JNKl.
  • Lower panel lanes 1-11 Hek293T cells transfected with a plasmid encoding HA-JNK2.
  • Lane 1 no inhibitor; lane 3) 7El 1 200 ⁇ M; lane 4) 7E5 200 ⁇ M; lane 5) 5328028 200 ⁇ M; lane 7) 5245976 200 ⁇ M; lane 8) JIP 10 ⁇ M; lane 9) SP600125 10 ⁇ M; and lane 12) no inhibitor GFP control.
  • JNKl or JNK2 were purified by immunoprecipitation with anti-HA antibodies and after a final wash, the immunoprecipitate was separated in different aliquots and incubated or not with the "candidate" JNK inhibitor.
  • Figure 15 schematically depicts the soluble assay for modulators of JIP/JNK interaction.
  • Figure 16 depicts the results of a statistical analysis of a soluble assay of the invention. Z was determined to be 0.8 indicating that the assay is very reliable.
  • FIGS 17A-C depict the LANCE assay results for JIPl, 24Dl and SPB07895, respectively.
  • Figures 18A and B depict the polypeptide sequence of JIP (SEQ ID NO:1) and JNK (SEQ ID NO:2), respectively.
  • Figures 19A-D depict the in vitro characterization of pep-JIP and BI-78D3: A) surface representation of JNKl in complex with ball and stick representation of pepJIPl (RPKRPTTLNLF) and the ATP mimic SP600125 (PDB-ID IUKI), surface generated with MOLCAD (Teschner et al., 1994) and color coded according to cavity depth (blue, shallow; yellow, deep); B) BI-78D3 chemical structure; and C) Displacement assay for BI-78D3. D) Kinase assay for BI-78D3. E) Double reciprocal plot of JNK kinase activity as function of ATF2 in the presence of various amounts of BI-78D3 or DMSO control.
  • Figures 20A-C depict docking studies and NMR analysis of BI-78D3: A) chemical structure and predicted binding mode of SP600125 derived azaindole-TEMPO compound; B) docked structure of BI-78D3 into the X-ray structure of JNKl (PDB ID IUKI) and predicted hydrogen bonding interactions between the compound and the residue Argl27 (displayed) are highlighted with dashed lines; and C) ID 1 H-NMR T lp spectra of BI-78D3 (500 ⁇ M) in the presence of 5 ⁇ M JNK2 (blue) or 5 ⁇ M JNK2 and 200 ⁇ M ATP-TEMPO (red) at 100 ms, NMR resonance assignments for the hydrogen nuclei of the small molecule are reported and signal reductions are as follows: Hl 46%, H2 56%, H3/H5 65%, and H4 72%.
  • Figures 21 A and B depict biological analysis of BI-78D3: A) TR-FRET analysis of ATF2 phosphorylation upon TNF-alpha stimulation of A549 cells in the presence of increasing BI-78D3; and B) BI-78D3 effect on serum alanine-aminotransferase levels after 7.5 hours of exposure to Conconavilin A as compared to DMSO control.
  • Figures 22 A and B set forth compounds with 100% displacement at 100 ⁇ M in the DELFIA assay for displacement of JIP peptide. Detailed Description of the Invention
  • a “label” as used herein is a molecule which is detectable or capable of detection, for instance, a radiolabel, biotin, a hapten, a fluorophore, e.g., coumarin, rhodamine, rhodols, CRG6, Texas Methyl Red, fluorescein, 7 aminocoumarin, and 7-hydroxycoumarin, 2-amino-4- methoxynapthalene, 1-hydroxypyrene, resorufin, phenalenones or benzphenalenones, acridinones, anthracenes, and derivatives of ⁇ - and ⁇ -napthol, fluorinated xanthene derivatives including fluorinated fluoresceins and rhodols, a bioluminescent molecule, or a chemiluminescent molecule.
  • the label may include a cleavable linker.
  • the label is a donor or
  • a "ligand” as used herein is a molecule that specifically binds to another molecule.
  • exemplary ligands are a HIS tag, GST, maltose binding protein, biotin, avidin, streptavidin, calmodulin binding protein, hemagglutinin and the like.
  • a “lanthanide,” “lanthanide series element” or “lanthanide series inner transition element” refers to Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium(Yb), or Lutetium (Lu).
  • lanthanides include, e.g., Ce(III), Ce(IV), Pr(III), Nd(III), Pm(III), Sm(II), Sm(III), Eu(II), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(II), Yb(III), and Lu(III).
  • the term "antibody” refers to a protein having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad of immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the basic immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (Y 11 ) refer to these light and heavy chains respectively.
  • Antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, FabFc 2 , Fab, Fv, Fd, (Fab D) 2 , an Fv fragment containing only the light and heavy chain variable regions, a Fab or (Fab) D 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, e.g., scFv, CDR-grafted antibodies and the like.
  • the heavy and light chain of a Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region.
  • the antibody may be of animal (especially mouse or rat) or human origin or may be chimeric or humanized. As used herein the term "antibody" includes these various forms.
  • JIP includes polypeptides having at least 80%, e.g., at least 85%, 90%, 95%, or more amino acid sequence identity to sequences having NCBI Accession Nos. Q9UQF2, Q9WV19, or Q9R237, and which bind JNK, and nucleic acid sequence encoding those polypeptides.
  • the JIP is JIP-I, e.g., human (SEQ ID NO:1), rodent, for instance, rabbit, mouse, rat, mink or guinea pig, or nonhuman primate JIP-I.
  • JNK includes polypeptides having at least 80%, e.g., at least 85%, 90%, 95%, or more amino acid sequence identity to sequences having NCBI Accession Nos. BAA85876, P45984, Q9WTU6 or Q91Y86, and which bind JIP, and nucleic acid sequence encoding those polypeptides.
  • the JNK is JNKl, e.g., human (SEQ ID NO:2), rodent, for instance, rabbit, mouse, rat, mink or guinea pig, or nonhuman primate JNKl.
  • the JNK is JNK2.
  • the JNK is a mutant JNK (e.g., JNK2), such as, a JNK with a one or two amino acid mutation.
  • JIP/JNK interaction is intended to mean an interaction, e.g., a physical or electrostatic interaction, between the JIP and JNK polypeptides or portions thereof.
  • the interaction can be ligand mediated or directly between the polypeptides.
  • modulator is intended to mean a compound, e.g., a small molecule, a peptide, a polypeptide, an antibody, or an antibody fragment, that has the ability to interfere, e.g., block, weaken or disrupt, the interaction between JIP and JNK.
  • Modulators of the invention can be reversible or irreversible modulators.
  • the term “obtaining” as in, e.g., "obtaining one or more reagents” is intended to include purchasing, synthesizing or otherwise acquiring the reagent or a material used in carrying out the methods of the invention.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic,
  • the pharmaceutically acceptable salts of the compounds useful in the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985), the disclosure of which is hereby incorporated by reference.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
  • One diastereomer of a compound disclosed herein may display superior activity compared with the other.
  • separation of the racemic material can be achieved by HPLC using a chiral column or by a resolution using a resolving agent such as camphonic chloride as in Thomas J .Tucker, et al., J. Med. Chem. 1994 37, 2437-2444.
  • a chiral compound of Formula I may also be directly synthesized using a chiral catalyst or a chiral ligand, e.g. Mark A. Huffman, et al., J. Org. Chem. 1995, 60, 1590-1594.
  • “Therapeutically effective amount” is intended to include an amount of a compound useful in the present invention or an amount of the combination of compounds claimed, e.g., to treat or prevent the disease or disorder, or to treat the symptoms of the disease or disorder, in a host.
  • the combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is advantageously demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased activity, or some other beneficial effect of the combination compared with the individual components.
  • metabolic disease is intended to mean a disease or disorder characterized by alteration of a normal metabolic process. These disorders are often the result of altered enzyme activity or production. Exemplary metabolic diseases include obesity and diabetes.
  • treating or “treat” includes (i) preventing a pathologic condition from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; (iii) relieving the pathologic condition; and/or diminishing symptoms associated with the pathologic condition.
  • subject refers to organisms to be treated by the methods of the present invention. Such organisms include, but are not limited to, mammals such as animals, preferably humans.
  • the term “subject” generally refers to an individual who will receive or who has received treatment (e.g., administration of a compound of the inventions) for obesity and/or diabetes.
  • Treatment e.g., administration of a compound of the inventions
  • stable compound and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated by the present invention.
  • Substituted is intended to indicate that one or more hydrogens on the atom indicated in the expression using “substituted” is replaced with a selection from the indicated group(s), provided that the indicated atom' s normal valency is not exceeded, and that the substitution results in a stable compound.
  • Suitable indicated groups include, e.g., alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.
  • 2 hydrogens on the atom are replaced.
  • virtual docking is intended to mean the process of testing the ability of a test agent to bind to a target protein, i.e., JNK, using a computer program algorithm. This research technique predicts whether one molecule will bind to another, usually a protein. Most commonly, virtual docking is done by modelling the interaction between two or more molecules: if the geometry of the pair is complementary and involves favorable biochemical interactions, one molecule will likely bind to the other in vitro or in vivo. A specific virtual docking approach is set forth in Example 1.
  • Interrupted is intended to indicate that in between two or more adjacent carbon atoms, and the hydrogen atoms to which they are attached (e.g., methyl (CH 3 ), methylene (CH 2 ) or methine (CH)), indicated in the expression using “interrupted” is inserted with a selection from the indicated group(s), provided that the each of the indicated atoms' normal valency is not exceeded, and that the interruption results in a stable compound.
  • Alkyl refers to a Ci-Ci8 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH3), 2-methyl-l -propyl (i-Bu, i-butyl, - CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2
  • the alkyl can optionally be substituted with one or more alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • the alkenyl can optionally be substituted with one or more alkyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • Alkylidenyl refers to a Cl -C 18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms.
  • the alkylidenyl can optionally be substituted with one or more alkyl, alkenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • the alkenylidenyl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • Alkylene refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or different carbon atoms of a parent alkane.
  • Typical alkylene radicals include, but are not limited to: methylene (-CH 2 -) 1,2-ethyl (-CH 2 CH 2 -), 1,3-propyl (-CH 2 CH 2 CH 2 -), 1,4-butyl (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • the alkylene can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • Alkenylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene.
  • the alkenylene can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or COOR X , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • alkoxy refers to the groups alkyl-O-, where alkyl is defined herein. Preferred alkoxy groups include, e.g., methoxy, ethoxy, w-propoxy, wo-propoxy, w-butoxy, tert- butoxy, seobutoxy, w-pentoxy, w-hexoxy, 1,2-dimethylbutoxy, and the like.
  • the alkoxy can optionally be substituted with one or more alkyl, alkylidenyl, alkenylidenyl, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and COOR X , wherein each R x and R y is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl).
  • Preferred aryls include phenyl, naphthyl and the like.
  • the aryl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and COOR X , wherein each R x and R y is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • aryl can optionally be a divalent radical, thereby providing an arylene.
  • aryl may be substituted with a divalentsubstituent forming multicyclic structure wherein at least one ring is aromatic, e.g., 2,3-dihydro-benzo[l,4]dioxin-6- yi-
  • cycloalkyl refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • the cycloalkyl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and COOR X , wherein each R x and R y is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • the cycloalkyl can optionally be at least partially unsaturated, thereby providing a cycloalkenyl. Additionally, the cycloalky
  • halo refers to fluoro, chloro, bromo, and iodo.
  • halogen refers to fluorine, chlorine, bromine, and iodine.
  • Haloalkyl refers to alkyl as defined herein substituted by 1-4 halo groups as defined herein, which may be the same or different. Representative haloalkyl groups include, by way of example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl, 2-bromooctyl, 3-bromo-6- chloroheptyl, and the like.
  • heteroaryl is defined herein as a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, thio, alkylthio, alkylsulfinyl, and alkylsulfonyl. Additionally, the heteroaryl can optionally be a divalent radical, thereby providing a heteroarylene. Examples of heteroaryl groups include, but are not limited to, 2/f-pyrrolyl, 3/f-indolyl,
  • heteroaryl denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from the group non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl.
  • heteroaryl denotes an ortho- fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, or tetramethylene diradical thereto.
  • the heteroaryl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and COOR X , wherein each R x and R y is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • heterocycle is a monocyclic, bicyclic, or tricyclic group containing one or more heteroatoms selected from the group oxygen, nitrogen, and sulfur.
  • heterocycle groups include 1,3- dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2/f-pyran, 2-pyrazoline, 4/f-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, and thiomorpholine.
  • the heterocycle can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and COOR X , wherein each R x and R y is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
  • the heterocycle can optionally be a divalent radical, thereby providing a heterocyclene.
  • nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing
  • the nitrogen heterocycle can be 3-methyl-5,6-dihydro-4H-pyrazino[3,2,l-jk]carbazol- 3-ium iodide.
  • Another class of heterocyclics is known as "crown compounds" which refers to a specific class of heterocyclic compounds having one or more repeating units of the formula [- (CH 2 -) a A-] where a is equal to or greater than 2, and A at each separate occurrence can be O, N, S or P.
  • Examples of crown compounds include, by way of example only, [-(CH 2 ) 3 -NH-] 3 , [- ((CH 2 ) 2 -O) 4 -((CH 2 ) 2 -NH) 2 ] and the like. Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
  • examples of acyloxy groups include, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Any alkyl group as defined above can be used to form an acyloxy group.
  • amino refers to -NH 2
  • alkylamino refers to -NR 2 , wherein at least one R is alkyl and the second R is alkyl or hydrogen.
  • nitro refers to -NO 2 .
  • trifluoromethyl refers to -CF 3 .
  • trifluoromethoxy refers to -OCF 3 .
  • cyano refers to -CN.
  • hydroxy or "hydroxyl” refers to -OH.
  • oxy refers to -O-.
  • nucleic acid base refers to a nitrogenous base that is planar, aromatic and heterocyclic. They are typically derivatives of either purine or pymidine. Suitable nucleic acid bases include, e.g., purine, pymidine, adenine, guanine, cytosine, uracil, and thymine.
  • the nucleic acid base can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y and/or C00R x , wherein each R x and R y is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.
  • any of the above groups which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
  • substituents within the compounds described herein are present to a recursive degree.
  • "recursive substituent” means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim.
  • One of ordinary skill in the art of medicinal chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis.
  • Recursive substituents are an intended aspect of the invention.
  • One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents.
  • the total number will be determined as set forth above.
  • the compounds described herein can be administered as the parent compound, a prodrug of the parent compound, or an active metabolite of the parent compound.
  • Pro-drugs are intended to include any covalently bonded substances which release the active parent drug or other formulas or compounds of the present invention in vivo when such pro-drug is administered to a mammalian subject.
  • Pro-drugs of a compound of the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation in vivo, to the parent compound.
  • Pro-drugs include compounds of the present invention wherein the carbonyl, carboxylic acid, hydroxy or amino group is bonded to any group that, when the pro-drug is administered to a mammalian subject, cleaves to form a free carbonyl, carboxylic acid, hydroxy or amino group.
  • Examples of pro-drugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention, and the like.
  • Metal refers to any substance resulting from biochemical processes by which living cells interact with the active parent drug or other formulas or compounds of the present invention in vivo, when such active parent drug or other formulas or compounds of the present are administered to a mammalian subject. Metabolites include products or intermediates from any metabolic pathway.
  • Metal pathway refers to a sequence of enzyme-mediated reactions that transform one compound to another and provide intermediates and energy for cellular functions.
  • the metabolic pathway can be linear or cyclic.
  • JNKl -3 isozymes are members of the mitogen activated protein kinases (MAPK), considered to be at the focal point of different signaling pathways activated by many distinct cell surface receptors and whose role is to transduce these signals to effector molecules that generate changes in gene expression by regulating transcription and mRNA stability (Hirosumi et al., 2002).
  • MAPK mitogen activated protein kinases
  • JNKs activate the transcription factors TCF/Elk-1, ATF2 and c-Jun by phosphorylation, leading to increased c-jun and fas gene transcription and increased API transcription factor activity (Hirosumi et al., 2002; Aibi et al., 1993; Derijard et al., 1994; Shanlian et al., 2002; Gupta et al., 1996; Kaneto et al., 2002; Shang et al., 2003).
  • the JNKs are also implicated in stabilization of inherently unstable mRNAs, as shown for IL-2 mRNA.
  • the JNKs are considered key signaling molecules in the "communication" between external stimuli (stress) and gene expression.
  • the JNKs are activated in response to several stresses, such as infection, pro-inflammatory cytokines, UV radiation, various xenobiotics and oxidative stress (Aibi et al., 1993; Derigard et al., 1994, Shanlian et al., 2002; Gupta et al., 1996; Kaneto et al., 2002, Shang et al., 2003; Dong et al., 2000; Yuan et al., 2001; Arkan et al., 2005; Lin, 2003; Hotamisligil et al., 1994a, Hotamisligil et al., 1994b; Ofei et al., 1996).
  • stresses such as infection, pro-inflammatory cytokines, UV radiation, various xenobiotics and oxidative stress (Aibi et al., 1993; Derigard et al., 1994, Shanlian et al., 2002; Gupta et al., 1996; Kane
  • JNKl is a potent regulator of insulin signaling and could be implicated in the pathogenesis of the metabolic syndrome (Hirosumi et al., 2002; Aguirre et al., 2000). Briefly, insulin stimulates glucose entry into cells by binding the insulin receptor (IR) at the cell surface. IR is a member of the tyrosine kinase receptor-family and after hormone binding, it autophosphorylates tyrosine residues on its cytoplasmic domain. This in turn leads to the recruitment of the scaffold proteins Insulin Receptor Substrates (IRS), likely via their phosphotyrosine binding domain (PTB). Several IRS molecules have been identified.
  • IR Insulin Receptor Substrates
  • IRSl and IRS2 have been clearly proven to play a major role in insulin signaling. Both IRSl and IRS2 are phosphorylated at several tyrosine residues by the IR, making possible the recruitment of other signaling molecules such as PI3 kinase (PI3K), which interacts with specific phosphotyrosine residues on IRS1/IRS2 via the SH2 domain of the regulatory P85 subunit. The signal is then transmitted to downstream signaling molecules and this cascade of events results in increased expression and activity of several enzymes involved in energy metabolism and in the increased translocation to the cytosolic membrane of the glucose transporter GLUT4, which increases glucose uptake in skeletal muscle and adipose tissue (Yang et al., 1998).
  • PI3K PI3 kinase
  • JNKl knock out mice were found to be resistant to diabetes in both dietary and genetic models of obesity (Hirosumi et al., 2002). It was proposed that JNKl elicits insulin resistance via specific phosphorylation of serine 307 present in the PTB domain of IRSl, which interferes with its recruitment to the IR, resulting in decreased IRS 1 tyrosine phosphorylation and impairment of PI3K recruitment in response to insulin (Whitmarsh et al., 1998; Dickens et al., 1997; Bonny et al., 2001).
  • JIPl specifically interacts with components of the JNK signaling modules by binding JNK, MKK7, and members of the mixed lineage kinase (MLK) group of MAP3K (Whitmarsh et al., 1998, Aquirrre et al., 2000; Momson et al., 2003; Willoughby et al., 2003).
  • JIPl and the p38 cognate, JIP2 can bind the MAPK phosphatase MKP7, indicating that these JIP scaffold complexes include both activating and inhibitory components of the JNK signaling pathway (Willoughby et al., 2003).
  • Deletion analysis of JIPl demonstrates that the sites of interaction with JNK, MKK7, and MLK protein kinases correspond to separate sites on JIPl (Whitmarsh et al., 1998).
  • JNK decreased the affinity of JIPl for MLK (Nihalani et al., 2001); the binding of FHF increased the binding of JIP2 to p38 MAPK (Fattorusso et al., 2005); the binding of AKT to JIPl decreased the binding of JNK to JIPl (Leone et al., 2003); and the binding of the phosphatase MKP7 to JIPl inhibited JNK activation caused by the JIPl scaffold complex (Willoughby et al., 2003).
  • One of the effective means of JNK inhibition employed the overexpression of JIP 1.
  • An 11-amino acid peptide inhibitor of activated JNKs based on amino acids 153-163 of JIPl (pepJIPl), inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 up to 90% at sub-micromolar concentrations and directly interacted with recombinant JNKs but not its substrates, as shown by surface plasmon resonance analysis and isothermal titration calorimetry (ITC) (Heo et al, 2004) Alanine scanning within pepJIPl identified 4 residues (Arg-156, Pro-
  • the crystal structure of JNKl in complex with pepJIPl provided insightful information on the nature of the JIPl /JNKl interaction (Heo et al., 2004).
  • three main interactions can be observed between the peptide and JNKl : a first hydrophobic cavity on JNKl accommodates the side-chain of Leu- 160 and Leu-162; a second shallower pocket is occupied by Pro-157; finally, intermolecular hydrogen bonding involving Arg-156 and Thrl58 was observed.
  • the identification of a lead compound represents the starting point for the development of new candidate drugs.
  • different strategies are pursued for hit identification and optimization processes.
  • One approach is based on a combination of medicinal chemistry guided by nuclear magnetic resonance (NMR) spectroscopy and structure-based techniques. These enable the identification of initial "weaker” hit compounds and guide the translation of such compounds into more potent and selective leads.
  • NMR nuclear magnetic resonance
  • the following selection criteria may be used: molecular weight ⁇ 500, number of hydrogen bond donors between 0 and 2, number of hydrogen bond acceptors between 2 and 9, number of rotatable bonds less then 8, and octanol/water repartition coefficient (LogP) ⁇ 5 (Oprea, 2000).
  • the goal in using these empirical drug-like property filters is to predict favorable outcome in ADMET (adsorption, distribution, metabolism, excretion, toxicity) studies, as well as final success as a drug in humans.
  • a library having a fairly small (about 600 compounds) but diverse set of low-molecular-weight scaffolds derived mainly from compounds commonly found in known drugs (Becattini et al., 2004) was employed.
  • This library was assembled and ID 1 H and 13 C NMR spectra were measured as a quality control and to assess solubility.
  • the library was designed also to optimize the detection of ligand-ligand interactions by selecting compounds with appropriate derivatizations of functional groups with proton NMR- detectable substituents. Furthermore, the introduction of heteroatoms in these substituents also results in large chemical shift dispersion between the compounds of the library, thus enabling the detection of ILOEs in complex mixtures.
  • solution-phase synthesis aided by resin-bound reagents and scavengers may be employed. These allow removal of excess reagents and byproducts by using filtration rather then using liquid-liquid extraction, chromatography or crystallization. Initially, the focus is on linkage via amide bonds, ethers or thioethers. Amide bond formation may be performed with the coupling reagent PS-carbodiimide (Argonaut) in presence of HOBt, subsequently scavenged post-reaction by the resin-bound PS- Trisamine.
  • Microwave assisted chemistry may also be used for various reactions (Forino et al., 2005; Tautz et al., 2004).
  • carbodiimmide catalyzed reactions both PS-CDI and water-soluble-CDI
  • yields varying from 25 to 50%.
  • uncatalyzed amidation of the acid was performed under solvent free conditions, using a slight excess of amine, excellent yields (> 80%) were obtained.
  • Milestone Microwave systems allow for up to 16 different reaction conditions at the same time.
  • one aspect of the invention provides a method or assay to identify/screen for a compound that modulates the JIP/JNK interaction.
  • the method comprises providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent; washing the solid support to remove molecules not associated with the solid support; separating the label from the ligand; and determining the amount of separated label.
  • the solid support can be any of the solid supports useful for high throughput screening known to those skilled in the art. These include but are not limited to a well of a multi well plate, bead, particle or resin, including a natural or synthetic polymer.
  • the invention also provides assays that do not use a solid support, e.g., a soluble assay to identify modulators of the JIP/JNK interaction.
  • the assay makes use of fluorescence resonance energy transfer (FRET) to determine if a test compound modulates the interaction of JIP and JNK.
  • FRET fluorescence resonance energy transfer
  • a donor and acceptor fluorophore are added to JIP and JNK thereby allowing the emission of acceptor specific fluorescence if binding occurs between JIP and JNK. This fluorescence emission occurs only when the donor and acceptor fluorophores are in close proximity to each other, e.g., when JIP and JNK are bound.
  • candidate compounds can be added to a solution prior to JIP/JNK complex formation or after the complex is formed, and the effect of the compound on the interaction can be monitored.
  • biotin is attached to the JIP and an acceptor fluorophore is attached to streptavidin thereby attaching a acceptor fluorophore to JIP through a biotin/ streptavidin interaction.
  • JNK is fused to GST and an donor fluorophore is attached to a GST antibody thereby providing a donor fluorophore JNK through the GST-antibody interaction.
  • the JNK/JIP interaction can be monitored via the emission from the acceptor fluorophore attached to JIP.
  • fluorophores e.g., donor and acceptor fluorophores that can be used in the FRET-based assays described herein.
  • the assays would be equally effective if the donor and acceptor molecules were placed on the other binding protein.
  • any of a number of known combinations of donor and acceptor labels can be used for FRET.
  • FRET fluorescence resonance spectrometry
  • the fluorescent donor and acceptor are Europium chelate and allophycocyanin, respectively.
  • the JNK/JIP interaction may be monitored when the JNK is a mutant JNK, wherein such mutation(s) are selected to determine particular active site involvement.
  • the methods of identification may further comprise the additional step of screening a potential modulator of JNK/JIP interaction against R127A and C167S mutants of JNK2 to determine if the compound is binding in the JIP pocket (i.e., a drop in binding affinity for the compound by these mutants, would indicate that a compound binds in the JIP pocket).
  • compounds identified by the methods of the invention which are useful to inhibit JIP/JNK interactions, include compounds of formula (II):
  • Q 2 is aryl, cycloalkyl, heteroaryl or heterocycle
  • Additional compounds useful to inhibit JIP/JNK interactions include compounds of formula (III):
  • Q is aryl, cycloalkyl, heteroaryl or heterocycle
  • Q 6 is aryl, cycloalkyl, heteroaryl or heterocycle
  • Z 1 is selected from the group consisting of phenyl, naphthyl, 2,3-dihydro- benzo[l,4]dioxin-6-yl, and benzo[l,3]dioxol-5-yl;
  • Z 2 is selected from H, thiazole (e.g., thiazol-2-yl), and benzylsulfonyl; and Z 1 and Z 2 may be independently substituted with one or more substituents selected from the group consisting of lower alkyl (e.g., t-butyl or methyl), CO 2 H, lower alkoxy (e.g., methoxy), halogen (e.g., F), NO 2 , and perfluoroalkoxy (e.g., OCF 3 ).
  • Z 2 is 5-nitrothiazol-2-yl.
  • Z 1 is 2,3-dihydro-benzo[l,4]dioxin-6-yl.
  • Additional compounds useful to inhibit JIP/JNK interactions include compounds of formula (V):
  • Z 1 is selected from the group consisting of phenyl, naphthyl, 2,3-dihydro- benzo[l,4]dioxin-6-yl, and benzo[l,3]dioxol-5-yl;
  • Z 2 is selected from H, thiazole (e.g., thiazol-2-yl), and benzylsulfonyl; and Z 1 and Z 2 may be independently substituted with one or more substituents selected from the group consisting of lower alkyl (e.g., t-butyl or methyl), CO 2 H, lower alkoxy (e.g., methoxy), halogen (e.g., F), NO 2 , and perfluoroalkoxy (e.g., OCF 3 ).
  • Z 1 is phenyl.
  • a compound of the invention is a compound of formula V, wherein Z 2 is H, Z 1 is not 2,3-dihydro-benzo[l,4]dioxin-6-yl or phenyl.
  • a specific value for Q 1 is aryl or heterocycle. Another specific value for Q 1 is aryl. Another specific value for Q 1 is heterocycle. Another specific value for Q 1 is phenyl. Another specific value for Q 1 is 2-furan. Another specific value for Q 1 is 2-(5-methyl) furan.
  • a specific value for Q 2 is absent, optionally substituted aryl or optionally substituted heterocycle. Another specific value for Q 2 is optionally substituted aryl. Another specific value for Q 2 is optionally substituted heterocycle. Another specific value for Q 2 is phenyl, 2-furan, N- (3-morpholino), 2-(5-methyl) furan, absent, o-NO 2 -phenyl or m-OMe-p-OH-phenyl. Another specific value for Q 2 is phenyl. Another specific value for Q 2 is 2-furan. Another specific value for Q 2 is N-(3-morpholino). Another specific value for Q 2 is 2-(5-methyl) furan. Another specific value for Q 2 is absent. Another specific value for Q 2 is o-NO 2 -phenyl. Another specific value for Q is m-OMe-p-OH-phenyl.
  • a specific value for X 4 is absent or O. Another specific value for X 4 is absent. Another specific value for X 4 is O.
  • R x is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl
  • N,N'-(propane-l,3-diyl)diacrylamide N,N'-(propane- l,3-diyl)diacetamide
  • N-propylacrylamide N,N'-(2,2-dimethylpropane-l,3-diyl)diacrylamide
  • N-(3-acetamidopropyl)acrylamide or N,N'- (propane- 1 ,3-diyl)diacrylamide.
  • Another specific value for X 5 is N,N'-(propane-l,3-diyl)diacrylamide. Another specific value for X 5 is N,N'-(propane-l,3-diyl)diacetamide. Another specific value for X 5 is N- propylacrylamide. Another specific value for X 5 is N,N'-(2,2-dimethylpropane-l,3- diyl)diacrylamide. Another specific value for X 5 is l,l'-(l,4-diazepane-l,4-diyl)diprop-2-ene-l- one. Another specific value for X is N-(3-acetamidopropyl)acrylamide. Another specific value for X 5 is N,N'-(propane-l,3-diyl)diacrylamide.
  • a specific value for X 6 is absent or O. Another specific value for X 6 is absent. Another specific value for X 6 is O.
  • a specific value for Q 3 is optionally substituted heteroaryl or optionally substituted cycloalkyl. Another specific value for Q 3 is optionally substituted heteroaryl. Another specific value for Q 3 is optionally substituted cycloalkyl. Another specific value for Q 3 is 6-( methyl 5- nitronicotinate); 2-(5-nitrothiazole); 7-(5-phenyl-[l,2,4]triazolo[l,5-a]pyrimidine); 4-(3-methyl- 1 -phenyl- lH-pyrazole) or 2-(thiazole). Another specific value for Q 3 is 6-( methyl 5-nitronicotinate). Another specific value for
  • Q 3 is 2-(5-nitrothiazole). Another specific value for Q 3 is 7-(5-phenyl-[l,2,4]triazolo[l,5- a]pyrimidine). Another specific value for Q 3 is 4-(3-methyl-l-phenyl-lH-pyrazole). Another specific value for Q 3 is 2-(thiazole).
  • a specific value for X 7 is absent or aryl. Another specific value for X 7 is absent. Another specific value for X 7 is aryl. Another specific value for X 7 is phenyl. Another specific value for X 7 is 1,2-phenyl. Another specific value for X 7 is 3,4'-(4-(4'-phenyl)-lH-l,2,4-triazol- 5(4H)-one). Another specific value for X 7 is l,2-(lH-benzo[d]imidazole). Another specific value for X 7 is 2,5-(l,3,4-thiadiazole).
  • Another specific value for Q 5 is absent.
  • Another specific value for Q 5 is absent.
  • Q is OR Z , wherein R z is alkylene, alkenylene, arylene, heteroarylene, heterocyclene, or cycloalkylene. Another specific value for Q is OCH 2 . A specific value for Q 6 is optionally substituted heteroaryl or optionally substituted cycloalkyl. Another specific value for Q 6 is optionally substituted heteroaryl. Another specific value for Q 6 is optionally substituted cycloalkyl. Another specific value for Q 6 is optionally substituted alkyl.
  • Q 6 Another specific value for Q 6 is 2-thiophene; m-CF 3 -phenyl; 2-(5-(pyridin-4- yl)-l,3,4-oxadiazole); 4-(3-(thiophen-2-yl)-lH-l,2,4-triazole-5(4H)-thione); orp-Cl-phenyl.
  • Another specific value for Q 6 is 2-thiophene.
  • Another specific value for Q 6 is m-CF 3 -phenyl.
  • Another specific value for Q 6 is 2-(5-(pyridin-4-yl)-l,3,4-oxadiazole).
  • Q 6 is or 4-(3-(thiophen-2-yl)-lH-l,2,4-triazole-5(4H)-thione). Another specific value for Q 6 is p-Cl-phenyl. Another specific value for Q 6 is phenyl. Another specific value for Q 6 is isopropyl.
  • Specific compounds of formula (III) include: methyl 5-nitro-6-(2-(thiophene-2-carboxamido)phenylthio)nicotinate; N-(2-(5-nitrothiazol-2-ylthio)phenyl)-3-(trifluoromethyl)benzamide; 2-(5-phenyl-[l,2,4]triazolo[l,5-a]pyrimidin-7-ylthio)-5-(pyridin-4-yl)-l,3,4- oxadiazole; (E)-4-((3-methyl-l-phenyl-lH-pyrazol-4-yl)methyleneamino)-3-(thiophen-2- yl)- IH- 1 ,2,4-triazole-5(4H)-thione; methyl 6-(2-(4-chlorobenzamido)phenylthio)-5-nitronicotinate; 4-(4-(benzyloxy)phenyl)-3-(5-nitrothia
  • the compounds of the invention may be selected from the exemplary compound listing shown below in the following tables (i.e., Tables I- VI). Importantly, it should be noted that the tabular listing below is used merely as a convenience, and each compound below should be considered a separate embodiment of the invention:
  • the compounds of the invention may be selected from Table IV.
  • Table IV
  • the compound is not 83A5 or 83A6.
  • particular compounds of the invention include those selected from the listing of compounds of Table V.
  • the compound may be selected from the compounds of Table
  • the compound of the invention is compound 83F6, 4-(4- Nitro-phenyl)-5-(5-nitro-thiazol-2-ylsulfanyl)-2,4-dihydro-[l,2,4]triazol-3-one.
  • the compounds of this invention are formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice.
  • Tablets will contain excipients, glidants, fillers, binders and the like.
  • Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.
  • the pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10.
  • the formulations both for veterinary and for human use, of the invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers therefore and optionally other therapeutic ingredients.
  • the carrier(s) are advantageously "acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.
  • the formulations include those suitable for the foregoing administration routes.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be administered as a bolus, electuary or paste.
  • a tablet is made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w and more preferably 0.5 to 10% w/w.
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in- water cream base.
  • the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogs.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
  • the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used.
  • compositions according to the present invention comprise one or more compounds of the invention together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
  • Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration.
  • tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
  • Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
  • excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate
  • granulating and disintegrating agents such as maize starch, or alginic acid
  • binding agents such as cellulose, microcrystalline cellulose, starch,
  • Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate).
  • a suspending agent
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p- hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives such as ethyl or n-propyl p- hydroxy-benzoate
  • coloring agents such as ethyl or n-propyl p- hydroxy-benzoate
  • flavoring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives.
  • a dispersing or wetting agent e.g., sodium tartrate
  • suspending agent e.g., sodium EDTA
  • preservatives e.g., sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate
  • the pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
  • Suitable emulsifying agents include naturally- occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • sweetening agents such as glycerol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic
  • a time -release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight:weight).
  • the pharmaceutical composition can be prepared to provide easily measurable amounts for administration.
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 ⁇ g of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
  • Formulations suitable for administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • the active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs.
  • Suitable formulations include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of a given condition.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • the invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route. Compounds of the invention can also be formulated to provide controlled release of the active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the invention also provided compositions comprising one or more compounds of the invention formulated for sustained or controlled release.
  • Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses), the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day. Typically, from about 0.01 to about 10 mg/kg body weight per day. More typically, from about .01 to about 5 mg/kg body weight per day. More typically, from about .05 to about 0.5 mg/kg body weight per day.
  • the daily candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.
  • One or more compounds of the invention are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that a preferred route may vary with for example the condition of the recipient.
  • An advantage of the compounds of this invention is that they are orally bioavailable and can be dosed orally.
  • the invention also provides kits comprising the reagents for performing the methods of the invention and instructions for use.
  • the kit provides JIP and JNK comprising fluorescent donor and acceptor moieties and instructions for use.
  • the donor and acceptor moieties are europium chelate and allophycocyanin, respectively.
  • Virtual docking strategies have been described to identify potential protein binders (Fattorusso et al., 2005; Leane et al., 2003; Kitada et al., 2003; Hajdaic et al., 1997 Forino et al., 2005). For instance, a number of in silico approaches have been used to identify inhibitors for Akt (Forino et al., 2005).
  • Virtual docking may be performed on Linux cluster constituted by a 40 slaves plus head node, each with 2 CPUs Intel® Xenon 2.4 GHz running FlexX (Biosolve) or by using FlexX as implemented in Sybyl 6.9 (TRIPOS) on a 10-R12000 CPUs challenge machine and generating several solutions per compound.
  • Top scoring compounds are selected by using CSCORE and visual inspection. Molecular models are built by using CORINA and Sybyl 6.9 (TRIPOS) and energy minimized by using the routine MAXIMIN or semi-empirical calculations (MOPAC). Cerius 2 (Accelrys, Inc.) may also be used. Initial docking studies with compounds are performed with FlexX as implemented in Sybyl 6.9 by using the three- dimensional structure of JNKl in complex with the JIPl peptide (PDB_ID IUKH) and the structure of the ternary complex between JNKl, JIPl and the ATP mimic SP600125 (PBDJD IUKI).
  • a virtual docking approach to identify modulators of JIP/JNK was analyzed using several strategies including FlexX (Kramer et al., 1999), GOLD (Ver donk et al., 2003) and CSCORE (Clark et al., 2002), where the top 100-200 scoring compounds from a 50,000 compound library were tested. Briefly, a 50,000 compound library (Chembridge, San Diego) was selected and all compounds docked against the JIPl binding site on JNKl (PDB_ID IUKH). For this task, FlexX was used. Initially 5 solutions were generated for each ligand and the compounds rank ordered by using the FlexX scoring function.
  • top scoring 4,000 compounds were then subsequently analyzed by using a consensus score between Chemscore (Eldridge et al., 1997) and Goldscore (Ver donk et al., 2003).
  • the top 1,000 compounds were visually analyzed using Sybyl and compounds to be tested selected according to the following criteria: occupancy of both hydrophobic pockets; formation of hydrogen bonding interactions (similar to those observed in pepJIPl -JNKl); drug-likeness (see above); and ease of synthesis.
  • Delfia ® dissociation enhanced lanthanide fluoroimmunoassay
  • Delfia chemistry has been applied with success using various assay formats in the detection of receptor-ligand interactions.
  • Delfia ® is a heterogeneous assay similar to an ELISA (enzyme linked immunosorbent assay).
  • proteins were produced from a pET- 19b (Novagen) plasmid construct containing the respective nucleotide sequence fused to a N- terminal poly- His tag or GST.
  • GST-JNK1/2 may be expressed in E. coli BL21 in LB media at 37 0 C, with an induction period of 3-4 hours with 1 mM IPTG. Approximate yields ranged from 13
  • ⁇ C-VaI labelled His-JNKl (His-JNK2) for binding studies was similarly produced in M9 media supplemented with 50 mg/L of ⁇ - 13 C-Val (Cambridge Isotopes) at the time of induction with IPTG.
  • soluble labelled protein was purified over a Hi-Trap chelating column (Amersham, Pharmacia), followed by ion-exchange purification with a MonoQ (Amersham, Pharmacia) column.
  • a schematic representation of Delfia ® is set forth in Figure 1.
  • a heterogeneous assay using a labeled JIPl peptide and a JNK fusion was employed to screen and characterize JIPl mimics.
  • a bio tin labeled pep JIPl is bound to streptavidin coated 96 -well plates.
  • GST-JNKl or GST- JNK2
  • GST-JNK2 is incubated so as to allow for binding to pepJIPl, and after washing to eliminate unbound GST-JNKl (or GST-JNK2), a solution containing a Eu labeled anti-GST antibody (Perkin Elmer) is added together with a test agent.
  • an enhancing solution is added to deliver the lanthanide from the antibody to the solution.
  • Residual Eu fluorescence is subsequently detected with a VictorV2 (Perkin Elmer) plate reader with excitation at 360 nm and emission at 620 nm. If the test compound is capable of dissociating GST-JNKl from the biotinylated-pepJIPl peptide, the antibody carrying the label (Eu) is washed out and no fluorescence is detected. Because of the relaxation properties of Eu, detection can be accomplished also in time-resolved mode (TRF), free from possible interferences by test compounds, matrices, plates, and the like. Also, the sensitivity of Eu is superior to that of most other fluorescent labels.
  • TRF time-resolved mode
  • the reproducibility of the Delfia ® assay was evaluated by varying the biotin-pepJIPl (Anaspec) concentration (from 1 pM to 50 nM) ( Figure 2A); GST-JNKl (and GST- JNK2) concentration (from 1 nM to 500 nM) ( Figure 2B); incubation times (from 30 minutes to 2 hours); and the number of washing steps (from 2 to 7).
  • the obtained IC 50 value of 3 ⁇ M is comparable with the K D value obtained by direct isothermal titration calorimetry experiments (about 0.6 ⁇ M).
  • 100 ⁇ L of a 100 ng/ml of biotin labeled pepJIPl was added to each well of a 96-well streptavidin coated plate (Perkin Elmer). After incubation for 1 hour, unbound peptide was eliminated with 4 washing steps. Subsequently, to each well 78 ⁇ L solution of Eu-antibody conjugate (25 ng/ml), 2 ⁇ L DMSO solution containing a test compound, and 10 ⁇ L solution containing GST-JNKl (or GST- JNK2) protein (100 nM), were added. After 2 hours of incubation, each well was washed 4 times to eliminate unbound JNKl and the Eu- antibody if displaced by the test compound.
  • enhancement solution Perkin Elmer
  • fluorescence read after 30 minutes incubation excitation wavelength, 360 nm; emission wavelength, 620 nm.
  • Controls include unlabeled pepJIPl peptide (Anaspec) and blanks received no compound.
  • Controls include unlabeled peptide and blanks receiving no compounds. Protein and peptide solutions were prepared in Delfia buffer (Perkin-Elmer). Upon identifying mixtures that gave >50% inhibition at 12.5 ⁇ M, dose response measurements were performed to filter out eventual false positives. The mixtures were deconvolved to identify individual test compounds having activity and the individual compounds were retested to identify actual hits.
  • a set of 31 potential pepJIPl mimics identified in a virtual docking approach were selected and tested further tested using Delfia®. Each compound was tested in duplicate; positive controls included pepJIPl (at 25 ⁇ M) and blanks received no proteins (6 wells each).
  • Exemplary compounds identified by the method are 5328208 and 5245976 as set forth in Figure 3.
  • TR-FRET assay was used as a secondary assay to further characterize the binding of the compounds identified through the virtual docking and Delfia assay described above.
  • Example 5 Fluorescence Polarization Assay A homogeneous assay may also be to identify modulators of JIP1/JNK.
  • the assay is based on displacement of fluorescence polarization (FP), which is also a mode of detection for the Victor V2 plate reader.
  • FP fluorescence polarization
  • a FITC-labeled-pepJIPl is synthesized (Anaspec) and its polarization measured at increasing protein concentrations (JNKl and JNK2) in 96-well plates. Upon binding, the initially rapid rotational correlation time of the FITC-pepJIPl approaches that of the macromolecule with concomitant increase of emitted polarized light.
  • Test compounds can be monitored for their ability to bind to the JIPl binding pocket of JNK by detecting a decrease of polarization due to the displacement of bound FITC-pepJIPl.
  • the advantage of FP versus Delfia® is that the sample preparation is simple and the assay does not require the use of tagged protein nor specific antibodies. In addition, being a homogeneous assay, FP is more suitable for testing several compounds (no time consuming washing steps).
  • the potential interference from fluorescence of the test compound may be at least partially solved by using pepJIPl tagged with red-shifted Alexa Fluor dyes available from Molecular Probes.
  • a FP assay may be used as rapid primary assay while Delfia ® may be used for hit validation in a secondary assay.
  • a 30,000 compound library (-16,000 compounds, Maybridge Corporation, Cornwell,
  • BI-78D3 was found to be 100-fold less active versus p38, a member of the MAPK family with high structural similarity to JNK. Furthermore, from Lineweaver-Burk analysis, BI-78D3 is a reversible inhibitor and is competitive with ATF2 for binding to JNKl with an apparent K(i) value of 200 nM ( Figure 19E). In addition, BI-78D3 does not inhibit the phosphorylation of a short peptide substrate lacking a D-domain (data not shown) further confirming that BI-78D3 is substrate competitive.
  • FIG. 20A were performed.
  • IC 50 data and structure-Activity Relationship (SAR) data were collected on compound analogues BI-78D3, BI-83C9, BI-83C7, BI-83C8, BI-83B3, BI-83C11, respectively, (see Table III).
  • SAR structure-Activity Relationship
  • the docked structure of BI-78D3 onto the binding surface of JNKl has many features in common with the recently solved X-ray structure of the ternary complex including pepJIPl, JNKl and the ATP mimic SP600125 (Heo et al., 2004; Figure 19A).
  • An in vitro kinase assay was used as a secondary assay to further characterize the binding of the BI-78D3, identified through the Delfia assay described in Example 4, and related compounds.
  • To assay in vitro kinase activity the LanthaScreen assay platform from Invitrogen was utilized. Time-resolved fluorescence resonance energy transfer assay (TR-FRET) was performed in 384 well plates. Each well received JNKl (100 ng/mL), ATF2 (200 nM), and ATP (1 ⁇ M) in 5OmM HEPES, 1OmM MgCl 2, ImM EGTA and 0.01% Brij-35, pH 7.5 and test compounds.
  • TR-FRET Time-resolved fluorescence resonance energy transfer assay
  • the kinase reaction was performed at room temperature for 1 hr. After this time, the terbium labeled antibody and EDTA were added into each well. After an additional hour incubation, the signal was measured at 520/495 nm emission ratio on a BMG Pherastar fluorescence plate reader.
  • the LanthaScreen kinase assay results for BI-78D3 and other compounds screened are shown in Table III and Table IV.
  • NMR-based approaches may also be used to derive potential JNK1/2 inhibitors.
  • Target mediated ligand-ligand transferred NOEs ILOEs
  • SAR by ILOE is analogous to the well known SAR by NMR strategy, and allows the design of possible bi- dentate inhibitors by screening a library of compound "scaffolds" in overlapping mixtures.
  • the approach is based on the detection of magnetization transfer between an initial hit and a second compound (a scaffold) occupying an adjacent site on the surface of the target, measured in the presence of a sub-stoichiometric amount of unlabeled protein.
  • a second compound a scaffold
  • ILOEs strong negative ligand- ligand NOEs
  • NMR -based assay is the saturation transfer difference (STD) (Mayer et al., 1999) or the related experiment WaterLOGSY (Dalrit et al., 2000) in which binding of a compound is detected by a direct transfer of magnetization (saturation) from the protein to the ligand.
  • STD saturation transfer difference
  • WaterLOGSY WaterLOGSY
  • Solubility at least 100 ⁇ M
  • off rates of the complex may be limiting factors (not slower than 1 s "1 , as general rule), as compounds with poor solubility ( ⁇ 50-100 ⁇ M) tend to be promiscuous and non-specific inhibitors.
  • an advantageous NMR-based assay may be the chemical shift mapping of compounds with selectively 13 C-labeled His-JNKl (His-JNK2). Such an assay may validate on the site of binding of compounds. Although these data do not provide precise structural information on the mode of binding (unless a differential chemical shift approach is used) they provide rapid insights on the site of binding and can be used to measure the dissociation constant of the complex by titration.
  • His-JNKl and His-JNK2 are labelled with ⁇ - 13 C-Val, because two such residues are present in the JIPl binding pocket and binding may be detected via 2D [ 13 C, 1 H]-HSQC spectra measured with 0.1 mM protein samples.
  • typical parameters include 1 H and 13 C sweep widths of 12 ppm and 5 ppm, respectively; 128 scans and 80 indirect acquisition points and a recycle delay of 1 second.
  • NMR probes may be used to confirm the predicted binding mode of BI- 78D3.
  • a recently developed probe, azaindole-TEMPO (Vasquez et al., submitted), brings a paramagnetic spin label into proximity of the JIP binding site of JNK by anchoring in the ATP binding site ( Figure 20A).
  • Azaindole-TEMPO is an ATP-mimic linked to 2,2,6,6- tetramethylpiperidine 1-oxyl, which provides the paramagnetic spin label ( Figure 20A). If a test compound binds in close proximity to azaindole-TEMPO, its NMR signal will be affected by the unpaired electron.
  • the signals of the aliphatic protons on the benzodioxan moiety are most affected while the signal of the proton on the thiazole ring is least affected (Hl in Figure 20B).
  • the data indicate that the benzodioxan moiety is closest to ATP-TEMPO.
  • paramagnetic spin probe data are consistent with the orientation of BI- 78D3 predicted by in silico docking ( Figure 20C).
  • Computational docking studies were performed with GOLD 2.1 (The Cambridge Crystallographic Data Centre, Cambridge, UK) (Eldridge et al., and Jones et al., 1997) and analyzed with Sybyl (Tripos, St. Louis).
  • Molecular surfaces were generated with MOLCAD (Teschner et al., 1994).
  • the X-ray coordinates of JNKl/pepJIPl/SP600125 (PDB-ID IUKI) were used to dock the compounds.
  • Molecular models were generated with CONCORD (Pearlman, 1998) and energy minimized with Sybyl.
  • Hits were validated and ranked in terms of potency (dose response curves with available assays, including NMR-based binding assays and chemical shift mapping), drug-likeness, and ease of synthesis.
  • Compound analogs were selected and pre-screened in silico against JNK and their binding properties predicted via a consensus score and visual inspection. Modeling studies served as a guide to prioritize the synthetic chemistry efforts.
  • NMR-based chemical shift mapping data served as experimental verification of the site of binding to provide further validation to the docked structures. The experimental results were and will be further used to verify and refine the models for further iterations.
  • bi-dentate compounds were confirmed to actually bind in the JIPl binding pocket of JNK and not bind in the ATP pocket.
  • ⁇ 13 C-Met labeled samples of JNKl and JNK2 and chemical shift mapping were employed.
  • Inter-ligand NOEs between the methyl group of the ester moiety of BI-7E11 and benzene hydrogen atoms adjacent to the amino group of BI7E5 were observed.
  • additional potential bi-dentate compounds may be designed by linking the respective scaffolds according to the NOEs data.
  • Typical trNOESY spectra are measured with 8 or 16 transients per increment with mixing times of 300 to 800 milliseconds, to maximize the detection of trNOEs and ILOEs. Pooling compounds in mixtures of 6 to 24 expedites the collection of the spectra for the 600 fragments. Analysis of the data and subsequent deconvolution of the spectra allows identification of weak ligands by means of positive trNOEs cross peaks. Similarly, compounds that bind GST-JNKl (GST- JNK2) in close proximity (less that 5 A) are identified by detecting intermolecular NOEs (ILOEs) and may serve as building blocks for producing linked compounds as described above.
  • GST-JNKl GST-JNKl
  • ILOEs intermolecular NOEs
  • Inter-nuclear distances are derived by ILOEs build-up rate measurements. When possible, eventual spin-diffusion effects are taken into account by measuring QUIET-NOESY (Pellechia et al., 2002) experiments. Binders are will be further validated by their ability to bind in the JIPl binding pocket, verified by, for instance, chemical shift mapping.
  • JNKl -2 activity To test the capacity of the putative inhibitors to block JNKl -2 activity, HEK293T cells were treated with 5 ⁇ g/ml anysomicin for 30 minutes to induce JNK activation. Protein extracts were prepared and JNKl -2 proteins were isolated by immunoprecipitation using monoclonal antibodies (Pharmigen). Different aliquots of the immunopurified JNKl -2 proteins were tested for kinase activity by incubation in kinase buffer in the presence of increasing concentrations of inhibitor, a fixed amount of purified GST-cJun substrate (residues 1-79), and [ 32 P] ⁇ -ATP. The products of the kinase reaction were resolved by SDS-PAGE and blotted onto PDF membrane.
  • the signals corresponding to the GSTcJun phosphorylated substrate were detected by phosphorimager and the PVDF membrane is further analyzed by immunoblot analysis with polyclonal anti-JNKl-2 antibodies (Pharmigen) to quantify the amount of JNKl -2 proteins in each kinase reaction ( Figure 14B).
  • the specificity of the inhibitors were tested by observing their effect on the closely related kinases p38 and ERK. Whereas p38 is also activated by the same stimuli that activate JNK, both ERK and p38 are closely related to JNK, have a very similar three-dimensional structure and are activated by the same group of upstream kinases, the MKKs.
  • ERK and p38 kinase assays are performed by immunoprecipitation using specific antibodies, myelin basic protein (UBI) as substrate for ERK and MAPK activate protein kinase 2 (GST-MAPKAPK2) as substrate for p38.
  • UBI myelin basic protein
  • GST-MAPKAPK2 MAPK activate protein kinase 2
  • the capacity of compounds to block the interaction of purified GST-JNKl and GSTJNK2 fusion proteins on purified substrates that are of relevance for type-2 diabetes was measured. More specifically, purified GST-JNKl or GST- JNK2 was incubated with His-tagged cJun or His-tagged IRS 1 fusion proteins.
  • HEK293T cells were transfected with plasmids encoding the kinases JNKl -2 and Myc-tagged substrates, Myc- cJun and Myc-IRSl. 24 hours after transfection, cells were treated with 10 ng/ml of TNF ⁇ for 30 minutes in the presence or absence of JNK inhibitors. JNK mediated phosphorylation of the cJun and IRS 1 substrate were measured by cMyc immunoprecipitation and immunoblot analyses using cJun phospho Ser63 antibodies (Cell Signaling) and anti IRSl Ser307 antibodies (Upstate).
  • JNK inhibits insulin signaling via IRSl Ser307 phosphorylation in TNF ⁇ treated hepatoma cells (Hirosumi et al., 2002). HepG2 hepatoma cells are treated with 10 ng/ml TNF ⁇ for 30 minutes in presence or absence of the JNK inhibitor, and controls are not treated with TNF ⁇ . Cell extracts are analyzed by immunoprecipitation with IRSl antibodies and immunoblotting using IRS 1 Ser307 specific antibodies (Upstate), then membranes are stripped and reprobed with IRSl antibody as a control to exclude an effect of the inhibitors on IRSl expression.
  • JNK inhibition was reported to protect pancreatic ⁇ -cells from the effect of hydrogen peroxide induced oxidative stress on insulin gene expression (Kaneto et al., 2002).
  • Primary pancreatic islets may be isolated from mice by collagenase-P digestion and Ficoll gradient purification (Shang et al., 2003).
  • Primary ⁇ -cell cultures are used to test the ability of the JNK inhibitors to protect pancreatic islets from the effects of H 2 O 2 on insulin gene expression. More specifically, the primary cell cultures are treated for 48 hours with 50 ⁇ M of H 2 O 2 in the presence or absence of the JNK inhibitor. Insulin and ⁇ -actin mRNA are measured by an Sl nuclease protection assay that we have developed (Westin et al., 2004).
  • the LanthaScreen technology may be used to test compounds in cell based assays for JNK inhibition.
  • Cell based kinase assays for c-Jun and ATF2 phosphorylation may be carried out using the LanthaScreen c-Jun (1-79) HeIa and LanthaScreen ATF2 (19-106) A549 cell lines (Invitrogen, Carlsbad, CA) which stably express GFP-c-Jun 1-79 and GFP-ATF2 19-106 respectively.
  • Phosphorylation is determined by measuring the time resolved FRET (TR-FRET)) between a terbium labeled phospho-specific antibody and the GFP-fusion protein (Robers et al., in press).
  • the cell-based LanthaScreen kinase assay was used to profile the properties of compound BI-78D3, which was isolated from the Delfia screen.
  • the cells were plated in white tissue culture treated 384 well plates at a density of 10000 cell per well in 32 ⁇ l assay medium (Opti-MEM ® , supplemented with 1% charcoal/dextran- treated FBS, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES pH 7.3, and lacking phenol red).
  • cells are pretreated for 60 min with compound (indicated concentration) followed by 30 min of stimulation with 2 ng/ml of TNF- ⁇ , which stimulates both JNK and p38.
  • the medium is then removed by aspiration and the cells were lysed by adding 20 ⁇ l of lysis buffer (20 mM TRIS-HCl pH 7.6, 5 mM EDTA, 1% NP-40 substitute, 5 mM NaF, 150 mM NaCl, 1 :100 protease and phosphatase inhibitor mix, SIGMA P8340 and P2850 respectively).
  • ConA Conconavilin A
  • BI-78D3 is a suitable candidate for further efficacy studies in a variety of animal models of JNK related diseases.
  • In vivo administration of a cell permeable peptidic JNK inhibitor (tat-pepJIPl peptide) was reported to be effective in treating glucose intolerance and type-2 diabetes in both dietary and genetic murine models of obesity (Kaneto, et al., 2004).
  • mice To test the efficacy of a JNK inhibitor found to be effective in cell culture in mice, the effects of the inhibitor to the tat- pepJIPl peptide are compound parallel in mice models of obesity (Hirosumi et al., 2002; Yan et al., 2001, Arikan et al., 2005). To test the effect of the compounds and to determine the optimal concentration needed to obtain an insulin sensitizing effect, 8 week old diabetic ob/ob C57B1/6J mice are injected once daily with different doses of the JNK inhibitor starting from an estimated blood concentration of the inhibitor equal to 5 times ED 50 dose in cultured cells up to 50 times the ED 50 .
  • mice 8 week old male diabetic ob/ob C57B1/6J mice are injected daily with either a saline solution or with 10 mg/Kg body weight of the tat- JIP peptide. Blood is collected every morning from tail vein. Glucose and insulin concentrations are measured using a glucometer (Roche) for glucose and an ELISA kit for insulin (Crystal Chem).
  • IGTT intraperitoneal glucose tolerance test
  • HTT insulin tolerance test
  • Insulin signaling in liver is measured by injection of 25 mIU insulin/kg of body weight through the portal vein, the whole liver will be collected and frozen in liquid nitrogen 2 minutes after injection (Hirosumi et al., 2002).
  • Insulin receptor ⁇ chain (IR ⁇ ), and IRSl tyrosine phosphorylation are measured by immunoprecipitation with either IRS 1 or IR ⁇ antibodies (Upstate) followed by immunoblotting analysis using phospho-tyrosine specific antibodies (Upstate).
  • IRSl Ser 307 phosphorylation is measured by western blot using IRSl phospho Ser 307 specific antibodies.
  • Insulin gene expression is measured by Sl nuclease protection using ⁇ - actin as control from whole pancreas.
  • the compound may also be tested in a dietary model of diabetes.
  • a group of 30 male C57B1/6J mice are placed on high fat high carbohydrate diet from week 4 to week 16 (Hirosumi et al., 2002), and another group of 30 mice is placed on standard chow diet. From each group, 10 mice are treated with control saline solution, 10 mice are injected with the tat- JIP peptide as previously described (Kaneto et al., 2004), and 10 mice are injected intraperitoneally with the optimal concentration of a test JNK inhibitor, and IGTT, IITT, measurements of insulin signaling and of insulin gene expression are then performed as described above.
  • Mass spectral data were acquired on a Shimadzu LCMS-2010EV for low resolution, and on an Agilent ESI-TOF for high resolution and low resolution. Purity of compounds was determined using a Waters HPLC. List of Abbreviations: equivalent (eqv), high performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), room temperature (rt). Purity of compounds was obtained in a HPLC Breeze from Waters Co. using an Atlantis T3 3 ⁇ m 4.6x150 mm reverse phase column.
  • the thiobiurea compound was synthesized from phenyl isothiocyanate in a similar procedure to that of compound 1.
  • 1 H NMR 300 MHz, DMSO-J 6 ) ⁇ 6.05 (s, 2H, NH 2 ), 7.09-
  • Compound 4 was synthesized from 2-(phenylcarbamothioyl)-hydrazinecarboxamide by the reported procedure (Joshua, C; Suni, M.; Nair, V. Tetrahedron 2001, 57, 2003) to afford the desired product as a white solid (75%).
  • Carboxylic acid 6b was obtained as a solid (72%).
  • JNK novel blockers of beta-cell death. Diabetes 50: 77-82 (2001).
  • Clark RD Strizhev A
  • Leonard JM Blake JF
  • Matthew J.B Consensus scoring for ligand/protein interactions. /. MoI. Graph. Model. 20, 281-295 (2002).
  • JNKl a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037 (1994).
  • JNK is required for effector T-cell function but not for T-cell activation. Nature, 405, 91-94 (2000).
  • Hotamisligil GS Spiegelman BM.
  • Tumor necrosis factor a a key component of the obesity- diabetes link. Diabetes, 43,1271-1278 (1994).
  • Kallunki, T. et al. JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes & development 8, 2996-3007 (1994). Kallunki T, Deng T, Hibi M and Michael Karin, c-Jun can recruit JNK to Phosphorylate

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Abstract

The invention provides methods, e.g., screening methods, for identifying modulators of the JNK/JIP interaction. The invention also provides kits comprising reagents for performing the methods of the invention. Also provided are compounds identified as modulators of the JNK/JIP interaction using the disclosed methods, pharmaceutical compositions and therepeutic methods using same.

Description

INHIBITORS OF JNK AND METHODS FOR IDENTIFYING INHIBITORS OF JNK
Related Applications This application claims priority to U.S. provisional application Ser. No. 60/905,933, filed March 8, 2007, and U.S. provisional application Ser. No. 61/002,736, filed November 8, 2007, the entire content of each of which is incorporated herein by this reference in their entireties.
Government Support
The invention described herein was made with support from the National Institutes of Health, Grant Nos. R21 DK073274 and R24 DK080263, and the Government has certain rights in the invention.
Background
Obesity and type 2 diabetes are the most prevalent and serious of the metabolic diseases, affecting more than 50% of adults in the USA (Must, 1999). Obesity activates the kinase JNK in liver, skeletal muscle, and adipose tissue. Recent in vivo data are in line with previous reports showing that activation of JNK by pro-inflammatory cytokines inhibits insulin signaling in mouse embryonic fibroblasts, 3T3-L1 and 32Dir cells, through phosphorylation of Ser307 of IRSl (Uysal et al., 1997). Insulin activates JNK in L6 myotubes, rat adipocytes and Rat-1 fibroblasts, indicating that JNK could act as an important negative feedback regulator of insulin signaling (Standaert et al., 1999).
JNK binds to scaffold proteins and substrates containing a D-domain having the consensus sequence R/KXXXXLXL (Kallunki et al., 1996; Yang et al., 1998). JNK-interacting protein- 1 (JIPl) is a scaffolding protein which enhances JNK signaling by creating a proximity effect between JNK and upstream kinases (Whitmarsh et al., 1998). The JNK-JIPl interaction is mediated by a specific, high affinity D-domain on JIPl. Overexpression of a minimal region of JIPl consisting of the single D-domain inhibited JNK signaling (Dickens et al., 1997; Bonny et al., 2001, Barr et al., 202). In particular, this peptide, pepJIPl, inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 and displayed a remarkable selectivity (no inhibition of the closely related Erk and p38 MAPKs) (Heo et al, 2004).
The mechanism of JNKl inhibition by pepJIPl is mainly due to the competition of pepJIPl with other D-domains of substrates or upstream kinases (Bonny et al, 2001; Ho et al, 2003). However, this is not the only mechanism through which pepJIPl inhibits the kinase. The x-ray structure of the catalytic cleft in the complex (Heo et al, 2004) shows that the ATP- binding site is distorted on binding of pepJIPl to JNKl, resulting in a reduced affinity of ATP to JNKl. In addition, pepJIPl inhibits JNKl phosphorylaton of myelin basic protein (MBP), which is a substrate without a docking site. Consistent with results in JNKl knock out mice (Hirosumi et al., 2002), recent in vivo data with a cell-permeable fusion of pepJIPl and HIV-TAT peptide showed that its administration to mouse models of insulin resistance and type-2 diabetes restored normoglycemia without causing hypoglycemia (Kaneto et al., 2004). However, peptide instability in vivo, short half-life, and costly and inefficient large-scale manufacturing and purification processes are all factors hampering the development of therapies for diabetes based on HIV-TAT-pepJIPl. Thus, there is a need for a method to identify modulators of JIP1/JNK.
Summary of the Invention
As described herein, assays were developed which allow for rapid screening of a number of test compounds to identify modulators of the JIP/JNK interaction. In one embodiment, the modulators are JIPl mimmetics. Additionally, using a combination of structure -based approaches, a number of compounds that bind to the JIPl binding pocket of JNK and thereby inhibit its function were identified. The invention also provides pharmaceutical compositions, kits comprising reagents for the disclosed methods, kits comprising compounds and/or pharmaceutical compositions of the invention and methods of treating patients having a metabolic disorder.
Specifically, in one aspect, the invention provides methods to identify modulators of the JIP/JNK interaction, by providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent, washing the solid support to remove molecules not associated with the solid support, separating the label from the ligand; and determining the amount of separated label.
In one embodiment, the polypeptide comprising JIPl or a fragment thereof is coupled to biotin. In another embodiment, the solid support comprises streptavidin.
In one embodiment, the polypeptide comprising JNK further comprises glutathione S transferase (GST) or a His tag. In yet another embodiment, the ligand is an antibody, e.g., an anti GST antibody. In a specific embodiment, the label is a lanthanide, e.g., europium. In one embodiment, the solid support is a multi-well plate. In one embodiment, the JNK is JNKl . In another embodiment, the JNK is JNK2. In one embodiment, the methods further include determining the amount of separated label in the associated molecules. In yet another embodiment, the methods further include determining the amount of separated label in the unassociated molecules.
-?- In one embodiment, the methods further comprise the step of pre-selecting test agents using virtual docking.
In one aspect, the invention provides methods to identify modulators of the JIP/JNK interaction, by providing a sample comprising a test agent, and a polypeptide comprising labeled JIPl or a fragment thereof, wherein the label is a fluorophore; and determining fluorescence polarization in the sample relative to a control sample that lacks the test agent.
In one embodiment, the JNK is JNKl . In another embodiment, the JNK is JNK2. In another embodiment, the fluorophore is fluorescein isothiocyanate (FITC).
In one embodiment, the methods further comprise the step of pre-selecting test agents using virtual docking
In one aspect, the invention provides methods to identify a modulator of the JIP/JNK interaction, by contacting a solid support comprising a polypeptide comprising JIP or a fragment thereof with a sample comprising a fusion protein comprising JNK or a fragment thereof, a labeled ligand for JIP, and a test agent, separating molecules not associated with the solid support comprising JIPl or the fragment thereof from molecules associated with the solid support, separating the label from the ligand, and determining the amount of separated label.
In one embodiment, the polypeptide comprising JIP or the peptide thereof is coupled to biotin. In another embodiment, the solid support comprises streptavidin. In another embodiment, the JNK fusion protein further comprises GST or a His tag. In another embodiment, the ligand is an antibody. In yet another embodiment, the label is a lanthanide.
In one embodiment, the solid support is a multi-well plate.
In one embodiment, the JNK is JNKl . In another embodiment, the JNK is JNK2.
In another embodiment, the methods further include determining the amount of separated label in the associated molecules. In yet another embodiment, the methods further include determining the amount of separated label in the unassociated molecules. In one embodiment, the methods further comprise the step of pre-selecting test agents using virtual docking.
In other aspects, the invention provides assays which do not use a solid support, e.g., soluble assays. Specifically, in one aspect, the invention provides methods for identifying a modulator of JIP/JNK interaction, by contacting a complex comprising polypeptides comprising JIP and JNK, or fragments thereof, each of which contains one of a donor and an acceptor fluorophore, with a test agent, and measuring the FRET between the donor and acceptor fluorophore, thereby identifying a modulator of the JIP/JNK interaction of JIP and JNK. In one aspect, a decrease in the FRET indicates that the test compound modulates the interaction. In one embodiment, the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex. In a specific embodiment, the fluorescent acceptor is attached to JIP.
In another embodiment, the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK. In a specific embodiment, the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
In specific embodiments, the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively. In another specific embodiment, the FRET is time resolved FRET.
In another embodiment, the methods of the invention also provide isolating and identifying the test compound.
In one embodiment, the methods further comprise the step of pre-selecting test agents using virtual docking. In one aspect, the invention provides methods for identifying modulators of JIP/JNK interaction by contacting a solution comprising a polypeptide comprising JIP, or a fragment thereof, wherein the polypeptide comprising JIP, or a fragment thereof, comprises a fluorescent donor or acceptor, with a test agent; adding to the solution a polypeptide comprising JNK, or a fragment thereof, comprising a donor or acceptor, wherein the JNK polypeptide comprises either a fluorescent donor or acceptor suitable to form a fluorescent donor/acceptor pair with the JIP fluorophore; and measuring the FRET between the donor and acceptor fluorophore, thereby identifying a modulator of the JIP/JNK interaction.
In one embodiment, a decrease in the FRET compared to a solution lacking the test compound indicates that the test compound modulates the interaction of JIP and JNK. In another embodiment, the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex. In a specific embodiment, the fluorescent acceptor is attached to JIP. In another embodiment, the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK. In another specific embodiment, the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
In a specific embodiment, the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively.
In one embodiment, the methods of the invention further provide for isolating and identifying the test compound. In one aspect, the invention provides methods for inhibiting JIP/JNK interaction in cells comprising contacting cells with an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
In one aspect, the invention provides methods for treating a metabolic disorder comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
In one aspect, the invention provides methods for treating diabetes, comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below. In one aspect, the invention provides methods for treating obesity comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV) or (V) further described herein below.
In one aspect, the invention provides compounds for the treatment of a metabolic disorder, wherein the compound has the structure set forth as formula (II), (III), (IV) or (V) further described herein below. In another embodiment, the invention provides compounds for the treatment of a metabolic disorder, wherein the compound has a structure set forth in Table I, Ia, II, Ha, III, IV, V, or VI set forth herein below. In related embodiments, the metabolic disease is obesity or diabetes.
In one aspect, the invention provides compounds having the structure set forth as formula (II), (III), (IV) or (V) further described herein below.
In another aspect the invention provides pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier therefor. In another embodiment, the pharmaceutical compositions are for the treatment of a metabolic disorder, e.g., obesity or diabetes. In yet another aspect, the invention provides a kit comprising one or more reagents useful in the methods of the invention and instructions for use.
In one aspect, the invention provides kits for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent donor and a JIP polypeptide, or fragment thereof, comprising a fluorescent acceptor and instructions for use.
In one aspect, the invention provides kits for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent acceptor and a JIP polypeptide, or fragment thereof, comprising a fluorescent donor and instructions for use. In one aspect, the invention provides kits comprising one or more compounds or pharmaceutical compositions of the invention and instructions for use. In another aspect, the invention provides a compound identified by any one of the methods described herein.
In particular aspects, the methods of the invention may comprise an additional step of screening a potential modulator of JNK/JIP interaction against a JNK2 mutant to determine active site binding location. For example, the additional step may comprise the step of screening a potential modulator of JNK/JIP interaction against R127A and C167S mutants of JNK2 to determine if the compound is binding in the JIP pocket.
Brief Description of the Drawings Figure 1 is a schematic representation of the use of Delfia® (Dissociation enhanced lanthanide fluorimmuno assay) to identify JIPl mimics.
Figures 2A-B set forth a (A) graph showing titration of a JIP peptide with Delfia® and JNK and (B) dinase assay with GST-cJun and JIPl.
Figure 3 sets forth exemplary compounds (5328208 and 5245976) identified with Delfia® and JNK as modulators of the JIP1/JNK interaction.
Figures 4A-B depicts NMR binding data for (A) 5328208 and (B) 5245976 in the presence of GST-JNK2.
Figure 5 depicts the results of a displacement assay for 5245976. Figure 6 sets forth analogs of 5245976 (6115184, 7246555 and 7261715) and their percent displacement.
Figure 7 depicts the results of a competition assay for 11E5, 11E6, 11E8 and 11E9. Figure 8 depicts the results of a kinase assay for 11E9. The IC50 of 11E9 was determined to be approximately 25μM.
Figure 9 sets forth analogs of 5245976. Figure 10 sets forth the structures of compounds identified after molecular docking,
NMR and Delfia® analyses (17A5, 17C6, 19E2 and 24Dl). Figure 10 also sets forth the percent inhibition for these compounds at 25μM. The percent inhibition is 80%, 83%, 80%, and 100% for 17A5, 17C6, 19E2 and 24Dl, respectively.
Figures HA-C set forth IC50 data for 17C6, 19E2 and 24Dl. Figure 12 sets forth compounds identified by NMR (7E5 and 7El 1).
Figure 13 sets fort TIr, STD, and WaterLogsy NMR data for 7E5 and 7El lat 400μM in the presence of 5μM JNK2.
Figures 14A-B sets forth the kinase assay results for 5245976. A) Effect of compounds on kinase activity for JNKl and JNK2. Upper panel lanes 1-11 : Hek293T cells transfected with a plasmid encoding HA-JNKl. Lower panel lanes 1-11 : Hek293T cells transfected with a plasmid encoding HA-JNK2. Lane 1) no inhibitor; lane 3) 7El 1 200 μM; lane 4) 7E5 200 μM; lane 5) 5328028 200 μM; lane 7) 5245976 200 μM; lane 8) JIP 10 μM; lane 9) SP600125 10 μM; and lane 12) no inhibitor GFP control. After lysis, and JNKl or JNK2 were purified by immunoprecipitation with anti-HA antibodies and after a final wash, the immunoprecipitate was separated in different aliquots and incubated or not with the "candidate" JNK inhibitor. B) Dose response data for 5245976.
Figure 15 schematically depicts the soluble assay for modulators of JIP/JNK interaction.
Figure 16 depicts the results of a statistical analysis of a soluble assay of the invention. Z was determined to be 0.8 indicating that the assay is very reliable.
Figures 17A-C depict the LANCE assay results for JIPl, 24Dl and SPB07895, respectively.
Figures 18A and B depict the polypeptide sequence of JIP (SEQ ID NO:1) and JNK (SEQ ID NO:2), respectively.
Figures 19A-D depict the in vitro characterization of pep-JIP and BI-78D3: A) surface representation of JNKl in complex with ball and stick representation of pepJIPl (RPKRPTTLNLF) and the ATP mimic SP600125 (PDB-ID IUKI), surface generated with MOLCAD (Teschner et al., 1994) and color coded according to cavity depth (blue, shallow; yellow, deep); B) BI-78D3 chemical structure; and C) Displacement assay for BI-78D3. D) Kinase assay for BI-78D3. E) Double reciprocal plot of JNK kinase activity as function of ATF2 in the presence of various amounts of BI-78D3 or DMSO control. Figures 20A-C depict docking studies and NMR analysis of BI-78D3: A) chemical structure and predicted binding mode of SP600125 derived azaindole-TEMPO compound; B) docked structure of BI-78D3 into the X-ray structure of JNKl (PDB ID IUKI) and predicted hydrogen bonding interactions between the compound and the residue Argl27 (displayed) are highlighted with dashed lines; and C) ID 1H-NMR Tlp spectra of BI-78D3 (500 μM) in the presence of 5 μM JNK2 (blue) or 5 μM JNK2 and 200 μM ATP-TEMPO (red) at 100 ms, NMR resonance assignments for the hydrogen nuclei of the small molecule are reported and signal reductions are as follows: Hl 46%, H2 56%, H3/H5 65%, and H4 72%.
Figures 21 A and B depict biological analysis of BI-78D3: A) TR-FRET analysis of ATF2 phosphorylation upon TNF-alpha stimulation of A549 cells in the presence of increasing BI-78D3; and B) BI-78D3 effect on serum alanine-aminotransferase levels after 7.5 hours of exposure to Conconavilin A as compared to DMSO control.
Figures 22 A and B set forth compounds with 100% displacement at 100 μM in the DELFIA assay for displacement of JIP peptide. Detailed Description of the Invention
Definitions
A "label" as used herein, is a molecule which is detectable or capable of detection, for instance, a radiolabel, biotin, a hapten, a fluorophore, e.g., coumarin, rhodamine, rhodols, CRG6, Texas Methyl Red, fluorescein, 7 aminocoumarin, and 7-hydroxycoumarin, 2-amino-4- methoxynapthalene, 1-hydroxypyrene, resorufin, phenalenones or benzphenalenones, acridinones, anthracenes, and derivatives of α- and β-napthol, fluorinated xanthene derivatives including fluorinated fluoresceins and rhodols, a bioluminescent molecule, or a chemiluminescent molecule. In one embodiment, the label may include a cleavable linker. In other embodiments the label is a donor or acceptor fluorophore.
A "ligand" as used herein is a molecule that specifically binds to another molecule. Exemplary ligands are a HIS tag, GST, maltose binding protein, biotin, avidin, streptavidin, calmodulin binding protein, hemagglutinin and the like. A "lanthanide," "lanthanide series element" or "lanthanide series inner transition element" refers to Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium(Yb), or Lutetium (Lu). Specific suitable lanthanides include, e.g., Ce(III), Ce(IV), Pr(III), Nd(III), Pm(III), Sm(II), Sm(III), Eu(II), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(II), Yb(III), and Lu(III).
As used herein, the term "antibody" refers to a protein having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (Y11) refer to these light and heavy chains respectively.
Antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, FabFc2, Fab, Fv, Fd, (Fab D)2, an Fv fragment containing only the light and heavy chain variable regions, a Fab or (Fab) D2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, e.g., scFv, CDR-grafted antibodies and the like. The heavy and light chain of a Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric or humanized. As used herein the term "antibody" includes these various forms.
As used herein, "JIP" includes polypeptides having at least 80%, e.g., at least 85%, 90%, 95%, or more amino acid sequence identity to sequences having NCBI Accession Nos. Q9UQF2, Q9WV19, or Q9R237, and which bind JNK, and nucleic acid sequence encoding those polypeptides. In one embodiment, the JIP is JIP-I, e.g., human (SEQ ID NO:1), rodent, for instance, rabbit, mouse, rat, mink or guinea pig, or nonhuman primate JIP-I.
As used herein, "JNK" includes polypeptides having at least 80%, e.g., at least 85%, 90%, 95%, or more amino acid sequence identity to sequences having NCBI Accession Nos. BAA85876, P45984, Q9WTU6 or Q91Y86, and which bind JIP, and nucleic acid sequence encoding those polypeptides. In one embodiment, the JNK is JNKl, e.g., human (SEQ ID NO:2), rodent, for instance, rabbit, mouse, rat, mink or guinea pig, or nonhuman primate JNKl. In other embodiment, the JNK is JNK2. In certain embodiments, the JNK is a mutant JNK (e.g., JNK2), such as, a JNK with a one or two amino acid mutation.
As used herein the term "JIP/JNK interaction" is intended to mean an interaction, e.g., a physical or electrostatic interaction, between the JIP and JNK polypeptides or portions thereof. The interaction can be ligand mediated or directly between the polypeptides.
As used herein the term "modulator" is intended to mean a compound, e.g., a small molecule, a peptide, a polypeptide, an antibody, or an antibody fragment, that has the ability to interfere, e.g., block, weaken or disrupt, the interaction between JIP and JNK. Modulators of the invention can be reversible or irreversible modulators. The term "obtaining" as in, e.g., "obtaining one or more reagents" is intended to include purchasing, synthesizing or otherwise acquiring the reagent or a material used in carrying out the methods of the invention.
As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
The pharmaceutically acceptable salts of the compounds useful in the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985), the disclosure of which is hereby incorporated by reference.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
One diastereomer of a compound disclosed herein may display superior activity compared with the other. When required, separation of the racemic material can be achieved by HPLC using a chiral column or by a resolution using a resolving agent such as camphonic chloride as in Thomas J .Tucker, et al., J. Med. Chem. 1994 37, 2437-2444. A chiral compound of Formula I may also be directly synthesized using a chiral catalyst or a chiral ligand, e.g. Mark A. Huffman, et al., J. Org. Chem. 1995, 60, 1590-1594.
"Therapeutically effective amount" is intended to include an amount of a compound useful in the present invention or an amount of the combination of compounds claimed, e.g., to treat or prevent the disease or disorder, or to treat the symptoms of the disease or disorder, in a host. The combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is advantageously demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased activity, or some other beneficial effect of the combination compared with the individual components.
As used herein, the term "metabolic disease" is intended to mean a disease or disorder characterized by alteration of a normal metabolic process. These disorders are often the result of altered enzyme activity or production. Exemplary metabolic diseases include obesity and diabetes. As used herein, "treating" or "treat" includes (i) preventing a pathologic condition from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; (iii) relieving the pathologic condition; and/or diminishing symptoms associated with the pathologic condition. As used herein, the term "subject" refers to organisms to be treated by the methods of the present invention. Such organisms include, but are not limited to, mammals such as animals, preferably humans. In the context of the invention, the term "subject" generally refers to an individual who will receive or who has received treatment (e.g., administration of a compound of the inventions) for obesity and/or diabetes. "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated by the present invention.
"Substituted" is intended to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated group(s), provided that the indicated atom' s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable indicated groups include, e.g., alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. When a substituent is keto (i.e., =0) or thioxo (i.e., =S) group, then 2 hydrogens on the atom are replaced. The term "virtual docking" is intended to mean the process of testing the ability of a test agent to bind to a target protein, i.e., JNK, using a computer program algorithm. This research technique predicts whether one molecule will bind to another, usually a protein. Most commonly, virtual docking is done by modelling the interaction between two or more molecules: if the geometry of the pair is complementary and involves favorable biochemical interactions, one molecule will likely bind to the other in vitro or in vivo. A specific virtual docking approach is set forth in Example 1.
"Interrupted" is intended to indicate that in between two or more adjacent carbon atoms, and the hydrogen atoms to which they are attached (e.g., methyl (CH3), methylene (CH2) or methine (CH)), indicated in the expression using "interrupted" is inserted with a selection from the indicated group(s), provided that the each of the indicated atoms' normal valency is not exceeded, and that the interruption results in a stable compound. Such suitable indicated groups include, e.g., non-peroxide oxy (-O-), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)-), imine (C=NH), sulfonyl (SO) or sulfoxide (SO2).
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents
"Alkyl" refers to a Ci-Ci8 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (i-Bu, i-butyl, - CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-l -butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (- CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.
The alkyl can optionally be substituted with one or more alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. The alkyl can optionally be interrupted with one or more non-peroxide oxy (-O-), thio (-S-), carbonyl (- C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2). Additionally, the alkyl can optionally be a divalent radical, thereby providing an alkylene.
"Alkenyl" refers to a C2-Ci8 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp2 double bond. Examples include, but are not limited to: ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2 CH2CH2CH2CH=CH2). The alkenyl can optionally be substituted with one or more alkyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. The alkenyl can optionally be interrupted with one or more non-peroxide oxy (-O-), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2). Additionally, the alkenyl can optionally be a divalent radical, thereby providing an alkenylene. "Alkylidenyl" refers to a Cl -C 18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methylidenyl (=CH2), ethylidenyl (=CHCH3), 1- propylidenyl (=CHCH2CH3), 2-propylidenyl (=C(CH3)2), 1-butylidenyl (=CHCH2CH2CH3), 2-methyl-l-propylidenyl (=CHCH(CH3)2), 2-butylidenyl (=C(CH3)CH2CH3), 1-pentyl (=CHCH2CH2CH2CH3), 2-pentylidenyl (=C(CH3)CH2CH2CH3), 3-pentylidenyl (=C(CH2CH3)2), 3-methyl-2-butylidenyl (=C(CH3)CH(CH3)2), 3-methyl- 1-butylidenyl (=CHCH2CH(CH3)2), 2 -methyl- 1-butylidenyl (=CHCH(CH3)CH2CH3), 1-hexylidenyl (=CHCH2CH2CH2CH2CH3),
2-hexylidenyl (=C(CH3)CH2CH2CH2CH3), 3-hexylidenyl (=C(CH2CH3)(CH2CH2CH3)), 3- methyl-2-pentylidenyl (=C(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentylidenyl (=C(CH3)CH2CH(CH3)2), 2-methyl-3-pentylidenyl (=C(CH2CH3)CH(CH3)2), and 3,3- dimethyl-2-butylidenyl (=C(CH3)C(CH3)3.
The alkylidenyl can optionally be substituted with one or more alkyl, alkenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, the alkylidenyl can optionally be interrupted with one or more non-peroxide oxy (- O-), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2). "Alkenylidenyl" refers to a C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp2 double bond. Examples include, but are not limited to: allylidenyl (=CHCH=CH2), and 5-hexenylidenyl (=CHCH2CH2CH2CH=CH2) .
The alkenylidenyl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, the alkenylidenyl can optionally be interrupted with one or more non-peroxide oxy (-O-), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2).
"Alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-) 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.
The alkylene can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, the alkylene can optionally be interrupted with one or more non-peroxide oxy (-0- ), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2). Moreover, the alkylene can optionally be at least partially unsaturated, thereby providing an alkenylene.
"Alkenylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2 -ethylene (-CH=CH-).
The alkenylene can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or COORX, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, The alkenylene can optionally be interrupted with one or more non-peroxide oxy (- O-), thio (-S-), carbonyl (-C(=O)-), carboxy (-C(=O)O-), sulfonyl (SO) or sulfoxide (SO2). The term "alkoxy" refers to the groups alkyl-O-, where alkyl is defined herein. Preferred alkoxy groups include, e.g., methoxy, ethoxy, w-propoxy, wo-propoxy, w-butoxy, tert- butoxy, seobutoxy, w-pentoxy, w-hexoxy, 1,2-dimethylbutoxy, and the like. The alkoxy can optionally be substituted with one or more alkyl, alkylidenyl, alkenylidenyl, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and COORX, wherein each Rx and Ry is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl.
The term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferred aryls include phenyl, naphthyl and the like.
The aryl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and COORX, wherein each Rx and Ry is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, the aryl can optionally be a divalent radical, thereby providing an arylene. Moreover, aryl may be substituted with a divalentsubstituent forming multicyclic structure wherein at least one ring is aromatic, e.g., 2,3-dihydro-benzo[l,4]dioxin-6- yi- The term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
The cycloalkyl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and COORX, wherein each Rx and Ry is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. The cycloalkyl can optionally be at least partially unsaturated, thereby providing a cycloalkenyl. Additionally, the cycloalkyl can optionally be a divalent radical, thereby providing a cycloalkylene.
The term "halo" refers to fluoro, chloro, bromo, and iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine. "Haloalkyl" refers to alkyl as defined herein substituted by 1-4 halo groups as defined herein, which may be the same or different. Representative haloalkyl groups include, by way of example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl, 2-bromooctyl, 3-bromo-6- chloroheptyl, and the like.
The term "heteroaryl" is defined herein as a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, thio, alkylthio, alkylsulfinyl, and alkylsulfonyl. Additionally, the heteroaryl can optionally be a divalent radical, thereby providing a heteroarylene. Examples of heteroaryl groups include, but are not limited to, 2/f-pyrrolyl, 3/f-indolyl,
4/f-quinolizinyl, 4n/f-carbazolyl, acridinyl, benzo[fe]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnaolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, naptho[2,3-fe], oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from the group non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl. In another embodiment heteroaryl denotes an ortho- fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, or tetramethylene diradical thereto.
The heteroaryl can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and COORX, wherein each Rx and Ry is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. The term "heterocycle" refers to a saturated or partially unsaturated ring system, containing at least one heteroatom selected from the group oxygen, nitrogen, and sulfur, and optionally substituted with alkyl or C(=O)ORb, wherein Rb is hydrogen or alkyl. Typically heterocycle is a monocyclic, bicyclic, or tricyclic group containing one or more heteroatoms selected from the group oxygen, nitrogen, and sulfur. A heterocycle group also can contain an oxo group (=0) attached to the ring. Non-limiting examples of heterocycle groups include 1,3- dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2/f-pyran, 2-pyrazoline, 4/f-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, and thiomorpholine.
The heterocycle can optionally be substituted with one or more alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and COORX, wherein each Rx and Ry is independently H, alkyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Additionally, the heterocycle can optionally be a divalent radical, thereby providing a heterocyclene.
Examples of nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles. In one specific embodiment of the invention, the nitrogen heterocycle can be 3-methyl-5,6-dihydro-4H-pyrazino[3,2,l-jk]carbazol- 3-ium iodide. Another class of heterocyclics is known as "crown compounds" which refers to a specific class of heterocyclic compounds having one or more repeating units of the formula [- (CH2-)aA-] where a is equal to or greater than 2, and A at each separate occurrence can be O, N, S or P. Examples of crown compounds include, by way of example only, [-(CH2)3-NH-]3, [- ((CH2)2-O)4-((CH2)2-NH)2] and the like. Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
The term "alkanoyl" refers to C(=O)R, wherein R is an alkyl group as previously defined.
The term "acyloxy" refers to -O-C(=O)R, wherein R is an alkyl group as previously defined. Examples of acyloxy groups include, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Any alkyl group as defined above can be used to form an acyloxy group.
The term "alkoxycarbonyl" refers to C(=O)OR, wherein R is an alkyl group as previously defined.
The term "amino" refers to -NH2, and the term "alkylamino" refers to -NR2, wherein at least one R is alkyl and the second R is alkyl or hydrogen.
The term "acylamino" refers to RC(=O)N, wherein R is alkyl or aryl. The term "imino" refers to -C=NH.
The term "nitro" refers to -NO2.
The term "trifluoromethyl" refers to -CF3.
The term "trifluoromethoxy" refers to -OCF3. The term "cyano" refers to -CN.
The term "hydroxy" or "hydroxyl" refers to -OH.
The term "oxy" refers to -O-.
The term "thio" refers to -S-.
The term "thioxo" refers to (=S). The term "keto" refers to (=0).
As used herein, "nucleic acid base" refers to a nitrogenous base that is planar, aromatic and heterocyclic. They are typically derivatives of either purine or pymidine. Suitable nucleic acid bases include, e.g., purine, pymidine, adenine, guanine, cytosine, uracil, and thymine.
The nucleic acid base can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NRxRy and/or C00Rx, wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. As to any of the above groups, which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
Selected substituents within the compounds described herein are present to a recursive degree. In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis.
Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in an claim of the invention, the total number will be determined as set forth above. The compounds described herein can be administered as the parent compound, a prodrug of the parent compound, or an active metabolite of the parent compound.
"Pro-drugs" are intended to include any covalently bonded substances which release the active parent drug or other formulas or compounds of the present invention in vivo when such pro-drug is administered to a mammalian subject. Pro-drugs of a compound of the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation in vivo, to the parent compound. Pro-drugs include compounds of the present invention wherein the carbonyl, carboxylic acid, hydroxy or amino group is bonded to any group that, when the pro-drug is administered to a mammalian subject, cleaves to form a free carbonyl, carboxylic acid, hydroxy or amino group. Examples of pro-drugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention, and the like.
"Metabolite" refers to any substance resulting from biochemical processes by which living cells interact with the active parent drug or other formulas or compounds of the present invention in vivo, when such active parent drug or other formulas or compounds of the present are administered to a mammalian subject. Metabolites include products or intermediates from any metabolic pathway.
"Metabolic pathway" refers to a sequence of enzyme-mediated reactions that transform one compound to another and provide intermediates and energy for cellular functions. The metabolic pathway can be linear or cyclic.
JNK Isozymes and JIPl
JNKl -3 isozymes are members of the mitogen activated protein kinases (MAPK), considered to be at the focal point of different signaling pathways activated by many distinct cell surface receptors and whose role is to transduce these signals to effector molecules that generate changes in gene expression by regulating transcription and mRNA stability (Hirosumi et al., 2002). JNKs activate the transcription factors TCF/Elk-1, ATF2 and c-Jun by phosphorylation, leading to increased c-jun and fas gene transcription and increased API transcription factor activity (Hirosumi et al., 2002; Aibi et al., 1993; Derijard et al., 1994; Shanlian et al., 2002; Gupta et al., 1996; Kaneto et al., 2002; Shang et al., 2003). The JNKs are also implicated in stabilization of inherently unstable mRNAs, as shown for IL-2 mRNA. Thus, the JNKs are considered key signaling molecules in the "communication" between external stimuli (stress) and gene expression. The JNKs are activated in response to several stresses, such as infection, pro-inflammatory cytokines, UV radiation, various xenobiotics and oxidative stress (Aibi et al., 1993; Derigard et al., 1994, Shanlian et al., 2002; Gupta et al., 1996; Kaneto et al., 2002, Shang et al., 2003; Dong et al., 2000; Yuan et al., 2001; Arkan et al., 2005; Lin, 2003; Hotamisligil et al., 1994a, Hotamisligil et al., 1994b; Ofei et al., 1996).
It was recently demonstrated in vivo that JNKl is a potent regulator of insulin signaling and could be implicated in the pathogenesis of the metabolic syndrome (Hirosumi et al., 2002; Aguirre et al., 2000). Briefly, insulin stimulates glucose entry into cells by binding the insulin receptor (IR) at the cell surface. IR is a member of the tyrosine kinase receptor-family and after hormone binding, it autophosphorylates tyrosine residues on its cytoplasmic domain. This in turn leads to the recruitment of the scaffold proteins Insulin Receptor Substrates (IRS), likely via their phosphotyrosine binding domain (PTB). Several IRS molecules have been identified. However, only IRSl and IRS2 have been clearly proven to play a major role in insulin signaling. Both IRSl and IRS2 are phosphorylated at several tyrosine residues by the IR, making possible the recruitment of other signaling molecules such as PI3 kinase (PI3K), which interacts with specific phosphotyrosine residues on IRS1/IRS2 via the SH2 domain of the regulatory P85 subunit. The signal is then transmitted to downstream signaling molecules and this cascade of events results in increased expression and activity of several enzymes involved in energy metabolism and in the increased translocation to the cytosolic membrane of the glucose transporter GLUT4, which increases glucose uptake in skeletal muscle and adipose tissue (Yang et al., 1998).
In recent studies, JNKl knock out mice were found to be resistant to diabetes in both dietary and genetic models of obesity (Hirosumi et al., 2002). It was proposed that JNKl elicits insulin resistance via specific phosphorylation of serine 307 present in the PTB domain of IRSl, which interferes with its recruitment to the IR, resulting in decreased IRS 1 tyrosine phosphorylation and impairment of PI3K recruitment in response to insulin (Whitmarsh et al., 1998; Dickens et al., 1997; Bonny et al., 2001). Data from an in vivo study with a JNK inhibitor are in line with the role of JNKl in type-2 diabetes and point attention to islet function (Kaneto et al., 2004). Recent studies where a dominant negative mutant of JNK (DN-JNK) was expressed in primary β-cells supported the hypothesis of a role of JNK in H2O2 mediated inhibition of insulin gene expression and suppression of GSIS. Islets transduced with an adenovirus expressing DN-JNK transplanted in streptozotocin treated mice were more efficient than islets transduced with a GFP adenovirus in controlling blood glucose levels.
The combination of high-throughput screening, kinase-specific libraries and structure- based drug design has facilitated the discovery of potential JNK inhibitors that target its ATP binding site (Manning et al., 2003). Determination of the X-ray structure of the members of the mitogen-activated protein kinase family, extracellular signal-regulated kinases, p38 and JNK3 has aided the design of potent inhibitors of the JNKs. These efforts have led to the optimization of a series of JNK inhibitors that show selectivity towards many other protein kinases, but certainly not all proteins in the cell (Manning et al., 2003). In fact, because of the large number of ATP-binding proteins involved in complex cross-talk within signaling events (> 500 if considering only protein kinases), it is difficult to predict potential adverse events that might arise from inhibitors that directly target the ATP site of JNK. In addition to screening strategies based on synthetic chemical libraries, it has become clear that domains of interacting partners can be exploited as signal transduction regulators. Indeed, an emerging feature of the signal transduction pathways, including the MAPK pathways, is their use of specific scaffolding proteins (Morrison et al., 2003). These scaffolds essentially lack enzymatic activity and have been proposed to act as the "directors" of each specific signaling pathway. The interaction of JNK with scaffold proteins offers a valid alternative target for JNK inhibition. JIPl specifically interacts with components of the JNK signaling modules by binding JNK, MKK7, and members of the mixed lineage kinase (MLK) group of MAP3K (Whitmarsh et al., 1998, Aquirrre et al., 2000; Momson et al., 2003; Willoughby et al., 2003). Interestingly, both JIPl and the p38 cognate, JIP2, can bind the MAPK phosphatase MKP7, indicating that these JIP scaffold complexes include both activating and inhibitory components of the JNK signaling pathway (Willoughby et al., 2003). Deletion analysis of JIPl demonstrates that the sites of interaction with JNK, MKK7, and MLK protein kinases correspond to separate sites on JIPl (Whitmarsh et al., 1998).
Nevertheless, binding studies demonstrate functional interactions between the proteins bound to JIP. For example, the binding of JNK decreased the affinity of JIPl for MLK (Nihalani et al., 2001); the binding of FHF increased the binding of JIP2 to p38 MAPK (Fattorusso et al., 2005); the binding of AKT to JIPl decreased the binding of JNK to JIPl (Leone et al., 2003); and the binding of the phosphatase MKP7 to JIPl inhibited JNK activation caused by the JIPl scaffold complex (Willoughby et al., 2003). One of the effective means of JNK inhibition employed the overexpression of JIP 1.
An 11-amino acid peptide inhibitor of activated JNKs, based on amino acids 153-163 of JIPl (pepJIPl), inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 up to 90% at sub-micromolar concentrations and directly interacted with recombinant JNKs but not its substrates, as shown by surface plasmon resonance analysis and isothermal titration calorimetry (ITC) (Heo et al, 2004) Alanine scanning within pepJIPl identified 4 residues (Arg-156, Pro-
157, Leu-160, and Leul62) as independently important for inhibition (Willoughby et al., 2003)). Intraperitoneal administration of a cell-permeable pepJIPl (10 mg/Kg) to C57BL/KsJ-db/db obese diabetic mice resulted in mice with a remarkably improved insulin resistance and glucose tolerance (Kaneto et al., 2004). Moreover, in vivo administration of the cell-permeable pepJIPl to both genetically and dietary obese mice restored normoglycemia but did not cause hypoglycemia in lean mice (Kaneto et al., 2004).Thus, in vitro and in vivo data with pepJIPl clearly point to the inhibition of JNK-JIPl interactions as a promising route for the treatment of type-2 diabetes.
Compound Design and Screening Recently, the interaction of JNK and JIP has been the subject of significant research that has confirmed the use of modulators of this interaction for the treatment of disease, e.g,. obesity or diabetes. Accordingly, the soluble and solid support assays described herein are useful in identifying modulators of the JIP/JNK interaction.
Specifically, the crystal structure of JNKl in complex with pepJIPl provided insightful information on the nature of the JIPl /JNKl interaction (Heo et al., 2004). In agreement with the alanine- scanning data, three main interactions can be observed between the peptide and JNKl : a first hydrophobic cavity on JNKl accommodates the side-chain of Leu- 160 and Leu-162; a second shallower pocket is occupied by Pro-157; finally, intermolecular hydrogen bonding involving Arg-156 and Thrl58 was observed. With such information in hand, it should be possible to derive small organic molecules capable of mimicking these interactions.
In the realm of drug discovery, the identification of a lead compound represents the starting point for the development of new candidate drugs. Given the nature of the JIPl target, different strategies are pursued for hit identification and optimization processes. One approach is based on a combination of medicinal chemistry guided by nuclear magnetic resonance (NMR) spectroscopy and structure-based techniques. These enable the identification of initial "weaker" hit compounds and guide the translation of such compounds into more potent and selective leads.
In this regard, some general strategies have developed aimed at the design of optimized inhibitors based either on structure-based iterative optimizations (SUBITO; Fattorusso et al., 2005; Pellechia et al., 2004) or on fragment linking approaches (SAR by ILOE; Becattani et al., 2004; Pellecchia et al., 2004). Other strategies take advantage of virtual docking and transfer nuclear Overhauser effect techniques, and are applicable even to "non-canonical" and otherwise less tractable protein sites, such as the JIPl binding pocket of JNK.
In designing compounds to be tested and/or synthesized, "drug-likeness" is addressed on empirical grounds. First, compounds with reactive functional groups such as halides, anhydrides, epoxides, aziridines, aliphatic esters and thioesters, phosphonates, sulphonates esters, imines, aldehydes, Michael acceptors, halopyrimidines, and the like, are generally not considered. Second, compounds with undesirable properties are generally eliminated. For instance, the following selection criteria may be used: molecular weight < 500, number of hydrogen bond donors between 0 and 2, number of hydrogen bond acceptors between 2 and 9, number of rotatable bonds less then 8, and octanol/water repartition coefficient (LogP) < 5 (Oprea, 2000). The goal in using these empirical drug-like property filters is to predict favorable outcome in ADMET (adsorption, distribution, metabolism, excretion, toxicity) studies, as well as final success as a drug in humans.
For an NMR-based approach (SAR by ILOE), a library having a fairly small (about 600 compounds) but diverse set of low-molecular-weight scaffolds derived mainly from compounds commonly found in known drugs (Becattini et al., 2004) was employed. This library was assembled and ID 1H and 13C NMR spectra were measured as a quality control and to assess solubility. The library was designed also to optimize the detection of ligand-ligand interactions by selecting compounds with appropriate derivatizations of functional groups with proton NMR- detectable substituents. Furthermore, the introduction of heteroatoms in these substituents also results in large chemical shift dispersion between the compounds of the library, thus enabling the detection of ILOEs in complex mixtures.
In performing chemical synthesis of linked compounds, solution-phase synthesis aided by resin-bound reagents and scavengers may be employed. These allow removal of excess reagents and byproducts by using filtration rather then using liquid-liquid extraction, chromatography or crystallization. Initially, the focus is on linkage via amide bonds, ethers or thioethers. Amide bond formation may be performed with the coupling reagent PS-carbodiimide (Argonaut) in presence of HOBt, subsequently scavenged post-reaction by the resin-bound PS- Trisamine. Similarly, basic reducing reactions including reductions of carbonyl compounds, azides and oximes, reductive amination, reduction of conjugated enones to unsaturated alcohols, may be performed with MP-Borohydride resin (Temoi et al., 1982). Synthesis of acyl or sulfonyl derivatives, including esters, amides, and sulfonamides may be performed by using PS-DMAP for "Catch and Release" reactions (Argonaut) (Flyn et al., 1998). When appropriate, solid-phase resins may be used to synthesize linked compounds. A simple approach is to use PS-Wang, PS- NH2 or PS-Cl resins (Argonaut) to link the first fragment, and perform subsequent steps on resin bound compound.
Microwave assisted chemistry may also be used for various reactions (Forino et al., 2005; Tautz et al., 2004). For example, using carbodiimmide catalyzed reactions (both PS-CDI and water-soluble-CDI) to couple primary amines and substituted anilines to the carboxylic acid derivatives gives good but not spectacular yields varying from 25 to 50%. However, in several cases, when the uncatalyzed amidation of the acid was performed under solvent free conditions, using a slight excess of amine, excellent yields (> 80%) were obtained. Milestone Microwave systems allow for up to 16 different reaction conditions at the same time.
For compound purification, precision packed RediSep columns and CombiFlash Companion flash-chromatography systems may be used. This combination yields more pure compound in less time than any glass column or other disposable columns. Standard analytical analyses of each compound such as high-resolution 1H and 13C NMR, MS and elemental analysis, are performed. Compounds with purity > 90-95% are used for in vitro binding/inhibition and subsequent cell based assays. Compounds for in vivo studies are further purified by preparative HPLC to a higher purity level. Thus, one aspect of the invention provides a method or assay to identify/screen for a compound that modulates the JIP/JNK interaction. The method comprises providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent; washing the solid support to remove molecules not associated with the solid support; separating the label from the ligand; and determining the amount of separated label. The solid support can be any of the solid supports useful for high throughput screening known to those skilled in the art. These include but are not limited to a well of a multi well plate, bead, particle or resin, including a natural or synthetic polymer.
In addition to the assays described above-using a solid support, the invention also provides assays that do not use a solid support, e.g., a soluble assay to identify modulators of the JIP/JNK interaction. In a particular embodiment, the assay makes use of fluorescence resonance energy transfer (FRET) to determine if a test compound modulates the interaction of JIP and JNK. A donor and acceptor fluorophore are added to JIP and JNK thereby allowing the emission of acceptor specific fluorescence if binding occurs between JIP and JNK. This fluorescence emission occurs only when the donor and acceptor fluorophores are in close proximity to each other, e.g., when JIP and JNK are bound. In order to screen potential modulators of this interaction, candidate compounds can be added to a solution prior to JIP/JNK complex formation or after the complex is formed, and the effect of the compound on the interaction can be monitored. In one specific embodiment of the soluble assay, biotin is attached to the JIP and an acceptor fluorophore is attached to streptavidin thereby attaching a acceptor fluorophore to JIP through a biotin/ streptavidin interaction. Additionally, JNK is fused to GST and an donor fluorophore is attached to a GST antibody thereby providing a donor fluorophore JNK through the GST-antibody interaction. In this embodiment, the JNK/JIP interaction can be monitored via the emission from the acceptor fluorophore attached to JIP. One of skill in the art would recognize that there are a multitude of fluorophores, e.g., donor and acceptor fluorophores that can be used in the FRET-based assays described herein. Moreover, the assays would be equally effective if the donor and acceptor molecules were placed on the other binding protein.
Any of a number of known combinations of donor and acceptor labels can be used for FRET. For example, Europium chelate and allophycocyanin, ABZ and DNP, DANSYL and Tyr(NO2), DANSYL and FAM, FAM and TMR, EDANS and DABCYL, and EDANS and DABSYL. In an exemplified embodiment, the fluorescent donor and acceptor are Europium chelate and allophycocyanin, respectively.
In additional embodiments, the JNK/JIP interaction may be monitored when the JNK is a mutant JNK, wherein such mutation(s) are selected to determine particular active site involvement. For example, in a specific embodiment the methods of identification may further comprise the additional step of screening a potential modulator of JNK/JIP interaction against R127A and C167S mutants of JNK2 to determine if the compound is binding in the JIP pocket (i.e., a drop in binding affinity for the compound by these mutants, would indicate that a compound binds in the JIP pocket).
Compounds Useful to Inhibit JIP/ JNK Interactions
Any compound identified by the methods of the invention as modulators of JIP/JNK interaction, is within the scope of the present invention.
In certain embodiments, compounds identified by the methods of the invention, which are useful to inhibit JIP/JNK interactions, include compounds of formula (II):
Y4 Y6
(H) wherein, Q1 is aryl, cycloalkyl, heteroaryl or heterocycle;
Q2 is aryl, cycloalkyl, heteroaryl or heterocycle;
X4 is absent, O, S, N(RX), OCH2O, C(=O), C(=O)O, OC(=O)O, OC(=O), C=NRX, C(=O)NRX, NRXC(=O), NRXSO2 ORZ, SO or SO2;
X is alkyl, alkenyl, aryl, cycloalkyl, heteroaryl, or heterocycle; X6 is absent, O, S, N(RX), OCH2O, C(=0), C(=0)0, 0C(=0)0, 0C(=0), C=NRX,
C(=O)NRX, NRXC(=O), NRXSO2, ORZ, SO or SO2; and wherein any alkyl or alkenyl of X5 is optionally interrupted with one or more O, S, N(RX), OCH2O, C(=0), C(=0)0, 0C(=0)0, 0C(=0), C=NRX, C(=O)NRX, NRXC(=O), NRXSO2, ORZ, SO or SO2; wherein any alkyl, alkenyl, aryl, cycloalkyl, heteroaryl, or heterocycle of X5 is optionally substituted on carbon with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, acyloxy, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NRxRy or COORX; each Rz is independently alkylene, alkenylene, arylene, heteroarylene, heterocyclene, or cycloalkylene; each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.
Additional compounds useful to inhibit JIP/JNK interactions include compounds of formula (III):
Q4 a* Q3 χ7 Q6
(III) wherein,
Q is aryl, cycloalkyl, heteroaryl or heterocycle; Q6 is aryl, cycloalkyl, heteroaryl or heterocycle; Q4 is absent, O, S, N(RX), OCH2O, C(=O), C(=O)O, OC(=O)O, OC(=O), C=NRX,
C(=O)NRX, NRXC(=O), NRXSO2, -CRX=N-, ORZ, SO or SO2;
X7 is absent, alkyl, alkenyl, aryl, cycloalkyl, heteroaryl, or heterocycle; and Q5 is absent, O, S, N(RX), OCH2O, C(=0), C(=0)0, 0C(=0)0, 0C(=0), C=NRX, C(=0)NRx, NRxC(=0), NRxS02 0Rz, SO or SO2; wherein any alkyl or alkenyl of X7 is optionally interrupted with one or more O, S,
N(RX), OCH2O, C(=0), C(=0)0, 0C(=0)0, 0C(=0), C=NRX, C(=O)NRX, NRXC(=O), NRXSO2> ORZ, SO or SO2; wherein any alkyl, alkenyl, aryl, cycloalkyl, heteroaryl, or heterocycle of X7 is optionally substituted on carbon with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, acyloxy, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NRxRy or COORX; each Rz is independently alkylene, alkenylene, arylene, heteroarylene, heterocyclene, or cycloalkylene; each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. Additional compounds useful to inhibit JIP/JNK interactions include compounds of formula (IV):
H
Figure imgf000029_0001
(IV) wherein
Z1 is selected from the group consisting of phenyl, naphthyl, 2,3-dihydro- benzo[l,4]dioxin-6-yl, and benzo[l,3]dioxol-5-yl;
Z2 is selected from H, thiazole (e.g., thiazol-2-yl), and benzylsulfonyl; and Z1 and Z2 may be independently substituted with one or more substituents selected from the group consisting of lower alkyl (e.g., t-butyl or methyl), CO2H, lower alkoxy (e.g., methoxy), halogen (e.g., F), NO2, and perfluoroalkoxy (e.g., OCF3). In a particular embodiment, Z2 is 5-nitrothiazol-2-yl. In a particular embodiment, Z1 is 2,3-dihydro-benzo[l,4]dioxin-6-yl.
Additional compounds useful to inhibit JIP/JNK interactions include compounds of formula (V):
Figure imgf000029_0002
(V)
wherein
Z1 is selected from the group consisting of phenyl, naphthyl, 2,3-dihydro- benzo[l,4]dioxin-6-yl, and benzo[l,3]dioxol-5-yl;
Z2 is selected from H, thiazole (e.g., thiazol-2-yl), and benzylsulfonyl; and Z1 and Z2 may be independently substituted with one or more substituents selected from the group consisting of lower alkyl (e.g., t-butyl or methyl), CO2H, lower alkoxy (e.g., methoxy), halogen (e.g., F), NO2, and perfluoroalkoxy (e.g., OCF3). In a particular embodiment, Z1 is phenyl. In certain embodiments, a compound of the invention is a compound of formula V, wherein Z2 is H, Z1 is not 2,3-dihydro-benzo[l,4]dioxin-6-yl or phenyl. Specific Ranges, Values and Embodiments
Specific ranges, values, and embodiments provided below are for illustration purposes only and do not otherwise limit the scope of the invention, as defined by the claims. For the compounds of formula (II):
A specific value for Q1 is aryl or heterocycle. Another specific value for Q1 is aryl. Another specific value for Q1 is heterocycle. Another specific value for Q1 is phenyl. Another specific value for Q1 is 2-furan. Another specific value for Q1 is 2-(5-methyl) furan.
A specific value for Q2 is absent, optionally substituted aryl or optionally substituted heterocycle. Another specific value for Q2 is optionally substituted aryl. Another specific value for Q2 is optionally substituted heterocycle. Another specific value for Q2 is phenyl, 2-furan, N- (3-morpholino), 2-(5-methyl) furan, absent, o-NO2-phenyl or m-OMe-p-OH-phenyl. Another specific value for Q2 is phenyl. Another specific value for Q2 is 2-furan. Another specific value for Q2 is N-(3-morpholino). Another specific value for Q2 is 2-(5-methyl) furan. Another specific value for Q2 is absent. Another specific value for Q2 is o-NO2-phenyl. Another specific value for Q is m-OMe-p-OH-phenyl.
A specific value for X4 is absent or O. Another specific value for X4 is absent. Another specific value for X4 is O.
A specific value for X5 is alkyl or alkenyl, optionally interrupted with one or more O, S, N(RX), OCH2O, C(=O), C(=O)O, OC(=O)O, OC(=O), C=NRX, C(=O)NRX, NRXC(=O), NRXSO2, SO or SO2, and optionally substituted on carbon with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, acyloxy, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, thio, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NRxRy or COORX; wherein each Rx and Ry is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy .
Another specific value for X is alkyl or alkenyl, optionally interrupted with one or more C(=O)NRX or NRXC(=O), wherein each Rx is independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl Another specific value for X5 is alkyl or alkenyl, optionally interrupted with one or more C(=0)NH or NHC(=0). Another specific value for X is N,N'-(propane-l,3-diyl)diacrylamide; N,N'-(propane- l,3-diyl)diacetamide; N-propylacrylamide; N,N'-(2,2-dimethylpropane-l,3-diyl)diacrylamide; l,l'-(l,4-diazepane-l,4-diyl)diprop-2-ene-l-one; N-(3-acetamidopropyl)acrylamide; or N,N'- (propane- 1 ,3-diyl)diacrylamide.
Another specific value for X5 is N,N'-(propane-l,3-diyl)diacrylamide. Another specific value for X5 is N,N'-(propane-l,3-diyl)diacetamide. Another specific value for X5 is N- propylacrylamide. Another specific value for X5 is N,N'-(2,2-dimethylpropane-l,3- diyl)diacrylamide. Another specific value for X5 is l,l'-(l,4-diazepane-l,4-diyl)diprop-2-ene-l- one. Another specific value for X is N-(3-acetamidopropyl)acrylamide. Another specific value for X5 is N,N'-(propane-l,3-diyl)diacrylamide.
A specific value for X6 is absent or O. Another specific value for X6 is absent. Another specific value for X6 is O.
Specific compounds of formula (II) include:
(2E,2'E)-N,N'-(propane-l,3-diyl)bis(3-(furan-2-yl)acrylamide); N,N' -(propane- l,3-diyl)bis(2-phenoxyacetamide); (E)-3-(5-methylfuran-2-yl)-N-(3-morpholinopropyl)acrylamide; (2E,2'E)-N,N'-(2,2-dimethylpropane-l,3-diyl)bis(3-(5-methylfuran-2- yl)acrylamide);
(2E,2'E)- 1 , T -( 1 ,4-diazepane- 1 ,4-diyl)bis(3-(5-methylfuran-2-yl)prop-2-en- 1 - one);
(E)-N-(3-acetamidopropyl)-3-(furan-2-yl)acrylamide; (E)-3-(furan-2-yl)-N-(3-(2-phenoxyacetamido)propyl)acrylamide;
(E)-3-(furan-2-yl)-N-(3-(2-(2nitrophenoxy)acetamido)propyl)acrylamide; and (E)-3-(furan-2-yl)-N-(3-((E)-3-(4-hydroxy-3- methoxyphenyl)acrylamido)propyl)acrylamide.
For the compounds of formula (III): A specific value for Q3 is optionally substituted heteroaryl or optionally substituted cycloalkyl. Another specific value for Q3 is optionally substituted heteroaryl. Another specific value for Q3 is optionally substituted cycloalkyl. Another specific value for Q3 is 6-( methyl 5- nitronicotinate); 2-(5-nitrothiazole); 7-(5-phenyl-[l,2,4]triazolo[l,5-a]pyrimidine); 4-(3-methyl- 1 -phenyl- lH-pyrazole) or 2-(thiazole). Another specific value for Q3 is 6-( methyl 5-nitronicotinate). Another specific value for
Q3 is 2-(5-nitrothiazole). Another specific value for Q3 is 7-(5-phenyl-[l,2,4]triazolo[l,5- a]pyrimidine). Another specific value for Q3 is 4-(3-methyl-l-phenyl-lH-pyrazole). Another specific value for Q3 is 2-(thiazole).
A specific value for Q4 is S or -CRX=N-, wherein Rx is H, alkyl, alkenyl, aryl, heteroaryl, heterocycle or cycloalkyl. Another specific value for Q4 is S or -CH=N-. Another specific value for Q4 is S. Another specific value for Q4 is -CRX=N-, wherein Rx is H, alkyl, alkenyl, aryl, heteroaryl, heterocycle or cycloalkyl. Another specific value for Q4 is -CH=N-.
A specific value for X7 is absent or aryl. Another specific value for X7 is absent. Another specific value for X7 is aryl. Another specific value for X7 is phenyl. Another specific value for X7 is 1,2-phenyl. Another specific value for X7 is 3,4'-(4-(4'-phenyl)-lH-l,2,4-triazol- 5(4H)-one). Another specific value for X7 is l,2-(lH-benzo[d]imidazole). Another specific value for X7 is 2,5-(l,3,4-thiadiazole).
A specific value for Q is absent or NRX(C=O), wherein Rx is H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Another specific value for Q5 is absent or NH(C=O). Another specific value for Q5 is absent. Another specific value for Q5 is NRX(C=O), wherein Rx is H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxyl. Another specific value for Q5 is absent. Another specific value for Q5 is NH(C=O). Another specific value for Q is ORZ, wherein Rz is alkylene, alkenylene, arylene, heteroarylene, heterocyclene, or cycloalkylene. Another specific value for Q is OCH2. A specific value for Q6 is optionally substituted heteroaryl or optionally substituted cycloalkyl. Another specific value for Q6 is optionally substituted heteroaryl. Another specific value for Q6 is optionally substituted cycloalkyl. Another specific value for Q6 is optionally substituted alkyl. Another specific value for Q6 is 2-thiophene; m-CF3-phenyl; 2-(5-(pyridin-4- yl)-l,3,4-oxadiazole); 4-(3-(thiophen-2-yl)-lH-l,2,4-triazole-5(4H)-thione); orp-Cl-phenyl. Another specific value for Q6 is 2-thiophene. Another specific value for Q6 is m-CF3-phenyl. Another specific value for Q6 is 2-(5-(pyridin-4-yl)-l,3,4-oxadiazole). Another specific value for Q6 is or 4-(3-(thiophen-2-yl)-lH-l,2,4-triazole-5(4H)-thione). Another specific value for Q6 is p-Cl-phenyl. Another specific value for Q6 is phenyl. Another specific value for Q6 is isopropyl. Specific compounds of formula (III) include: methyl 5-nitro-6-(2-(thiophene-2-carboxamido)phenylthio)nicotinate; N-(2-(5-nitrothiazol-2-ylthio)phenyl)-3-(trifluoromethyl)benzamide; 2-(5-phenyl-[l,2,4]triazolo[l,5-a]pyrimidin-7-ylthio)-5-(pyridin-4-yl)-l,3,4- oxadiazole; (E)-4-((3-methyl-l-phenyl-lH-pyrazol-4-yl)methyleneamino)-3-(thiophen-2- yl)- IH- 1 ,2,4-triazole-5(4H)-thione; methyl 6-(2-(4-chlorobenzamido)phenylthio)-5-nitronicotinate; 4-(4-(benzyloxy)phenyl)-3-(5-nitrothiazol-2-ylthio)-lH-l,2,4-triazol-5(4H)-one; 2-(l-isopropyl-lH-benzo[d]imidazol-2-ylthio)thiazole; 2-isopropyl-5-(5-nitrothiazol-2-ylthio)-l,3,4-thiadiazole; 4-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-3-(5-nitromiazol-2-ylthio)-l # -1,2,4- triazol-5(4/f)-one;
3-(5-nitrothiazol-2-ylthio)-4-phenyl-lH-l,2,4-triazol-5(4H)-one; and 5-nitro-2-(l -phenyl- lH-tetrazo 1-5 -ylthio)thiazo Ie.
Specific compounds of formula (II) are shown in Tables I and Ia. Specific compounds of formula (III) are shown in Tables II, Ha, and III. Specific compounds of formulae (IV) and (V) are shown in Tables III, IV, V, and VI.
In particular embodiments, the compounds of the invention may be selected from the exemplary compound listing shown below in the following tables (i.e., Tables I- VI). Importantly, it should be noted that the tabular listing below is used merely as a convenience, and each compound below should be considered a separate embodiment of the invention:
Figure imgf000033_0001
(H)
Figure imgf000033_0002
Table Ia
Figure imgf000034_0001
Q4 tf
Q
(III)
Table II
Figure imgf000035_0001
Table Ha
Figure imgf000036_0001
Table Ha (cont'd)
Figure imgf000037_0001
Table III
Figure imgf000037_0002
Table III (cont'd) 3.3
Figure imgf000038_0001
Table III (cont'd)
Figure imgf000039_0001
In additional particular embodiments, the compounds of the invention may be selected from Table IV. Table IV
Figure imgf000040_0001
Table IV (cont'd)
Figure imgf000041_0001
Table IV (cont'd)
Figure imgf000042_0001
Table IV (cont'd)
Figure imgf000043_0001
Table IV (cont'd)
Figure imgf000044_0001
Table IV (cont'd)
Figure imgf000045_0001
Table IV (cont'd)
Figure imgf000046_0001
Table IV (cont'd)
Figure imgf000047_0001
Table IV (cont'd)
Figure imgf000048_0001
Table IV (cont'd)
Figure imgf000049_0001
In a specific embodiments of the invention, the compound is not 83A5 or 83A6. In certain embodiments, particular compounds of the invention include those selected from the listing of compounds of Table V.
Table V
Figure imgf000050_0001
Table V (cont'd)
Figure imgf000051_0001
Table V (cont'd)
Figure imgf000052_0001
Table V (cont'd)
Figure imgf000053_0001
Table V (cont'd)
Figure imgf000054_0001
Table V (cont'd)
Figure imgf000055_0001
Table V (cont'd)
Figure imgf000056_0001
In particular embodiments, the compound may be selected from the compounds of Table
VI:
Table VI
Figure imgf000056_0002
Table VI (cont'd)
Figure imgf000057_0001
Table VI (cont'd)
Figure imgf000058_0001
In a particular, embodiment, the compound of the invention is compound 83F6, 4-(4- Nitro-phenyl)-5-(5-nitro-thiazol-2-ylsulfanyl)-2,4-dihydro-[l,2,4]triazol-3-one.
Pharmaceutical Formulations
The compounds of this invention are formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10.
While it is possible for the active ingredients to be administered alone it may be preferable to present them as pharmaceutical formulations. The formulations, both for veterinary and for human use, of the invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers therefore and optionally other therapeutic ingredients. The carrier(s) are advantageously "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof. The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.
A tablet is made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.
For administration to the eye or other external tissues e.g., mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w and more preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in- water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogs. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used.
Pharmaceutical formulations according to the present invention comprise one or more compounds of the invention together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed. Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p- hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally- occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.
The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time -release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight:weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
Formulations suitable for administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of a given condition.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient. It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route. Compounds of the invention can also be formulated to provide controlled release of the active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the invention also provided compositions comprising one or more compounds of the invention formulated for sustained or controlled release.
Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses), the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day. Typically, from about 0.01 to about 10 mg/kg body weight per day. More typically, from about .01 to about 5 mg/kg body weight per day. More typically, from about .05 to about 0.5 mg/kg body weight per day. For example, the daily candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.
One or more compounds of the invention (herein referred to as the active ingredients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that a preferred route may vary with for example the condition of the recipient. An advantage of the compounds of this invention is that they are orally bioavailable and can be dosed orally. The invention also provides kits comprising the reagents for performing the methods of the invention and instructions for use. In one embodiment, the kit provides JIP and JNK comprising fluorescent donor and acceptor moieties and instructions for use. In one particular embodiment, the donor and acceptor moieties are europium chelate and allophycocyanin, respectively.
Exemplification
The invention will be further described by the following nonlimiting examples.
Example 1 Virtual Docking Approaches
Virtual docking strategies have been described to identify potential protein binders (Fattorusso et al., 2005; Leane et al., 2003; Kitada et al., 2003; Hajdaic et al., 1997 Forino et al., 2005). For instance, a number of in silico approaches have been used to identify inhibitors for Akt (Forino et al., 2005). Virtual docking may be performed on Linux cluster constituted by a 40 slaves plus head node, each with 2 CPUs Intel® Xenon 2.4 GHz running FlexX (Biosolve) or by using FlexX as implemented in Sybyl 6.9 (TRIPOS) on a 10-R12000 CPUs challenge machine and generating several solutions per compound. Top scoring compounds are selected by using CSCORE and visual inspection. Molecular models are built by using CORINA and Sybyl 6.9 (TRIPOS) and energy minimized by using the routine MAXIMIN or semi-empirical calculations (MOPAC). Cerius 2 (Accelrys, Inc.) may also be used. Initial docking studies with compounds are performed with FlexX as implemented in Sybyl 6.9 by using the three- dimensional structure of JNKl in complex with the JIPl peptide (PDB_ID IUKH) and the structure of the ternary complex between JNKl, JIPl and the ATP mimic SP600125 (PBDJD IUKI). As a general strategy, for each compound, 10 solutions are generated and compounds are the analyzed according to CSCORE which includes drug score, chemscore and Gold score functions. Further studies are performed with GOLD. GOLD is implemented on a dual-CPU 3.2 GHz Linux workstation. The binding energies of the docked compounds can be correlated with the experimental inhibition constants by using an empirical binding energy scoring function, similar to those recently obtained (Fattorusso et al., 2005; Forino et al., 2005).
A virtual docking approach to identify modulators of JIP/JNK was analyzed using several strategies including FlexX (Kramer et al., 1999), GOLD (Ver donk et al., 2003) and CSCORE (Clark et al., 2002), where the top 100-200 scoring compounds from a 50,000 compound library were tested. Briefly, a 50,000 compound library (Chembridge, San Diego) was selected and all compounds docked against the JIPl binding site on JNKl (PDB_ID IUKH). For this task, FlexX was used. Initially 5 solutions were generated for each ligand and the compounds rank ordered by using the FlexX scoring function. The top scoring 4,000 compounds were then subsequently analyzed by using a consensus score between Chemscore (Eldridge et al., 1997) and Goldscore (Ver donk et al., 2003). The top 1,000 compounds were visually analyzed using Sybyl and compounds to be tested selected according to the following criteria: occupancy of both hydrophobic pockets; formation of hydrogen bonding interactions (similar to those observed in pepJIPl -JNKl); drug-likeness (see above); and ease of synthesis.
Example 2 Competitive Assay for JIP Mimics
One choice for the development of a robust assay for pepJIPl mimics is the Delfia® (dissociation enhanced lanthanide fluoroimmunoassay). Delfia chemistry has been applied with success using various assay formats in the detection of receptor-ligand interactions. Delfia® is a heterogeneous assay similar to an ELISA (enzyme linked immunosorbent assay). To employ Delfia® to identify modulators of the JIP/JNK interaction, proteins were produced from a pET- 19b (Novagen) plasmid construct containing the respective nucleotide sequence fused to a N- terminal poly- His tag or GST. GST-JNK1/2 may be expressed in E. coli BL21 in LB media at 37 0C, with an induction period of 3-4 hours with 1 mM IPTG. Approximate yields ranged from 13
2 to 10 mg per litre of culture, γ C-VaI labelled His-JNKl (His-JNK2) for binding studies was similarly produced in M9 media supplemented with 50 mg/L of γ-13C-Val (Cambridge Isotopes) at the time of induction with IPTG. Following cell lysis, soluble labelled protein was purified over a Hi-Trap chelating column (Amersham, Pharmacia), followed by ion-exchange purification with a MonoQ (Amersham, Pharmacia) column. A schematic representation of Delfia® is set forth in Figure 1.
A heterogeneous assay using a labeled JIPl peptide and a JNK fusion was employed to screen and characterize JIPl mimics. In one embodiment of an assay to detect modulators of the JIP/JNK interaction, a bio tin labeled pep JIPl is bound to streptavidin coated 96 -well plates. GST-JNKl (or GST- JNK2) is incubated so as to allow for binding to pepJIPl, and after washing to eliminate unbound GST-JNKl (or GST-JNK2), a solution containing a Eu labeled anti-GST antibody (Perkin Elmer) is added together with a test agent. After a second washing step, an enhancing solution is added to deliver the lanthanide from the antibody to the solution. Residual Eu fluorescence is subsequently detected with a VictorV2 (Perkin Elmer) plate reader with excitation at 360 nm and emission at 620 nm. If the test compound is capable of dissociating GST-JNKl from the biotinylated-pepJIPl peptide, the antibody carrying the label (Eu) is washed out and no fluorescence is detected. Because of the relaxation properties of Eu, detection can be accomplished also in time-resolved mode (TRF), free from possible interferences by test compounds, matrices, plates, and the like. Also, the sensitivity of Eu is superior to that of most other fluorescent labels.
The reproducibility of the Delfia® assay was evaluated by varying the biotin-pepJIPl (Anaspec) concentration (from 1 pM to 50 nM) (Figure 2A); GST-JNKl (and GST- JNK2) concentration (from 1 nM to 500 nM) (Figure 2B); incubation times (from 30 minutes to 2 hours); and the number of washing steps (from 2 to 7). For a typical displacement curve with the non-biotinylated pepJIPl as a test inhibitor, the obtained IC50 value of 3 μM is comparable with the KD value obtained by direct isothermal titration calorimetry experiments (about 0.6 μM).
In another embodiment, 100 μL of a 100 ng/ml of biotin labeled pepJIPl was added to each well of a 96-well streptavidin coated plate (Perkin Elmer). After incubation for 1 hour, unbound peptide was eliminated with 4 washing steps. Subsequently, to each well 78 μL solution of Eu-antibody conjugate (25 ng/ml), 2 μL DMSO solution containing a test compound, and 10 μL solution containing GST-JNKl (or GST- JNK2) protein (100 nM), were added. After 2 hours of incubation, each well was washed 4 times to eliminate unbound JNKl and the Eu- antibody if displaced by the test compound. Subsequently, 200 μL of enhancement solution (Perkin Elmer) was added to each well, and fluorescence read after 30 minutes incubation (excitation wavelength, 360 nm; emission wavelength, 620 nm). Controls include unlabeled pepJIPl peptide (Anaspec) and blanks received no compound.
In another embodiment, 100 μL of a 100 ng/ml solution of biotin-labeled pepJIPl (Biotin- lc-KRPKRPTTLNLF, where Ic indicates a hydrocarbon chain of 6 methylene groups) was added to each well of 96-well streptavidin-coated plates (Perkin-Elmer). After incubation for 1 hour, unbound bio tin-pep JIPl was removed by 3 washing steps. Subsequently to each well was added 87 μL of Eu-labeled anti-GST antibody solution (300 ng/ml; 1.9 nM), 2.5 μL DMSO solution containing test compound, and 10 μL solution of GST-JNK2 (or GST-JNKl) for a final protein concentration of 10 nM. After 1 hour of incubation at 0 0C, each well was washed 5 times to eliminate unbound protein and the Eu-antibody if displaced by a test compound. Subsequently, 200 μL of enhancement solution (Perkin-Elmer) was added to each well and fluorescence measured after 10 min incubation (excitation wavelength, 340 nm; emission wavelength, 615 nm). Controls include unlabeled peptide and blanks receiving no compounds. Protein and peptide solutions were prepared in Delfia buffer (Perkin-Elmer). Upon identifying mixtures that gave >50% inhibition at 12.5 μM, dose response measurements were performed to filter out eventual false positives. The mixtures were deconvolved to identify individual test compounds having activity and the individual compounds were retested to identify actual hits.
Example 3
A set of 31 potential pepJIPl mimics identified in a virtual docking approach were selected and tested further tested using Delfia®. Each compound was tested in duplicate; positive controls included pepJIPl (at 25 μM) and blanks received no proteins (6 wells each).
Negative controls (6 wells) in which no inhibitors were added were also included. The
JIP/JNK Delfia® was highly reproducible (Z' factor > 0.7; see Zhang et al. (1999) J. Biomol
Screen for an explanation of Z' factors) and highly sensitive (over 30,000 RFUs between controls). Exemplary compounds identified by the method are 5328208 and 5245976 as set forth in Figure 3.
Thus, by using a combination of structure -based design followed by experimental verification via Delfia®, low micro molar pepJIPl mimics were identified.
To optimize hits, a first round of iterations of Structure- Activity Relationship (SAR) data on compound analogues was collected (see Figures 6 and 9). These studies provided information on ligand binding in a series of closely related compounds, and such data was suitable for application of QSAR (quantitative structure-activity relationship) analysis (Hansen et al., 1995). These analogs may be tested in competition and kinase assays as was done for the compounds set forth in Figures 7 and 8. Figure 10 shows compounds identified after a second round of molecular docking, NMR and Delfia® analysis and sets forth the percent each compound inhibited in the Delfia® analysis at 25μM. Figures 1 IA-C shows ICsodata for compounds 17C6, 19E2, and 24Dl, respectively.
Example 4 TR-FRET Assay
The TR-FRET assay was used as a secondary assay to further characterize the binding of the compounds identified through the virtual docking and Delfia assay described above.
The following reagents were dissolved in Lance detection buffer [Perkin Elmer] with 0.1% BSA: GST-JNKl 10 nMBiotinylated pepJIPl (KRPKRPTTLNLF); 150 nM Eu anti-GST 8 nM; and Streptavidin APC 20 nM in 96 well black plates (50 μl final volume). GST-JNK, biotin-pepJIPl, Eu, streptavidin APC, with no inhibitor was used as a positive control. For a negative control, Eu anti-GST and streptavidin APC GST-protein, biotinylated- peptide, and inhibitors were incubated for 30 minutes at room temperature followed by addition of the Eu anti-GST/ APC-streptavidin mixture. The plate was read after 90 minutes at 4 0C (emission: D340, absosption: D665).
LANCE assay results are shown in Figures 17A-C for JIPl, 24Dl and SPB07895, respectively.
Example 5 Fluorescence Polarization Assay A homogeneous assay may also be to identify modulators of JIP1/JNK. The assay is based on displacement of fluorescence polarization (FP), which is also a mode of detection for the Victor V2 plate reader. Briefly, a FITC-labeled-pepJIPl is synthesized (Anaspec) and its polarization measured at increasing protein concentrations (JNKl and JNK2) in 96-well plates. Upon binding, the initially rapid rotational correlation time of the FITC-pepJIPl approaches that of the macromolecule with concomitant increase of emitted polarized light. Test compounds can be monitored for their ability to bind to the JIPl binding pocket of JNK by detecting a decrease of polarization due to the displacement of bound FITC-pepJIPl. The advantage of FP versus Delfia® is that the sample preparation is simple and the assay does not require the use of tagged protein nor specific antibodies. In addition, being a homogeneous assay, FP is more suitable for testing several compounds (no time consuming washing steps). The potential interference from fluorescence of the test compound may be at least partially solved by using pepJIPl tagged with red-shifted Alexa Fluor dyes available from Molecular Probes. Examples of dyes that could be used are Alexa Fluor 555 (ex/em = 555nm/565nm) or Alexa Fluor 610 (ex/em = 610nm/628nm). Thus, a FP assay may be used as rapid primary assay while Delfia® may be used for hit validation in a secondary assay. Example 6
Identification of 4-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-3-(5-nitrothiazol-2-ylthio)-l H - l,2,4-triazol-5(4//)-one (BI-78D3) and related compounds
A 30,000 compound library (-16,000 compounds, Maybridge Corporation, Cornwell,
UK; 14,000 compounds, ASDI Biosciences, Newark, DE), prepared in mixtures of 20 compounds per sample, was screened using the Delfia® assay for dissociation of GST- JNK2 from biotinylated-pepJIPl peptide. Upon identifying mixtures that gave >50% inhibition at 12.5 μM, dose response measurements were performed to filter out eventual false positives. The mixtures were deconvolved to identify individual test compounds having activity and the individual compounds were retested to identify actual hits. The hits from the Delfia assay were further tested for their ability to inhibit JNKl phosphorylation of ATF2 in the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) based LanthaScreen kinase assay (Invitrogen, Carlsbad, CA) and screened for effects on cell viability and morphology by propidium iodide staining to eliminate cytotoxic compounds. These efforts lead to the identification of 4-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-3-(5-nitrothiazol-2-ylthio)-l H -1,2,4- triazol-5(4/f)-one (BI-78D3; Figure 19B). Other compounds identified in the Delfia assay and having 100% displacement of bio tinylated-pep JIPl peptide at lOOμM are set forth in Figures 22A and B. BI-78D3 is able to compete with pepJIPl for JNKl binding (IC50 = 600 nM; Figure
19C), and inhibit the in vitro kinase activity of JNKl (IC50 = 280 nM; Figure 19D), both with a Hillslope of ~1. Using the same in vitro LanthaScreen kinase assay and the same ATF2 substrate, BI-78D3 was found to be 100-fold less active versus p38, a member of the MAPK family with high structural similarity to JNK. Furthermore, from Lineweaver-Burk analysis, BI- 78D3 is a reversible inhibitor and is competitive with ATF2 for binding to JNKl with an apparent K(i) value of 200 nM (Figure 19E). In addition, BI-78D3 does not inhibit the phosphorylation of a short peptide substrate lacking a D-domain (data not shown) further confirming that BI-78D3 is substrate competitive.
To characterize the binding properties of BI-78D3, computational modeling studies supported by structure activity relationship (SAR) data (Table III) and experimental NMR
(Figure 20A) were performed. IC50 data and structure-Activity Relationship (SAR) data were collected on compound analogues BI-78D3, BI-83C9, BI-83C7, BI-83C8, BI-83B3, BI-83C11, respectively, (see Table III). The docked structure of BI-78D3 onto the binding surface of JNKl has many features in common with the recently solved X-ray structure of the ternary complex including pepJIPl, JNKl and the ATP mimic SP600125 (Heo et al., 2004; Figure 19A). For example, these studies predict that the benzodioxan moiety of BI-78D3 occupies a region corresponding to the highly conserved leucines of pepJIPl (Barr et al., 2002). In fact, the removal of the benzodioxan moiety results in a two-fold reduction in activity in both the DELFIA and kinase assay (BI-83C9; Table III). In addition, an extensive network of hydrogen bonding interactions is predicted between the triazole, nitro group, and thiazole of BI-78D3 with JNKl R 127, an interaction that is thought to be important for pep JIPl binding to JNKl (Heo et al., 2004). Although substitution of a triazole with a tetrazole seems to be tolerated (cf. BI-83C9 and BI-83C7; Table III) modifications to the nitro or thiazole result in a marked reduction in inhibitory activity (cf. BI-83C8, BI-83B3, and BI-83C11; Table III).
Example 7
In vitro Kinase Assay
An in vitro kinase assay was used as a secondary assay to further characterize the binding of the BI-78D3, identified through the Delfia assay described in Example 4, and related compounds. To assay in vitro kinase activity, the LanthaScreen assay platform from Invitrogen was utilized. Time-resolved fluorescence resonance energy transfer assay (TR-FRET) was performed in 384 well plates. Each well received JNKl (100 ng/mL), ATF2 (200 nM), and ATP (1 μM) in 5OmM HEPES, 1OmM MgCl 2, ImM EGTA and 0.01% Brij-35, pH 7.5 and test compounds. The kinase reaction was performed at room temperature for 1 hr. After this time, the terbium labeled antibody and EDTA were added into each well. After an additional hour incubation, the signal was measured at 520/495 nm emission ratio on a BMG Pherastar fluorescence plate reader. The LanthaScreen kinase assay results for BI-78D3 and other compounds screened are shown in Table III and Table IV.
Example 8
NMR-based techniques
NMR-based approaches may also be used to derive potential JNK1/2 inhibitors. Target mediated ligand-ligand transferred NOEs (ILOEs) allow detection of a pair of relatively weak binders (high micromolar to millimolar) that may be subsequently chemically linked to give a more potent lead compound (sub-micro molar). This approach, named SAR by ILOE, is analogous to the well known SAR by NMR strategy, and allows the design of possible bi- dentate inhibitors by screening a library of compound "scaffolds" in overlapping mixtures.
The approach is based on the detection of magnetization transfer between an initial hit and a second compound (a scaffold) occupying an adjacent site on the surface of the target, measured in the presence of a sub-stoichiometric amount of unlabeled protein. Briefly, in a mixture of compounds in the presence of GST-JNK (10 μM), only those compounds that bind appreciably to the protein exhibit strong negative NOEs (positive cross-peaks), and if two ligands bind simultaneously and in adjacent sites on the protein surface, strong negative ligand- ligand NOEs (ILOEs) can be observed. Compounds that display ligand-ligand interactions may be used to design covalently linked compounds with increased activity. A subset of the "scaffold" library (about 600 compounds representing the scaffolds most commonly found in drugs) was tested to identify possible pairs of "weak" JNK binders that may be chemically linked to yield more potent lead compounds.
Mixtures of compounds from the scaffold library (0.4 to 0.9 mM each) were prepared and tested in the presence of 5-10 μM GST-JNKl, GST- JNK2 and GST alone as control. trNOEs and ILOEs were subsequently detected by means of 2d [1H, 1H]-NOESY spectra, typically acquired wth 8-32 scans for each of 400 indirect points and mixing times varying between 300 to 800 msec, to maximize the detection of trNOEs and ILOEs (Becattini et al., 2004; Zerbe, 2003). Two compounds displaying inter-ligand NOEs, BI-7E5 and BI-7E11 in the presence of GST-JNKl and GST- JNK2 but not GST alone or in absence of any protein, were identified. Initial displacement assays with ATP suggested that the compounds were not binding in the ATP binding pocket. Additional assays, including NMR displacement assays and chemical shift mapping with selectively labeled JNKl are performed to verify that compounds BI-7E5 and BI-7E11 do bind in the JIPI binding pocket.
A more rapid approach than the trNOE is to measure compounds 1H transverse relaxation rates in presence of sub-stochiometric amounts of protein targets (GST- JNK1/2). These assays exploit the different rotational correlation time of the small molecule in solution versus when bound to a large macromolecule. Typical Tlp (Hajduk et al., 1997) experiments (10 to 300 msec spin-lock duration) are performed on 100 μM of each compound, in the presence and absence of 10 μM GST-JNKl (GST-JNK2). All experiments are performed with either a 500 MHz or 600 MHz Bruker Avance spectrometer, both equipped with TXI probes.
Another simple NMR -based assay is the saturation transfer difference (STD) (Mayer et al., 1999) or the related experiment WaterLOGSY (Dalrit et al., 2000) in which binding of a compound is detected by a direct transfer of magnetization (saturation) from the protein to the ligand. These techniques have the advantage of being less prone to false positives. Solubility (at least 100 μM) and off rates of the complex may be limiting factors (not slower than 1 s"1, as general rule), as compounds with poor solubility (< 50-100 μM) tend to be promiscuous and non-specific inhibitors. Standard and WaterLOGSY NMR spectra of ImM 5328298 and 5245976 in the presence of 10 μMGST-JNK2 were obtained and are set forth in Figure 4A and 4B, respectively. In addition to ligand based assays, an advantageous NMR-based assay may be the chemical shift mapping of compounds with selectively 13C-labeled His-JNKl (His-JNK2). Such an assay may validate on the site of binding of compounds. Although these data do not provide precise structural information on the mode of binding (unless a differential chemical shift approach is used) they provide rapid insights on the site of binding and can be used to measure the dissociation constant of the complex by titration. For chemical shift mapping, His-JNKl and His-JNK2 are labelled with γ-13C-Val, because two such residues are present in the JIPl binding pocket and binding may be detected via 2D [13C,1 H]-HSQC spectra measured with 0.1 mM protein samples. For the 2D [13C,1 H]- HSQC, typical parameters include 1H and 13C sweep widths of 12 ppm and 5 ppm, respectively; 128 scans and 80 indirect acquisition points and a recycle delay of 1 second. The use of selective methyl labelling is superior to "traditional" uniform 15N labelling in particular for proteins with MW > 30 kDa (Pellecchia et al., 2002; Beccattini et al., 2004; Zerbe et al., 2003, Pellecchia et al., 2004). Using VaI labelling, the labor consuming and cost intensive steps related to resonance assignments is bypassed and problems of spectral overlap are largely eliminated. Resonance specific assignments are obtained by chemical shift mapping with pepJIPl. Eventual ambiguities can be resolved by single point mutations.
In addition, NMR probes may be used to confirm the predicted binding mode of BI- 78D3. A recently developed probe, azaindole-TEMPO (Vasquez et al., submitted), brings a paramagnetic spin label into proximity of the JIP binding site of JNK by anchoring in the ATP binding site (Figure 20A). Azaindole-TEMPO is an ATP-mimic linked to 2,2,6,6- tetramethylpiperidine 1-oxyl, which provides the paramagnetic spin label (Figure 20A). If a test compound binds in close proximity to azaindole-TEMPO, its NMR signal will be affected by the unpaired electron. The effect, manifested in a reduction in signal intensity in the ID 1H-NMR spectrum of the test compound, is distance dependent thus allowing a rough estimate of the binding mode of the test compound with respect to azaindole-TEMPO. NMR spectra were acquired on a 600 MHz Bruker Avance spectrometer equipped with a Bruker TXI probe. All ID 1H experiments carried out with samples containing unlabeled His-tagged JNK2 at a concentration of 5 μM. ID Tlp experiments were measured with a spin-lock time of 100 (or 50) ms, a recycle delay of 1.5 sec, and water suppression based on the Watergate sequence. As shown in Figure 2OB, the signals of BI-78D3 are attenuated in the presence of azaindole- TEMPO. The attenuation is strong evidence of binding to JNK in the JIP binding site.
Moreover, the signals of the aliphatic protons on the benzodioxan moiety (H4 in Figure 20B) are most affected while the signal of the proton on the thiazole ring is least affected (Hl in Figure 20B). The data indicate that the benzodioxan moiety is closest to ATP-TEMPO.
Additionally, the paramagnetic spin probe data are consistent with the orientation of BI- 78D3 predicted by in silico docking (Figure 20C). Computational docking studies were performed with GOLD 2.1 (The Cambridge Crystallographic Data Centre, Cambridge, UK) (Eldridge et al., and Jones et al., 1997) and analyzed with Sybyl (Tripos, St. Louis). Molecular surfaces were generated with MOLCAD (Teschner et al., 1994). The X-ray coordinates of JNKl/pepJIPl/SP600125 (PDB-ID IUKI) were used to dock the compounds. Molecular models were generated with CONCORD (Pearlman, 1998) and energy minimized with Sybyl. Ten solutions were generated and subsequently ranked according to Chemscore. The top 5 solutions converged to a similar pose that was used to represent the docked geometry of BI-78D3. Peptide and SP600125 poses reported in Figure 19 correspond to those obtained directly from the X-ray coordinates.
Example 9
In silico and Iterative Structure Based Optimization
Structure based iterative optimization was used to bring compounds of the benzothiazole series to nanomolar binding affinity. About 500,000 compounds from several commercially available repositories (Maybridge, UK; Chembridge, USA; Asinex, Russia; ChemDiv, USA; SIGMA rare, USA; ASDI, USA) were screened in silico. By using a similar ranking strategy, top scoring compounds (up to 1,000) were selected and tested for their ability to displace pepJIPl-JNK interactions in the assays described herein (see for example, Figures 5 and 6). A hit was defined as a compound with a percent inhibition larger then 50% when tested at 20 μM. Hits were validated and ranked in terms of potency (dose response curves with available assays, including NMR-based binding assays and chemical shift mapping), drug-likeness, and ease of synthesis. Compound analogs were selected and pre-screened in silico against JNK and their binding properties predicted via a consensus score and visual inspection. Modeling studies served as a guide to prioritize the synthetic chemistry efforts. NMR-based chemical shift mapping data served as experimental verification of the site of binding to provide further validation to the docked structures. The experimental results were and will be further used to verify and refine the models for further iterations. In the design/selection of closely related compound analogs, R substituents from all areas of a Craig-diagram in the π/σ space (Hansch et al., 1995) were sampled. This allowed the use of traditional QSAR analysis (Hansch et al., 1995) to complement the docking studies. For the SAR by ILOE approach, compounds such as BI-7E5 and BI-7E11 (Figures 12-
13) were confirmed to actually bind in the JIPl binding pocket of JNK and not bind in the ATP pocket. In silico design of possible bi-dentate compounds suggested that the compounds bind to the JIPl binding pocket of JNK. To address this point experimentally, ε13C-Met labeled samples of JNKl and JNK2 and chemical shift mapping were employed. Inter-ligand NOEs between the methyl group of the ester moiety of BI-7E11 and benzene hydrogen atoms adjacent to the amino group of BI7E5 were observed. Hence, additional potential bi-dentate compounds may be designed by linking the respective scaffolds according to the NOEs data.
Typical trNOESY spectra are measured with 8 or 16 transients per increment with mixing times of 300 to 800 milliseconds, to maximize the detection of trNOEs and ILOEs. Pooling compounds in mixtures of 6 to 24 expedites the collection of the spectra for the 600 fragments. Analysis of the data and subsequent deconvolution of the spectra allows identification of weak ligands by means of positive trNOEs cross peaks. Similarly, compounds that bind GST-JNKl (GST- JNK2) in close proximity (less that 5 A) are identified by detecting intermolecular NOEs (ILOEs) and may serve as building blocks for producing linked compounds as described above. Inter-nuclear distances are derived by ILOEs build-up rate measurements. When possible, eventual spin-diffusion effects are taken into account by measuring QUIET-NOESY (Pellechia et al., 2002) experiments. Binders are will be further validated by their ability to bind in the JIPl binding pocket, verified by, for instance, chemical shift mapping.
Example 10
In Vitro and Cell Based Testing
To test the capacity of the putative inhibitors to block JNKl -2 activity, HEK293T cells were treated with 5 μg/ml anysomicin for 30 minutes to induce JNK activation. Protein extracts were prepared and JNKl -2 proteins were isolated by immunoprecipitation using monoclonal antibodies (Pharmigen). Different aliquots of the immunopurified JNKl -2 proteins were tested for kinase activity by incubation in kinase buffer in the presence of increasing concentrations of inhibitor, a fixed amount of purified GST-cJun substrate (residues 1-79), and [32P]γ-ATP. The products of the kinase reaction were resolved by SDS-PAGE and blotted onto PDF membrane. The signals corresponding to the GSTcJun phosphorylated substrate were detected by phosphorimager and the PVDF membrane is further analyzed by immunoblot analysis with polyclonal anti-JNKl-2 antibodies (Pharmigen) to quantify the amount of JNKl -2 proteins in each kinase reaction (Figure 14B). The specificity of the inhibitors were tested by observing their effect on the closely related kinases p38 and ERK. Whereas p38 is also activated by the same stimuli that activate JNK, both ERK and p38 are closely related to JNK, have a very similar three-dimensional structure and are activated by the same group of upstream kinases, the MKKs. ERK and p38 kinase assays are performed by immunoprecipitation using specific antibodies, myelin basic protein (UBI) as substrate for ERK and MAPK activate protein kinase 2 (GST-MAPKAPK2) as substrate for p38. To further ascertain the specificity of the inhibitors and to study their mechanism of action, the capacity of compounds to block the interaction of purified GST-JNKl and GSTJNK2 fusion proteins on purified substrates that are of relevance for type-2 diabetes was measured. More specifically, purified GST-JNKl or GST- JNK2 was incubated with His-tagged cJun or His-tagged IRS 1 fusion proteins. The ability of the inhibitors to block enzyme-substrate interaction is tested by GST pull down and immunoblot analysis with cJun or IRS 1 antibodies, and by His-tag pull down using Ni-affinity beads and immunoblot analysis with JNKl -2 antibodies (see Figure 14A).
The compounds identified through virtual docking and Delfia assay described above and confirmed to work as JNK inhibitors in vitro were tested for their ability to inhibit JNK in living cells. These experiments provide information regarding the ability of the inhibitors to permeate the cell membranes, their stability and effectiveness in the intracellular milieu. HEK293T cells were transfected with plasmids encoding the kinases JNKl -2 and Myc-tagged substrates, Myc- cJun and Myc-IRSl. 24 hours after transfection, cells were treated with 10 ng/ml of TNFα for 30 minutes in the presence or absence of JNK inhibitors. JNK mediated phosphorylation of the cJun and IRS 1 substrate were measured by cMyc immunoprecipitation and immunoblot analyses using cJun phospho Ser63 antibodies (Cell Signaling) and anti IRSl Ser307 antibodies (Upstate).
The ability of the different compounds to inhibit JNK may be also tested in more physiological cellular models. JNK inhibits insulin signaling via IRSl Ser307 phosphorylation in TNFα treated hepatoma cells (Hirosumi et al., 2002). HepG2 hepatoma cells are treated with 10 ng/ml TNFα for 30 minutes in presence or absence of the JNK inhibitor, and controls are not treated with TNFα. Cell extracts are analyzed by immunoprecipitation with IRSl antibodies and immunoblotting using IRS 1 Ser307 specific antibodies (Upstate), then membranes are stripped and reprobed with IRSl antibody as a control to exclude an effect of the inhibitors on IRSl expression. JNK inhibition was reported to protect pancreatic β-cells from the effect of hydrogen peroxide induced oxidative stress on insulin gene expression (Kaneto et al., 2002). Primary pancreatic islets may be isolated from mice by collagenase-P digestion and Ficoll gradient purification (Shang et al., 2003). Primary β-cell cultures are used to test the ability of the JNK inhibitors to protect pancreatic islets from the effects of H2O2 on insulin gene expression. More specifically, the primary cell cultures are treated for 48 hours with 50 μM of H2O2 in the presence or absence of the JNK inhibitor. Insulin and β-actin mRNA are measured by an Sl nuclease protection assay that we have developed (Westin et al., 2004).
The LanthaScreen technology may be used to test compounds in cell based assays for JNK inhibition. Cell based kinase assays for c-Jun and ATF2 phosphorylation may be carried out using the LanthaScreen c-Jun (1-79) HeIa and LanthaScreen ATF2 (19-106) A549 cell lines (Invitrogen, Carlsbad, CA) which stably express GFP-c-Jun 1-79 and GFP-ATF2 19-106 respectively. Phosphorylation is determined by measuring the time resolved FRET (TR-FRET)) between a terbium labeled phospho-specific antibody and the GFP-fusion protein (Robers et al., in press). The cell-based LanthaScreen kinase assay was used to profile the properties of compound BI-78D3, which was isolated from the Delfia screen. The cells were plated in white tissue culture treated 384 well plates at a density of 10000 cell per well in 32 μl assay medium (Opti-MEM®, supplemented with 1% charcoal/dextran- treated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES pH 7.3, and lacking phenol red). After overnight incubation, cells are pretreated for 60 min with compound (indicated concentration) followed by 30 min of stimulation with 2 ng/ml of TNF-α, which stimulates both JNK and p38. The medium is then removed by aspiration and the cells were lysed by adding 20 μl of lysis buffer (20 mM TRIS-HCl pH 7.6, 5 mM EDTA, 1% NP-40 substitute, 5 mM NaF, 150 mM NaCl, 1 :100 protease and phosphatase inhibitor mix, SIGMA P8340 and P2850 respectively). The lysis buffer included 2 nM of the terbium labeled anti-pc-Jun (pSer73) or anti-pATF2 (pThr71) detection antibodies (Invitrogen). After allowing the assay to equilibrate for 1 hour at room temperature, TR-FRET emission ratios were determined on a BMG Pherastar fluorescence plate reader (excitation at 340 nm, emission 520 nm and 490 nm; 100 μs lag time, 200 μs integration time, emission ratio = Em520 / Em 490).
In this assay BI-78D3 is able to inhibit TNF-α stimulated phosphorylation of c-Jun in cells (EC50 = 12.4 μM; Figure 21A) in a cellular context. Because the cell-based system employed makes use of a GFP-c-Jun stable expression system, the high levels of GFP-c-Jun in these cells, which are higher than endogenous levels of c-Jun, may have an inflationary effect on the EC50 values obtained with this assay when testing substrate competitive compounds. Given that TNF-α stimulates both JNK and p38 and that ATF2 is a substrate for both kinases, one would predict that a JNK selective compound such as BI-78D3 would not affect the ability of p38 to phosphorylate ATF2. Indeed, ATF2 phosphorylation was not affected in the same assay further demonstrating the selectivity of BI-78D3.
Example 11 In Vivo Assays
The link between Conconavilin A (ConA) induced liver damage and JNK function was reported by studies employing JNKl"'" and JNK2"Λ mice (Maeda et al., 2003; Wang et al., 2006). To test the efficacy of the JNK-inhibitory properties of BI-78D3 at blocking the effects of ConA on mouse liver function, ConA (Sigma) and BI-78D3 were injected i.v. at 10 mg/kg into 6 to 8 weeks old male BL/6 mice. For partial hepatectomy, mice were anesthetized with isofluorane and subjected to mid ventral laparotomy followed by removal of the left lateral and median lobes. Animals were sacrificed, blood was collected by cardiac puncture, and livers were surgically removed. Serum was separated and analyzed for alanine-aminotranferase levels (Sigma) following treatment with test compounds. The ability of BI-78D3 to abrogate ConA induced liver damage as measured by serum alanine-aminotranferase levels in vivo (Figure 21B) is consistent with its proposed function as an effective JNK inhibitor. Liquid chromatography/mass spectrometry bio-availability analysis demonstrates that BI-78D3 has a half-life of 54 minutes in a microsome stability assay and favorable plasma stability. Taken together the in vivo and in vitro findings suggest that BI-78D3 is a suitable candidate for further efficacy studies in a variety of animal models of JNK related diseases. In vivo administration of a cell permeable peptidic JNK inhibitor (tat-pepJIPl peptide) was reported to be effective in treating glucose intolerance and type-2 diabetes in both dietary and genetic murine models of obesity (Kaneto, et al., 2004). To test the efficacy of a JNK inhibitor found to be effective in cell culture in mice, the effects of the inhibitor to the tat- pepJIPl peptide are compound parallel in mice models of obesity (Hirosumi et al., 2002; Yan et al., 2001, Arikan et al., 2005). To test the effect of the compounds and to determine the optimal concentration needed to obtain an insulin sensitizing effect, 8 week old diabetic ob/ob C57B1/6J mice are injected once daily with different doses of the JNK inhibitor starting from an estimated blood concentration of the inhibitor equal to 5 times ED50 dose in cultured cells up to 50 times the ED50. As controls, 8 week old male diabetic ob/ob C57B1/6J mice are injected daily with either a saline solution or with 10 mg/Kg body weight of the tat- JIP peptide. Blood is collected every morning from tail vein. Glucose and insulin concentrations are measured using a glucometer (Roche) for glucose and an ELISA kit for insulin (Crystal Chem).
When an evident effect of the inhibitor leading to blood glucose reduction is observed, an intraperitoneal glucose tolerance test (IGTT) and an insulin tolerance test (HTT) are performed (Hirosumi et al., 2002). Briefly, for IGTT, a group of mice is injected intraperitoneally with a bolus of 1 g/kg of body weight of glucose. Blood is collected from the tail at different time points for a total time course of 0-2 hours and glucose concentration is measured using a glucometer. For IITT, mice are injected intraperitoneally with 2 IU insulin/kg of body weight, blood collection, and measurement of glucose concentration are performed as for IGTT. Insulin signaling in liver is measured by injection of 25 mIU insulin/kg of body weight through the portal vein, the whole liver will be collected and frozen in liquid nitrogen 2 minutes after injection (Hirosumi et al., 2002). Insulin receptor β chain (IRβ), and IRSl tyrosine phosphorylation are measured by immunoprecipitation with either IRS 1 or IRβ antibodies (Upstate) followed by immunoblotting analysis using phospho-tyrosine specific antibodies (Upstate). IRSl Ser 307 phosphorylation is measured by western blot using IRSl phospho Ser 307 specific antibodies. Insulin gene expression is measured by Sl nuclease protection using β- actin as control from whole pancreas.
The compound may also be tested in a dietary model of diabetes. A group of 30 male C57B1/6J mice are placed on high fat high carbohydrate diet from week 4 to week 16 (Hirosumi et al., 2002), and another group of 30 mice is placed on standard chow diet. From each group, 10 mice are treated with control saline solution, 10 mice are injected with the tat- JIP peptide as previously described (Kaneto et al., 2004), and 10 mice are injected intraperitoneally with the optimal concentration of a test JNK inhibitor, and IGTT, IITT, measurements of insulin signaling and of insulin gene expression are then performed as described above.
Example 12 Synthetic Examples of Compounds of the Invention
Materials
All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and solvents were purchased as the highest grade available and used without further purification. Thin-layer chromatography (TLC) analysis of reaction mixtures was performed using Merck silica gel 60 F254 TLC plates, and visualized using ultraviolet light. 1H NMR data were collected using a 300 MHz Varian instrument and recorded in deuteriochloroform (CDCl3) or dimethyl sulfoxide- d6 (DMSO-J6)- Chemical shifts (δ) are reported in parts per million (ppm) referenced to 1H (Me4Si at 0.00). Mass spectral data were acquired on a Shimadzu LCMS-2010EV for low resolution, and on an Agilent ESI-TOF for high resolution and low resolution. Purity of compounds was determined using a Waters HPLC. List of Abbreviations: equivalent (eqv), high performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), room temperature (rt). Purity of compounds was obtained in a HPLC Breeze from Waters Co. using an Atlantis T3 3μm 4.6x150 mm reverse phase column.
Methods
Scheme 1
Figure imgf000079_0001
3b
Scheme 2
Figure imgf000079_0002
Scheme 3
Figure imgf000079_0003
6b 2-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-ylcarbamothioyl)hydrazinecarboxamide (l)
Figure imgf000080_0001
To a suspension of sodium acetate (425 mg, 5.18 mmol) and semicarbazide (578 mg,
5.18 mmol) in CH3CN (8 mL) was added 2,3-dihydro-l,4-benzodioxin-6-yl-isothiocyante and the reaction mixture was stirred for 24 h. The solvent was removed by rotary evaporation and the resulting crude residue was dissolved in 2M NaOH (50 mL) and filtered through activated charcoal. The filtrate was acidified with IN HCl (50 mL). The precipitate was collected and washed with H2O (3 x 15 mL), and hexanes (2 x 20 mL) to afford 1 as an off white solid (1.25 g,
90%). 1H NMR (300 MHz, DMSO-J6) δ 4.21 (s, 4H), 6.03 (s, 2H, NH2), 6.77 (d, J = 8.4 Hz, IH), 6.86 (dd, J = 2.4, 8.7 Hz, IH), 7.06 (d, J = 2.4 Hz, IH), 7.97 (s, IH, NH), 9.28 (s, IH, NH),
9.49 (s, IH, NH); MS m/z 559 (2M+Na) +, 537 (2M+H)+, 291 (M+Na) +, 269 (M+H)+, 101, 79,
64, 56; HRMS (ESI-TOF) calcd for C10H12N4O3S 269.0703 (M+H), found 269.0703.
2-(phenylcarbamothioyl)hydrazinecarboxamide
Figure imgf000080_0002
The thiobiurea compound was synthesized from phenyl isothiocyanate in a similar procedure to that of compound 1. 1H NMR (300 MHz, DMSO-J6) δ 6.05 (s, 2H, NH2), 7.09-
7.17 (m, IH), 7.31 (t, J = 7.8 Hz, 2H), 7.51 (d, J = 7.8 Hz, 2H), 8.02 (s, IH, NH), 9.39 (s, IH,
NH), 9.65 (s, IH, NH); MS m/z 443 (2M+Na) +, 421 (2M+H)+, 233 (M+Na) +, 211 (M+H)+, 194, 109, 107, 87, 85, 74; HRMS (ESI-TOF) calcd for C8H10N4OS 211.0648 (M+H), found 211.0650.
4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)-3-mercapto-li/-l,2,4-triazol-5(4i/)-one (2)
Figure imgf000080_0003
Thiobiurea 1 (237 mg, 0.88 mmol) in 2M NaOH (8.8 mL) was stirred at reflux for 4.5 h. The reaction mixture was allowed to cool to rt and then acidified with IN HCl. The resulting precipitate was collected by filtration and washed with H2O (3 x 10 mL), hexanes (2 xlO mL) and 1 :1 hexanes: diethyl ether (10 mL) and dried in vacuo to afford 2 as a white solid (164 mg, 74%). 1H NMR (300 MHz, DMSO-J6) δ 4.27 (s, 4H), 6.80 (dd, J = 2.1, 8.7 Hz, IH), 6.87 (d, J = 2.1 Hz, IH), 6.94 (d, J = 8.7 Hz, IH); LCMS m/z 252 (M+H); MS 525 (2M+Na) +, 503 (2M+H)+, 274 (M+Na) +, 252 (M+H)+, 160, 102, 85, 74; HRMS (ESI-TOF) calcd for C10H9N3O3S 252.0437 (M+H), found 252.0440; purity 96% by HPLC. 3-mercapto-4-phenyl-l//-l,2,4-triazol-5(4H)-one (4)
Figure imgf000081_0001
Compound 4 was synthesized from 2-(phenylcarbamothioyl)-hydrazinecarboxamide by the reported procedure (Joshua, C; Suni, M.; Nair, V. Tetrahedron 2001, 57, 2003) to afford the desired product as a white solid (75%). 1H NMR (300 MHz, DMSO-J6) δ 7.34-7.40 (m, 2H, 2NH), 7.42-7.54 (m, 5H); MS m/z 409 (2M+Na) +, 387 (2M+H)+, 216 (M+Na) +, 194 (M+H)+, 159, 107, 87, 85, 74, 67; HRMS (ESI-TOF) calcd for C8H7N3OS 194.0383 (M+H), found 194.0383.
General Procedure for Compounds 3(a, b)
To a solution of triazole 2 (1 eqv) in MeOH (0.3 mM) was added NaOMe (1 eqv) and stirred. After 5 min. was added the appropriate brominated compound (1 eqv) and the reaction mixture was stirred until deemed complete by TLC. Upon completion the reaction mixture was suspended in H2O (10 mL) and filtered. The solid was washed with H2O (3 x 10 mL), IN HCl (10 mL), H2O (10 mL), hexanes (10 mL) and dried in vacuo to afford the desired product.
4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)-3-(5-nitrothiazol-2-ylthio)-li/-l,2,4-triazol-5(4i/)- one (3a) (BI-78D3)
Figure imgf000081_0002
The reaction mixture of 2 and 2-bromo-5-nitrothiazole was stirred for 4 h. Desired product BI-78D3 (3a) was obtained as an off white solid (96.5 mg, 65%). 1H NMR (300 MHz, CDCl3) δ 4.32 (s, 4H), 6.82 (dd, / = 2.4, 8.7 Hz, IH), 6.86 (d, / = 2.4 Hz, IH), 6.99 (d, J = 8.7 Hz, IH), 8.41 (s, IH); MS m/z 380 (M+H)+, 359, 241, 179, 107, 101, 85, 79, 64, 63; HRMS (ESI-TOF) calcd for C13H9N5O5S2 380.0118 (M+H), found 380.0111 ; purity 99.0% by HPLC.
4-(2,3-dihydrobenzo[Z>][l,4]dioxin-6-yl)-3-(5-nitrothiophen-2-ylthio)-li/-l,2,4-triazol-
5(4J/)-one (3b) (BI-83C11)
Figure imgf000081_0003
The reaction mixture of 2 and 2-bromo-5-thiophene was stirred for 16 h. Desired product 3b was obtained as a yellow solid (57 mg, 56%). 1H NMR (300 MHz, DMSO-J6) δ 4.28 (s, 4H), 6.83 (d, J = 8.1 Hz, IH), 6.98-6.91 (m, 2H), 7.18 (d, J = 4.5 Hz, IH), 8.00 (d, J = 4.5 Hz, IH); MS m/z 401 (M+Na) +, 379 (M+H)+, 359, 354, 345, 295, 274, 159, 126, 98, 85, 74, 68; HRMS (ESI-TOF) calcd for C14H10N4O5S2 379.0165 (M+H)+, found 379.0162; purity 98.6% by HPLC.
3-(5-nitrothiazol-2-ylthio)-4-phenyl-li/-l,2,4-triazol-5(4i/)-one (5) (BI-83C9)
Figure imgf000082_0001
Compound 5 was synthesized from thiazole 4 and 2-bromo-5-nitrothiazole in a similar procedure to that of compounds 3. The reaction mixture was stirred for 4 h. Desired product 5 was obtained as an off-white solid (70%). 1H NMR (300 MHz, DMSO-J6) δ 7.36-7.53 (m, 6H), 8.67 (s, IH); MS m/z 665 (2M+Na) +, 344 (M+Na) +, 322 (M+H)+, 160, 88, 85, 74, 56; HRMS (ESI-TOF) calcd for C11H7N5O3S2 322.0063 (M+H), found 322.0063; purity 99.3% by HPLC.
General Procedure for Compounds 6(a, b)
To a solution of sodium 1 -phenyl- 1/f-tetrazo Ie -5-thiolate (1 eqv) in MeOH (0.3 mM) was added the appropriate bromo-thiazole (1 eqv) and the reaction mixture was stirred until deemed complete by TLC. Upon completion the reaction mixture was suspended in H2O (10 mL) and filtered. The solid was washed with H2O (3 x 10 mL), IN HCl (10 mL), H2O (10 mL), hexanes (10 mL) and dried in vacuo to afford the desired product.
5-nitro-2-(l-phenyl-li/-tetrazol-5-ylthio)thiazole (6a) (BI-83C7)
Figure imgf000082_0002
The crude solid was recrystallized from MeOH to afford 6a as a white solid (125.2 mg,
78%). 1H NMR (300 MHz, CDCl3) δ 7.58-7.63 (m, 2H), 7.66-7.69 (m, 3H), 8.48 (s, IH); MS m/z 329 (M+Na) +, 307 (M+H)+, 295, 279, 159, 142, 126, 117, 98, 88, 85, 74; HRMS (ESI-TOF) calcd for C10H6N6O2S2 307.0066 (M+H), found 307.0066; purity 92.2% by HPLC. 4-methyl-2-(l-phenyl-li/-tetrazol-5-ylthio)thiazole-5-carboxylic acid (6b) (BI-83C8)
Figure imgf000083_0001
Carboxylic acid 6b was obtained as a solid (72%). 1H NMR (300 MHz, DMSO-J6) δ 2.41 (s, 3H), 7.42-7.63 (m, 4H), 7.79 (d, J = 7.2 Hz, IH); MS m/z 342 (M+Na) +, 320 (M+H)+, 292, 224, 173, 179, 159, 128, 117, 85, 74; HRMS (ESI-TOF) calcd for C12H9N5O2S2 320.027 (M+H), found 320.0270; purity 99.3% by HPLC.
5-(benzylthio)-l-phenyl-lf/-tetrazole (7) (BI-83B3)
Figure imgf000083_0002
To a mixture of l-phenyl-l/f-tetrazole-5-thiol (500 mg, 2.75 mmol) and K2CO3 (570 mg, 4.13 mmol) in dry DMF (10 mL) was added benzyl bromide (343 μL, 2.89 mmol) and the reaction was stirred at 8OC under nitrogen for 4.5 h. Upon completion the reaction mixture was suspended in H2O (30 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with H2O (3 x 50 mL), brine (3 x 50 mL) and dried over MgSO4. The crude yellow oil was purified by flash chromatography (10% EtOAc:hexanes) to afford 7 as a white solid (720mg, 98%). 1H NMR (300 MHz, CDCl3) δ 4.63 (s, 2H), 7.26-7.40 (m, 3H), 7.41-7.48 (m, 2H), 7.50-7.59 (m, 5H); MS m/z 559 (2M+Na) +, 291 (M+Na) +, 269 (M+H)+, 159, 121, 102, 91, 85, 79, 85, 64, 56; HRMS (ESI-TOF) calcd for C14H12N4S 269.0855 (M+H), found 269.0856; purity 99.3% by HPLC.
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Incorporation by Reference
All publications, patents and patent applications are incorporated herein by reference.
Equivalents
In the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration. Nevertheless, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention. Likewise, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims

Claims

WHAT IS CLAIMED:
1. A method to identify a modulator of the JIP/JNK interaction, comprising: providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent; washing the solid support to remove molecules not associated with the solid support; separating the label from the ligand; and determining the amount of separated label.
2. The method of claim 1 wherein the polypeptide comprising JIPl or a fragment thereof is coupled to biotin.
3. The method of claim 1 wherein the solid support comprises streptavidin.
4. The method of claim 1 wherein the polypeptide comprising JNK further comprises glutathione S transferase (GST) or a His tag.
5. The method of claim 1 wherein the ligand is an antibody.
6. The method of claim 5, wherein the antibody is an anti GST antibody.
7. The method of claim 1 wherein the label is a lanthanide.
8. The method of claim 7, wherein the lanthanide is europium.
9. The method of claim 1 wherein the solid support is a multi-well plate.
10. The method of claim 1 wherein the JNK is JNKl .
11. The method of claim 1 wherein the JNK is JNK2
12. The method of claim 1 wherein the amount of separated label in the associated molecules is determined.
13. The method of claim 1 wherein the amount of separated label in the unassociated molecules is determined.
14. The method of claim 1, further comprising the step of pre- selecting test agents using virtual docking.
15. A method to identify a modulator of the JIP/JNK interaction, comprising: providing a sample comprising an agent a polypeptide comprising labeled JIPl or a fragment thereof, wherein the label is a fluorophore; and determining fluorescence polarization in the sample relative to a control sample that lacks the agent.
16. The method of claim 15 wherein JNK is JNKl .
17. The method of claim 15 wherein JNK is JNK2.
18. The method of claim 15 wherein the fluorophore is fluorescein isothiocyanate
(HTC).
19 A method to identify a modulator of the JIP/JNK interaction, comprising: contacting a solid support comprising a polypeptide comprising JIP or a fragment thereof with a sample comprising a fusion protein comprising JNK or a fragment thereof, a labeled ligand for JIP, and a test agent; separating molecules not associated with the solid support comprising JIPl or the fragment thereof from molecules associated with the solid support; separating the label from the ligand; and determining the amount of separated label.
20. The method of claim 19 wherein the polypeptide comprising JIP or the peptide thereof is coupled to biotin.
21. The method of claim 19 wherein the solid support comprises streptavidin.
22. The method of claim 19 wherein the polypeptide comprising JNK further comprises GST or a His tag.
23. The method of claim 19 wherein the ligand is an antibody.
24. The method of claim 19 wherein the label is a lanthanide.
25. The method of claim 19 wherein the solid support is a multi-well plate.
26. The method of claim 19 wherein the JNK is JNKl .
27. The method of claim 19 wherein the JNK is JNK2
28. The method of claim 19 wherein the presence or amount of separated label in the associated molecules is detected or determined.
29. The method of claim 19 wherein the presence or amount of separated label in the unassociated molecules is detected or determined.
30. The method of claim 19, further comprising the step of pre-selecting test agents using virtual docking.
31. A method for identifying a modulator of JIP/JNK interaction, comprising;
contacting a complex comprising polypeptides comprising JIP and JNK, or fragments thereof, each of which contains one of a donor and an acceptor fluorophore with a test agent; and measuring the FRET between the donor and acceptor fluorophore; thereby identifying a modulator of the JIP/JNK interaction of JIP and JNK.
32. The method of claim 31 , wherein a decrease in the FRET indicates that the test compound modulates the interaction.
33. The method of claim 31, wherein the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex.
34. The method of claim 31 , wherein the fluorescent acceptor is attached to JIP.
35. The method of claim 31, wherein the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK.
36. The method of claim 35, wherein the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
37. The method of claim 31 , wherein the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively.
38. The method of claim 31, wherein the FRET is time resolved FRET.
39. The method of claim 31 , further comprising isolating and identifying the test compound.
40. The method of claim 31, further comprising the step of pre-selecting test agents using virtual docking.
41. A method for identifying a modulator of JIP/JNK interaction, comprising;
contacting a solution comprising a polypeptide comprising JIP, or a fragment thereof, wherein the polypeptide comprising JIP, or a fragment thereof, comprises a fluorescent donor or acceptor, with a test agent; adding to the solution a polypeptide comprising JNK, or a fragment thereof, comprising a donor or acceptor, wherein the JNK polypeptide comprises either a fluorescent donor or acceptor suitable to form a fluorescent donor/acceptor pair with the JIP fluorophore; measuring the FRET between the donor and acceptor fluorophore; thereby identifying a modulator of the JIP/JNK interaction.
42. The method of claim 41, wherein a decrease in the FRET compared to a solution lacking the test compound indicates that the test compound modulates the interaction of JIP and JNK.
43 The method of claim 41 , wherein the fluorescent acceptor is attached to the JIP or JNK by a stereptavidin/biotin complex.
44. The method of claim 43, wherein the fluorescent acceptor is attached to JIP.
45. The method of claim 41, wherein the fluorescent donor is attached to an antibody that specifically recognizes a polypeptide comprising JIP or JNK.
46. The method of claim 45, wherein the fluorescent donor is attached to an antibody that specifically recognizes GST and the polypeptide comprising JNK is a JNK/GST fusion polypeptide.
47. The method of claim 41, wherein the fluorescent donor and acceptor are europium chelate and allophycocyanin, respectively.
48. The method of claim 41, further comprising isolating and identifying the test compound.
49. A method to identify a modulator of the JIP/JNK interaction, comprising: providing a mixture comprising a solid support comprising a polypeptide comprising JIPl or a fragment thereof, a polypeptide comprising JNK or a fragment thereof, a labeled ligand for JIPl, and a test agent; washing the solid support to remove molecules not associated with the solid support; separating the label from the ligand; and determining the amount of separated label.
50. A method to identify a modulator of the JIP/JNK interaction, comprising: preselecting a test compound capable of binding to JIP, JNK, or a JIP/JNK complex by virtual docking; providing a sample comprising an a preselected test compound a polypeptide comprising labeled JIPl or a fragment thereof, wherein the label is a fluorophore; and determining fluorescence polarization in the sample relative to a control sample that lacks the test agent.
51. A method to identify a modulator of the JIP/JNK interaction, comprising: preselecting a test compound capable of binding to JIP, JNK, or a JIP/JNK complex by virtual docking; contacting a solid support comprising a polypeptide comprising JIP or a fragment thereof with a sample comprising a fusion protein comprising JNK or a fragment thereof , a labeled ligand for JIP, and a preselected test agent; separating molecules not associated with the solid support comprising JIPl or the fragment thereof from molecules associated with the solid support; separating the label from the ligand; and determining the amount of separated label.
52. A method for identifying a modulator of JIP/JNK interaction, comprising; preselecting a test compound capable of binding to JIP, JNK, or a JIP/JNK complex by virtual docking; contacting a complex comprising polypeptides comprising JIP and JNK, or fragments thereof, each of which contains one of a donor and an acceptor fluorophore with a preselected test agent; and measuring the FRET between the donor and acceptor fluorophore; thereby identifying a modulator of the JIP/JNK interaction of JIP and JNK.
53. A method for identifying a modulator of JIP/JNK interaction, comprising; preselecting a test agent capable of binding to JIP, JNK, or a JIP/JNK complex by virtual docking; contacting a solution comprising a polypeptide comprising JIP, or a fragment thereof, wherein the polypeptide comprising JIP, or a fragment thereof, comprises a fluorescent donor or acceptor, with a preselected test agent; adding to the solution a polypeptide comprising JNK, or a fragment thereof, comprising a donor or acceptor, wherein the JNK polypeptide comprises either a fluorescent donor or acceptor suitable to form a fluorescent donor/acceptor pair with the JIP fluorophore; measuring the FRET between the donor and acceptor fluorophore; thereby identifying a modulator of the JIP/JNK interaction.
54. A method to inhibit JIP/JNK interaction in cells comprising contacting cells with an effective amount of a compound of formula (II), (III), (IV), or (V).
55. The method of claim 54 wherein the compound is 4-(2,3-dihydrobenzo[b][l,4]dioxin-6- yl)-3-(5-nitrothiazol-2-ylthio)-l H -l,2,4-triazol-5(4/f)-one.
56. The method of claim 54 wherein the compound is 3-(5-nitrothiazol-2-ylthio)-4-phenyl- l/M,2,4-triazol-5(4/f)-one.
57. The method of claim 54 wherein the compound is 5-nitro-2-(l-phenyl-l/f-tetrazol-5- ylthio)thiazole.
58. A method to treat a metabolic disorder comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV), or (V) to thereby treat said subject for a metabolic disorder.
59. The method of claim 58 wherein the compound is 4-(2,3-dihydrobenzo[b][l,4]dioxin-6- yl)-3-(5-nitrothiazol-2-ylthio)-l H -l,2,4-triazol-5(4/f)-one.
60. The method of claim 58 wherein the compound is 3-(5-nitrothiazol-2-ylthio)-4-phenyl- IH- 1 ,2,4-triazol-5(4/f)-one.
61. The method of claim 58 wherein the compound is 5-nitro-2-(l-phenyl-l/f-tetrazol-5- ylthio)thiazole.
62. A method to treat diabetes, comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV), or (V).
63. The method of claim 62 wherein the compound is 4-(2,3-dihydrobenzo[b][l,4]dioxin-6- yl)-3-(5-nitrothiazol-2-ylthio)-l H -l,2,4-triazol-5(4/f)-one.
64. The method of claim 62 wherein the compound is 3-(5-nitrothiazol-2-ylthio)-4-phenyl- IH- 1 ,2,4-triazol-5(4/f)-one.
65. The method of claim 62 wherein the compound is 5-nitro-2-(l-phenyl-l/f-tetrazol-5- ylthio)thiazole.
66. A method to treat obesity comprising administering to a subject in need thereof an effective amount of a compound of formula (II), (III), (IV), or (V).
67. The method of claim 66 wherein the compound is 4-(2,3-dihydrobenzo[b][l,4]dioxin-6- yl)-3-(5-nitrothiazol-2-ylthio)-l H -l,2,4-triazol-5(4/f)-one.
68. The method of claim 66 wherein the compound is 3-(5-nitrothiazol-2-ylthio)-4-phenyl- IH- 1 ,2,4-triazol-5(4/f)-one.
69. The method of claim 66 wherein the compound is 5-nitro-2-(l-phenyl-l/f-tetrazol-5- ylthio)thiazole.
70. A compound for the treatment of a metabolic disorder, wherein the compound has the structure set forth as formula (II), (III), (IV) or (V).
71. A compound for the treatment of a metabolic disorder, wherein the compound has a structure set forth in Table I, Ia, II, Ha, III, IV, V, or VI.
72. The compound of claims 70 or 71, wherein the metabolic disease is obesity or diabetes.
73. A compound having the structure set forth as formula (II) or (III).
74. A compound having the structure set forth as formula (IV) or (V).
75. A pharmaceutical composition comprising the compound of any one of claims 70-75.
76. The pharmaceutical composition of claim 75 for the treatment of a metabolic disorder.
77. The pharmaceutical composition of claim 76, wherein the metabolic disease is obesity or diabetes.
78. A kit comprising one or more of the reagents recited in the method of any one of claims 1-53 and instructions for use.
79. A kit for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent donor and a JIP polypeptide, or fragment thereof, comprising a fluorescent acceptor and instructions for use.
80. A kit for identifying modulators of the JIP/JNK interaction comprising a JNK polypeptide, or fragment thereof, comprising a fluorescent acceptor and a JIP polypeptide, or fragment thereof, comprising a fluorescent donor and instructions for use.
81. A kit comprising the compound of any one of claims 70-74 or the pharmaceutical composition of any one of claims 15-11 , and instructions for use.
82. The method of any one of claims 1 -53 further comprising obtaining one or more of the reagents recited in the method of any one of claims 1-53.
83. A compound identified by any one of the methods of claim 1, 15, 19, 31, 41, 49, or 50-
53.
84. The method of any one of claim 1, 15, 19, 31, 41, 49, or 50-53, further comprising the step of screening a potential modulator of JNK/JIP interaction against a JNK2 mutant to determine active site binding location.
85. The method of claim 84, further comprising the step of screening a potential modulator of JNK/JIP interaction against R127A and C167S mutants of JNK2 to determine if the compound is binding in the JIP pocket.
86. The method of any one of claim 54, 58, 62, or 66, wherein the compound is 4-(4-nitro- phenyl)-5-(5-nitro-thiazol-2-ylsulfanyl)-2,4-dihydro-[l,2,4]triazol-3-one.
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