EP1309721A2 - Modulating multiple lineage kinase proteins - Google Patents

Modulating multiple lineage kinase proteins

Info

Publication number
EP1309721A2
EP1309721A2 EP01961958A EP01961958A EP1309721A2 EP 1309721 A2 EP1309721 A2 EP 1309721A2 EP 01961958 A EP01961958 A EP 01961958A EP 01961958 A EP01961958 A EP 01961958A EP 1309721 A2 EP1309721 A2 EP 1309721A2
Authority
EP
European Patent Office
Prior art keywords
carbons
group
inclusive
alkyl
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01961958A
Other languages
German (de)
English (en)
French (fr)
Inventor
Anna Maroney
Kevin M. Walton
Craig A. Dionne
Nicola Neff
Ernest Knight, Jr.
Marcie A. Glicksman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cephalon LLC
Original Assignee
Cephalon LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cephalon LLC filed Critical Cephalon LLC
Publication of EP1309721A2 publication Critical patent/EP1309721A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • the present invention is directed, in part, to methods for modulating members ofthe multiple lineage kinase (MLK) family, methods for identifying compounds which modulate a multiple lineage kinase protein and either promote cell survival or promote cell death, methods for identifying compounds which may be useful in the treatment of neurodegenerative disorders and/or inflammation, and methods of treating neurodegenerative disorders with compounds which inhibit a multiple lineage kinase protein.
  • MLK multiple lineage kinase
  • the MLK family comprises a group of proteins in which the protein sequence ofthe kinase domains of the family members closely resemble the MAPKKKs but have greater similarity to each other than to other MAPKKKs.
  • MLK family members comprise a portion of very complex kinase cascades such as, for example, the stress-signaling cascade, which involves modulation of, inter alia, the c-Jun N-terminal kinase (JNK), which in turn modulates, inter alia, transcription factors including c-Jun, ATF2, and ELK-1.
  • JNK is described in U.S. Patents 5,534,426, 5,593,884, 5,605,808, and WO 95/03324, each of which is incorporated herein by reference in its entirety.
  • the MLK family includes, in part, the following groups: 1) multiple lineage kinase 1 (MLK1); 2) multiple lineage kinase 2 (MLK2); 3) multiple lineage kinase 3 (MLK3); 4) leucine zipper bearing kinase (LZK); 5) dual leucine zipper bearing kinase (DLK); and 6) multiple lineage kinase 6 (MLK6).
  • MLK1 has a catalytic domain similar to both kinases specific for Tyr and Ser/Thr. Dorow, etal.,Eur. J. Biochem., 1993, 213, 701-710.
  • MLK2 also has a catalytic domain similar to both kinases specific for Tyr or Ser/Thr. Dorow, et al. , Eur.
  • MLK2 is also known as MST. Katoh, et al, Oncogene, 1995, 10, 1447-1451.
  • MLK3 comprises a protein that, in addition to the kinase domain, contains two leucine zippers with an adj acent carboxy-terminal basic region, and a proline rich region. Ing, etal, Oncogene, 1994, 9, 1745-1750.
  • MLK3 is also known as SPRK (Gallo, et al, J. Biol. Chem., 1994, 269, 15092-15100), and PTK1 (Ezoe, et al, Oncogene, 1994, 9,
  • LZK is a leucine zipper bearing kinase. Sakuma, etal, J. Biol. Chem., 1991, 272, 28622-28629.
  • DLK has a kinase domain and two putative leucine zipper motifs. Holzman, et al, J. Biol. Chem., 1994, 269, 30808-30817.
  • DLK is also known as ZPK (Reddy, et al, Biochem. Biophys. Res. Comm., 1994, 202, 613-620) andMUK(Hirai, etal, Oncogene, 1996, 12, 641-650).
  • Members ofthe MLK family are also described in, for example, U.S. Patents
  • motoneuron apoptosis is inhibited by a derivative of K-252a, an indolocarbazole which also modulates the stress-signaling cascade.
  • K-252a an indolocarbazole which also modulates the stress-signaling cascade.
  • the present invention provides methods for identifying compounds which modulate activity of a multiple lineage kinase protein and promote cell survival comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound, determining whether the compound decreases activity ofthe multiple lineage kinase protein, and determining whether the compound promotes cell survival.
  • the present invention also provides methods for identifying compounds which modulate activity of a multiple lineage kinase protein and promote cell death comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound, determining whether the compound increases activity ofthe multiple lineage kinase protein, and determining whether the compound promotes cell death.
  • the present invention also provides methods for identifying compounds which may be useful in treating neurodegenerative disorders comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases activity ofthe multiple lineage kinase protein.
  • the present invention also provides methods for identifying compounds which may be useful in treating inflammation comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases activity ofthe multiple lineage kinase protein.
  • the present invention also provides methods for treating a mammal having or suspected of having a neurodegenerative disorder comprising administering to said mammal a compound which inhibits or reduces multiple lineage kinase protein activity.
  • the present invention also provides methods for treating a mammal having inflammation comprising administering to said mammal a compound which inhibits or reduces multiple lineage kinase protein activity.
  • the present invention also provides methods for modulating the activity of a multiple linage kinase protein comprising contacting the protein or a cell containing the protein with a compound having formula II:
  • ring B and ring F independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: an unsaturated 6-membered carbocyclic aromatic ring in which from 1 to 3 carbon atoms may be replaced by nitrogen atoms; an unsaturated 5-membered carbocyclic aromatic ring; and an unsaturated 5-membered carbocyclic aromatic ring in which either one carbon atom is replaced with an oxygen, nitrogen, or sulfur atom; two carbon atoms are replaced with a sulfur and a nitrogen atom, an oxygen and a nitrogen atom, or two nitrogen atoms; or three carbon atoms are replaced with three nitrogen atoms;
  • R 1 is selected from the group consisting of:
  • substituted or unsubstituted alkyl having from 1 to 4 carbons substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl;
  • R 9 is selected from the group consisting of alkyl, aryl and heteroaryl;
  • R 10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons;
  • R u and R 12 are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or R u and R 12 together form a linking group of the formula
  • X 1 is selected from the group consisting of -O-, -S-, and -CH 2 -;
  • Y is selected from the group consisting of -O-, -S-, -N(R 10 )-, -N + (O-)(R 10 )-, -N(OR 10 )-, and -CH 2 -;
  • R 17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl;
  • a 1 and A 2 are selected from the group consisting of H, H; H, OR 2 ; H, -SR 2 ; H, -N(R 2 ) 2 ; and a group wherein A 1 and A 2 together form a moiety selected from the group consisting of
  • the present invention also provides methods for modulating the activity of a multiple linage kinase protein comprising contacting the protein or a cell containing the protein with a compound having formula III:
  • R 2 is selected from the group consisting of H, Br, CI, I, CH 2 S(CH 2 ) 2 N(CH 3 ) 2 , NHCONHC 2 H 5 , CH 2 SC 2 H 5 , CH 2 OCH 2 OCH 2 CH 3 , CH 2 S[3-(l,2,4-triazine)], CH 2 CH 2 SCH 3 , and CH 2 OH;
  • X is selected from the group consisting of H, CH 2 OH, CH 2 NH-SerineH, CO 2 CH 3 ,
  • R is selected from the group consisting of OH, and OCH 3 .
  • the present invention also provides methods of modulating the activity of a multiple linage kinase protein comprising contacting the protein or a cell containing the protein with a compound having formula IV:
  • Figure 1 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles .
  • Figure 2 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles.
  • Figure 3 is a schematic drawing showing a preparation of resin-bound indenopyrrolocarbazoles.
  • Figure 4 is a schematic drawing showing the preparation of protected, soluble indenopyrrolocarbazoles .
  • Figure 5 is a schematic drawing showing the preparation of intermediate V.
  • Figure 6 is a schematic drawing showing the preparation of bridged indenopyrrolocarbazoles using method A.
  • Figure 7 is a schematic drawing showing the preparation of bridged indenopyrrolocarbazoles using method B.
  • Figure 8 is a schematic drawing showing the preparation of B ring-substituted bridged indenopyrrolocarbazoles.
  • Figure 9 is a schematic drawing showing the derivatization ofthe E ring of bridged indenopyrrolocarbazoles .
  • Figure 10 shows a graph of two separate experiments depicting the amount of viable neuronally differentiated PC- 12 cells remaining after 5 days of culturing in the absence of
  • Figure 11B shows 32 P-labeled phosphorylated myelin basic protein product formed as a result of a kinase reaction catalyzed by baculoviras-expressed FLAG-MLK-3 (mixture of full-length and kinase domain) or GST-MLK-3 kinase domain.
  • Figure 12 is an immunoblot analysis showing the phosphorylation of kinase-dead GST- SEK-1 by baculoviras-expressed FLAG-MLK-3 (mixture of full-length and kinase domain) as detected by a phospho-specific SEK-1 antibody.
  • Figure 13 shows the phosphorylation of myelin basic protein by bacterially-expressed GST-MLK-3 kinase domain using the (o) multiscreen trichloroacetic acid precipitation assay, or the (•) phosphocellulose membrane method.
  • Figure 14 shows a saturation binding curve of [ 3 H]K252a incubated with lysate of MLK-3 baculo virus infected insect cells.
  • Figure 15A shows the amount of 3 P -labelled c-jun in an immunoprecipitation/kinase reaction from cells overexpressing MLK-3, MLK-2 or DLK and treated with either 0.025% DMSO (control) or 500 nM K-252a.
  • Figure 15B shows a graph quantifying the percent activity remaining in immunoprecipitate/kinase reactions from samples described in FIG. 15 A. Columns represent the average of duplicate samples where the error bar indicates the range ofthe mean.
  • Figure 15C shows the amount of 32 P -labelled c-jun in an immunoprecipitation/kinase reaction from cells overexpressing HA- JNK 1 alone or with MEKKl at various amounts of cDNA as indicated and treated with either 0.025% DMSO (control) or 500 nM of Compound
  • Figure 16 shows that Compound III-3 promotes neuronal survival in a concentration-dependent fashion.
  • Dissociated neurons were cultured from sympathetic ganglia (SG) (A), dorsal root ganglia (DRG) (B), ciliary ganglia (CG) (C), and motoneurons (MN) (D), in the presence or absence ofthe indicated trophic factors.
  • Cells were counted 48 h after plating as described in materials and methods. Data represent means ⁇ SD of triplicate or quadruplicate determinations. Shown is one of three experiments.
  • Figure 17 shows phase-contrast micrographs of cultures of El 2 DRG (A, E), E9 sympathetic (B, F), E8 ciliary (C, G) and E5.5 motor neurons (D, H) after 48 h in culture (24 h for ciliary neurons) in the presence ofthe respective neurotrophic factor (20 ng/ml NGF for sympathetic and sensory neurons 10 ng/ml CNTF for ciliary neurons, 30 ⁇ g/ml muscle extract
  • Figure 18 shows a photomicrograph of dorsal root ganglia explants in vitro.
  • Explants from chick DRG (E9) were plated in 96-well plates medium containing 0.05% BSA. After a 2 h attachment period, additions were made: (A) control DMSO; (B) 20 ng/ml NGF; (C) 250 nM Compound III-3. Forty-eight h later, medium was removed and explants were fixed with 4% paraformaldehyde in phosphate-buffered saline.
  • Figure 19 shows the number of chick lumbar motor neurons surviving on E10 after daily treatment (E5-9) with specified doses of Compound 111-3.
  • Presented data are the mean +S.D. of 5-6 animals/treatment group. The reported experiment was repeated two times. The data are from one representative experiment and represent one side of the lumbar column. *p ⁇ 0.01 , **p ⁇ 0.001 , Student t test between Compound III-3 and control groups with Bonferroni correction.
  • Figure 20 shows the number of motor neurons in the female rat spinal nucleus ofthe bulbocavernosus (SNB) surviving on PN10 or PN60 after daily treatment (PNl-5) with Compound III-3, or control vehicle (5% SolutolTM).
  • SNB spinal nucleus ofthe bulbocavernosus
  • PNl-5 Compound III-3
  • control vehicle 5% SolutolTM
  • rats were sacrificed and the region of the spinal cord containing the SNB was dissected and processed for histology ; Cresylecht violet-stained motor neurons were then counted in serial section of the lumbar 5 -sacral 1 region of the spinal cord as described previously (Wingfield, et al , Steroids, 1975, 26, 311-327).
  • Experimental data are the means + S.E.M. from 4-8 animals/treatment group.
  • Figure 21 shows loss of ChAT immunoreactivity after hypoglossal axotomy in the adult rat after treatment with Compound III-3.
  • Figure 22 shows inhibition ofthe MLK-3 pathway demonstrates in vivo efficacy and blockage of phosphorylation events downstream.
  • Figure 22A shows increase of substantia nigra tyrosine hydroxylase immunoreactive neurons after MPTP lesion upon systematic administration of Compound III-3.
  • Figure 22B is a representative immunoblot showing MPTP induced increase in levels of phosphorylated MKK4.
  • Figure 22C depicts a representative immunoblot and ELIS A showing attenuation of MPTP induced phosphorylated MKK4 in the presence of Compound ffl-3.
  • Figure 23 shows the induction of IL-2 in Jurkat cells.
  • Figure 23 A shows the time course of IL-2 induction.
  • Figure 23B shows inhibition of IL-2 induction by Compound III-3.
  • Figure 23C shows inhibition of IL-2 induction by Compound III-3 and Compound 1-4.
  • Apoptosis refers to a specific morphological form of cell death characterized by fragmentation of cells and their nuclei into membrane-bound particles. Apoptosis can be triggered by, for example, treatment with apoptosis-inducing compounds such as etoposide, staurosporine, tumor necrosis factor- , ceramide, and the like, or by conditions such as x- irradiation.
  • apoptosis-inducing compounds such as etoposide, staurosporine, tumor necrosis factor- , ceramide, and the like, or by conditions such as x- irradiation.
  • cell death refers to death of cells by apoptosis, necrotic, or other means widely known to those skilled in the art.
  • Cell death can be characterized, for example, as a decrease in total cell numbers of cells or a decrease in cell viability compared to untreated control populations of cells.
  • Compounds which "promote cell death” result in a decrease in cell numbers or a decrease in cell viability as compared to control populations.
  • compounds which "promote cell survival” result in an increase in cell numbers or cell viability, or which slow or reduce the rate of cell death.
  • reacts selectively or “binds specifically” describe compounds which physically or chemically interact directly with an MLK protein.
  • compounds which do not “react selectively” or “bind specifically” may effect proteins downstream or upstream ofthe MLK protein, and thus may effect the activity of MLK proteins, but do not physically or chemically interact directly with an MLK protein.
  • modulates refers to increasing or decreasing an activity of a particular protein or substrate thereof.
  • the present invention is directed, in part, to methods for identifying compounds which modulate activity of a MLK protein and promote either cell survival or cell death.
  • Compounds which result in increased MLK protein activity may promote cell death, whereas compounds which result in decreased MLK protein activity may promote cell survival.
  • the MLK protein can be any protein identified as belonging to the MLK class of proteins.
  • the MLK protein is selected from the group consisting of MLK1 , MLK2,
  • MLK3 SPRK, PTKl
  • LZK LZK
  • DLK ZPK, MUK
  • MLK6 MLK6 which are described above.
  • the methods identif compounds which directly interact or bind with the MLK protein as determined by binding assays, kinase assays, or other equivalent assays.
  • a cell or cells containing the MLK protein is contacted with the test compound.
  • the contacting can take place in buffers or media well known to those skilled in the art. Alternately, the contacting can take place in vivo, in which an animal, such as, for example, a mouse or other suitable animal known to those skilled in the art, is contacted by administering a pharmaceutical composition comprising the test compound and pharmaceutically acceptable salt, carrier, or diluent.
  • varying numbers of cells and concentrations of test compounds can be used. Whether the test compound increases or decreases activity ofthe MLK protein is determined. In addition, whether the test compound promotes cell survival or cell death is also determined.
  • the cells which are contacted with the test compounds can be any mammalian cell.
  • the cell is a neuronal cell.
  • the cell is involved in a neurodegenerative disease.
  • a neurodegenerative disease a "neurodegenerative disease," a "neurodegenerative disease"
  • neuronal disorder and a “neurodegenerative condition” are interchangeable and are used to describe any disease or disorder involving neuronal cells or cells involved in the neuronal system, including, but not limited to, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and concussive or penetrating injuries to the brain or spinal cord.
  • MLK protein activity can be determined by a number of techniques.
  • MLK activity can be determined by measuring the activity of a substrate ofthe MLK protein.
  • the substrate is a member ofthe mitogen activated protein kinase kinase family or mitogen activated protein kinase family or substrates further down the pathway which includes, but is not limited to, a protein selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38 , p38 ⁇ , p38 ⁇ , p38 ⁇ , MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, and also general substrates of Ser/Thr protein kinases such as myelin basic protein (MBP).
  • MBP myelin basic protein
  • MLK protein activity can also be determined by an in vitro kinase assay. In vitro kinase assays are well known to the skilled artisan. Other techniques for measuring protein activity are known to those skilled in the art and are intended to be covered by the present invention.
  • test compound modulates, i.e., increases or decreases, MLK protein activity.
  • Whether or not the test compound promotes cell survival or cell death can be determined in a number of ways.
  • promotion of cell survival or cell death is determined by using cells at risk of dying and comparing the amount of cells which were contacted with the test compound and remain alive with the amount of cells which were not contacted with the test compound and remain alive.
  • the cells are primary embryonic motoneuron cells which are pre-programmed to die. Primary embryonic motoneuron cells are described in Maroney, et al. , J. Neurosci.
  • normal cells, or wild-type cells are converted to be cells at risk of dying by overexpressing the MLK protein, as described below in the Examples, and then contacted with the test compound.
  • Cells overexpressing MLK proteins may die unless rescued by the test compound.
  • Overexpression of MLK proteins can be accomplished using vectors capable of expressing the particular protein inside a cell.
  • Expression vectors are well known to those skilled in the art. In addition, metliods of preparing expression vectors are also well l ⁇ iown to those skilled in the art. Expression vectors which express any ofthe MLK proteins can be prepared in a manner similar to those described in the Examples. A greater number of living cells in the population of overexpressing cells treated with the test compound as compared to the number of living cells in the population of overexpressing cells which were not treated with the test compound is indicative of a test compound which promotes cell survival. In contrast, a lesser number of living cells in the population of overexpressing cells treated with the test compound as compared to the number of living cells in the population of overexpressing cells which were not treated with the test compound is indicative of a test compound which promotes cell death.
  • promotion of cell survival is determined by observing or measuring a decrease in apoptosis.
  • Cytoplasmic slirinkage and nuclear condensation are associated with apoptosis.
  • one skilled in the art can measure a decrease in apoptosis by measuring or observing a decrease in cytoplasmic shrinkage and/or nuclear condensation.
  • one skilled in the art can measure apoptosis by employing conventional staining techniques.
  • normal, wild-type neuronal cells can be used to identify compounds which promote cell death. Normal neuronal cells will survive unless they are induced to die by the test compound. A lesser number of living cells in the population of normal cells treated with the test compound as compared to the number of living cells in the population of normal cells which were not treated with the test compound is indicative of a test compound which promotes cell death. In contrast, a greater or equal number of living cells in the population of normal cells treated with the test compound as compared to the number of living cells in the population of normal cells which were not treated with the test compound is not indicative of a test compound which promotes cell death.
  • the present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having formula G (denoted formula I herein) set forth in U.S. Patent No. 5,705,511, which is assigned to the assignee ofthe present application and is incorporated herein by reference in its entirety.
  • the present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having formula III below:
  • R 2 is selected from the group consisting of H, Br, CI, I, CH 2 S(CH 2 ) 2 N(CH 3 ) 2 , NHCONHC 2 H 5 , CH 2 SC 2 H 5 , CH 2 OCH 2 OCH 2 CH 3 , CH 2 S[3-(l,2,4-triazine)], CH 2 CH 2 SCH 3 , and CH 2 OH;
  • R is selected from the group consisting of OH, and OCH 3 .
  • Z and Z 2 are H, X is CO 2 CH 3 , R x is NHCONHC 2 H 5 , R 2 is CH 2 CH 2 (2-Pyridyl), and R is OH.
  • Z x and Z 2 are H, X is CO 2 CH 3 ; R and R 2 are CH 2 OCH 2 OCH 2 CH 3 , and R is OH; or Z x and Z 2 are H, X is CO 2 CH 3 , R and R 2 are CH 2 SCH 2 CH 3 , and R is OH; or Z l5 Z 2 , R l5 and R 2 are H, X is CO 2 CH 3 ; and R is OH; or Z l5 Z 2 , R token and R 2 are H, X is CO 2 (CH 2 ) 4 CH 3 , and R is OH; or Z l5 Z 2 , and R l3 are H, R 2 is CH 2 OH, X is CO 2 CH 3 , and R OH.
  • the present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having formula II below:
  • ring B and ring F independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring in which from 1 to 3 carbon atoms may be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring in which either 1) one carbon atom is replaced with an oxygen, nitrogen, or sulfur atom;
  • R 11 and R 12 are each independently selected from the group consisting of H and alkyl having from 1 to 4 carbons; or
  • R 11 and R 12 together form a linking group of the formula -(CH 2 ) 2 -X 1 -(CH 2 ) 2 -, wherein X 1 is selected from the group consisting of -O-, -S-, and -CH 2 -;
  • each alkyl, alkenyl, or alkynyl group is unsubstituted; or
  • Y is selected from the group consisting of -O-, -S-, -N(R 10 )-, -N + (O-)(R 10 )-, -N(OR 10 )- , and -CH 2 -;
  • R 17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl;
  • B 1 and B 2 are selected from the group consisting of H, H; H, -OR 2 ; H, -SR 2 ; H, -N(R ) 2 ; and a group wherein B 1 and B 2 together form a moiety selected from the group consisting of
  • the present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having formula IV below:
  • R x is H or Br;
  • R 2 is H;
  • the present invention also provides methods for identifying compounds which may be useful in treating neurodegenerative disorders comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases activity of the multiple lineage kinase protein.
  • the cells, and extracts therefrom include those described above.
  • Compounds which are found by the present methods i.e., those compounds which inhibit or reduce the activity of a multiple lineage kinase protein may be useful to treat neurodegenerative disorders.
  • the protein is preferably selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, leucine zipper bearing kinase, dual leucine zipper bearing kinase, and multiple lineage kinase 6.
  • the cell is contacted in vitro or in vivo.
  • the protein activity is determined by measuring the activity or phosphorylation state of a substrate of said protein.
  • the substrate is selected from the group consisting of JNK1 , JNK2, JNK3 , ERK1, ERK2, p38 ⁇ , p38 ⁇ , p38 ⁇ , p38 ⁇ , MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK 1, and the mammalian homolog of AEX-3, as well as general Ser/Thr substrates, such as, for example, myelin basic protein (MBP).
  • the protein activity may also be determined by measuring the activity of a substrate ofthe protein, amount of a substrate ofthe protein, or mRNA encoding the substrate ofthe protein.
  • Protein activity may also be determined by an in vitro kinase assay or binding assay.
  • Cells are preferably primary embryonic motoneuron cells, cells which overexpress a multiple lineage kinase protein, or a neuronal cell, but can be any cell or extract therefrom.
  • compounds which directly bind the multiple lineage kinase protein are identified, as described above.
  • the present invention also provides methods for identifying compounds which may be useful in treating inflammation comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases activity ofthe multiple lineage kinase protein.
  • the cells, and extracts therefrom include those described above.
  • Compounds which are found by the present methods i.e., those compounds which inhibit or reduce the activity of a multiple lineage kinase protein may be useful to treat im ⁇ ammation.
  • the protein is preferably selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, leucine zipper bearing kinase, dual leucine zipper bearing kinase, and multiple lineage kinase
  • the cell is contacted in vitro or in vivo.
  • the protein activity is determined by measuring the activity or phosphorylation state of a substrate of said protein.
  • the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38 ⁇ , p38 ⁇ , p38 ⁇ , p38 ⁇ , MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, as well as general Ser/Thr substrates, such as, for example, myelin basic protein (MBP).
  • MBP myelin basic protein
  • the protein activity may also be determined by measuring the activity of a substrate ofthe protein, amount of a substrate of the protein, or mRNA encoding the substrate of the protein. Protein activity may also be determined by an in vitro kinase assay or binding assay.
  • Cells are preferably primary embryonic motoneuron cells, cells which overexpress a multiple lineage kinase protein, or a neuronal cell, but can be any cell or extract therefrom.
  • Cells also include, but are not limited to, those involved in inflammation such as, for example, lymphocytes, macrophages and other white blood cells well known to those skilled in the art.
  • compounds which directly bind the multiple lineage kinase protein are identified.
  • the present invention also provides methods for treating a mammal having or suspected of having a neurodegenerative disorder comprising administering to the mammal a compound which inhibits or reduces multiple lineage kinase protein activity.
  • a compound which inliibits or reduces multiple lineage kinase protein activity includes, but is not limited to, compounds having formula I, II, III, and IV.
  • Preferred compounds include those described above with respect to the method for screening compounds which modulate the activity of a multiple lineage kinase protein and either promote cell survival or cell death.
  • a preferred mammal is a human.
  • An individual may be suspected of having a neurodegenerative disease if the individual has symptoms of a particular neurodegenerative disease, is in a high-risk group, or has a family history of a neurodegenerative disease.
  • the present invention also provides methods for treating a mammal having inflammation comprising administering to said mammal a compound which inhibits or reduces multiple lineage kinase protein activity.
  • a compound which inhibits or reduces multiple lineage kinase protein activity includes, but is not limited to, compounds having formula I, II, III, and IV.
  • Preferred compounds include those described above with respect to the method for screening compounds which modulate the activity of a multiple lineage kinase protein and either promote cell survival or cell death.
  • a preferred mammal is a human.
  • the contacting with compounds having formulas I-IV can take place in buffers or media, which are well known to those skilled in the art. Alternately, the contacting can take place by administration of a pharmaceutical composition containing the test compound and a pharmaceutically acceptable salt, carrier, or diluent to a suitable animal or mammal, such as, for example, a mouse or other suitable animal known to those skilled in the art. In addition, varying numbers of cells and concentrations of compounds can be used.
  • the cells which are contacted with the test compounds can be any mammalian cell. Preferably, the cell is a neuronal cell.
  • the cell is involved in a neurodegenerative disease, such as, for example, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and concussive or penetrating injuries to the brain or spinal cord.
  • a neurodegenerative disease such as, for example, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and concussive or penetrating injuries to the brain or spinal cord.
  • a neurodegenerative disease such as, for example, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and concussive or penetrating injuries to the brain or spinal cord.
  • Patents 5,741,8098, 5, 621,100, 5,621,101, 5,461,146, and 5,756,494, and WO 97/46567 each of which is inco ⁇ orated herein by reference in its entirety.
  • Compounds having formula IV, and methods of making the same, are described in U.S. Patents 5,741,8098, 5, 621,100, 5,621,101, 5,461,146, and 5,756,494, and WO 97/46567, each of which is inco ⁇ orated herein by reference in its entirety.
  • Compounds having formula II include diasteriomers and enantiomers around the carbon atoms to which the substituents R 2 , R 7 , and R 8 are attached.
  • Preferred bridged indenopyrrolocarbazoles are represented by formula II:
  • R 1 is H.
  • R 2 is H, hydroxyl, or substituted or unsubstituted alkyl.
  • R 3 , R 4 , R 5 , and R 6 are independently H, substituted or unsubstituted alkyl, halogen, substituted or unsubstituted alkoxy, substituted or unsubstituted amino, or substituted or unsubstituted aryl.
  • R 7 and R 8 are independently H, or substituted or unsubstituted alkyl.
  • Y is O.
  • Z is a bond,
  • n and n are independently 1 or 2.
  • Y is O
  • Z is a bond or O
  • m and n are independently 1 or 2.
  • Some especially preferred embodiments of the compounds of formula II are compounds 11-1, 11-2, II-3, II-4a, II-4b, II-5, II-6, II-7a, II-7b, II-8, II-9, 11-10, 11-11. and 11-12 set forth in Table 1, infra.
  • Carbocyclic refers to cyclic groups in which the ring portion is composed solely of carbon atoms.
  • heterocyclo and heterocyclic refer to cyclic groups in which the ring portion includes at least one heteroatom such as O, N, or S.
  • alkyl means a straight-chain, cyclic, or branched alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, hexyl, octyl, cyclopropyl, and cyclopentyl.
  • alkyl moiety of alkyl-containing groups such as alkoxy, alkoxycarbonyl, and alkylaminocarbonyl groups, has the same meaning as alkyl defined above.
  • Lower alkyl groups which are preferred, are alkyl groups as defined above which contain 1 to 4 carbons.
  • alkenyl is intended to include straight-chain or branched hydrocarbon chains having at least one carbon-carbon double bond.
  • alkenyl groups include ethenyl and propenyl groups.
  • alkynyl is intended to include straight-chain or branched hydrocarbon chains having at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl and propynyl groups.
  • acyl moiety of acyl-containing groups such as acyloxy groups is intended to include a straight-chain or branched alkanoyl group having 1 to 6 carbon atoms, such as formyl, acetyl, propanoyl, butyryl, valeryl, pivaloyl or hexanoyl.
  • aryl means a group having 6 to 12 carbon atoms such as phenyl, biphenyl and naphthyl. Preferred aryl groups include unsubstituted or substituted phenyl and naphthyl groups.
  • heteroaryl denotes an aryl group in which one or more ring carbon atom is replaced by a hetero (i.e., non-carbon) atom such as O, N or S.
  • Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, friazolyl, tetrazolyl, quinolyl, isoquinolyl, benzoimidazolyl, thiazolyl, pyrazolyl, and benzothiazolyl groups.
  • aralkyl (or “arylalkyl”) is intended to denotes a group having from 7 to 15 carbons, consisting of an alkyl group that bears an aryl group.
  • aralkyl groups include benzyl, phenethyl, benzhydryl and naphthylmethyl groups.
  • Alkyl groups and alkyl moieties contained within substituent groups such as aralkyl, alkoxy, arylalkoxy, hydroxyalkoxy, alkoxy-alkoxy, hydroxy-alkylthio, alkoxy-alkylthio, alkylcarbonyloxy, hydroxyalkyl and acyloxy groups may be substituted or unsubstituted.
  • a substituted alkyl group has 1 to 3 independently-selected substituents, preferably hydroxy, lower alkoxy, lower alkoxy-alkoxy, substituted or unsubstituted arylalkoxy-lower alkoxy, substituted or unsubstituted heteroarylalkoxy-lower alkoxy, substituted or unsubstituted arylalkoxy, substituted or unsubstituted heterocycloalkoxy, halogen, carboxyl, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, dioxolane, dioxane, dithiolane, dithione, furan, lactone, or lactam.
  • Substituted aryl, substituted heteroaryl and substituted aralkyl groups each have 1 to 3 independently-selected substituents that are preferably lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, and halogen.
  • Heterocyclic groups formed with a nitrogen atom include pyrrolidinyl, piperidinyl, piperidino, mo ⁇ holinyl, mo ⁇ holino, thiomo ⁇ holino, N-methylpiperazinyl, indolyl, isoindolyl, imidazole, imidazoline, oxazoline, oxazole, triazole, thiazoline, thiazole, pyrazole, pyrazolone, and triazole groups.
  • Heterocyclic groups formed with an oxygen atom includes furan, tetrahydrofuran, pyran, and tetrahydropyran groups.
  • Haldroxyalkyl groups are alkyl groups that have a hydroxyl group appended thereto. Halogens include fluorine, chlorine, bromine and iodine.
  • heteroarylalkyl means an arylaklyl group that contains a heteroatom.
  • oxy denotes the presence of an oxygen atom.
  • alkoxy are alkyl groups that are attached through an oxygen atom
  • carbonyloxy are carbonyl groups that are attached through an oxygen atom.
  • heterocycloalkoxy means an alkoxy group that has a heterocyclo group attached to the alkyl moiety thereof
  • arylalkoxy means an alkoxy group that has an aryl group attached to the alkyl moiety thereof.
  • alkyloxy-alkoxy denotes an alkoxy group that contains an alkyloxy substituent attached to its alkyl moiety.
  • alkoxy-alkylthio means an alkylthio group (i.e., a group of formula -S-alkyl) that contains an alkoxy substituent attached to its alkyl moiety.
  • hydroxy-alkylthio means an alkylthio group (i.e., a group of formula -S-alkyl) that contains a hydroxy substituent attached to its alkyl moiety.
  • the term “monosaccharide” has its accustomed meaning as a simple sugar.
  • amino acid denotes a molecule containing both an amino group and a carboxyl group.
  • amino acids include -amino acids; i.e., carboxylic acids of general formula HOOC-CH(NH 2 )-(side chain).
  • Side chains of amino acids include naturally occurring and non-naturally occurring moieties.
  • Non-naturally occurring (i.e., unnatural) amino acid side chains are moieties that are used in place of naturally occurring amino acid side chains in, for example, amino acid analogs. See, for example, Lehninger, Biochemistry, Second Edition, Worth Publishers, Inc, 1975, pages 73-75, inco ⁇ orated by reference herein.
  • Preferred ⁇ -amino acids include glycine, alanine, proline, glutamic acid, and lysine, having the D configuration, the L configuration, or as a racemate.
  • Functional groups present on the compounds of formula II may contain protecting groups.
  • the amino acid sidechain substituents ofthe compounds of formula II can be substituted with protecting groups such as benzyloxycarbonyl or t-butoxycarbonyl groups.
  • protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention.
  • One such protecting group is the benzyloxycarbonyl (Cbz; Z) group.
  • Other preferred protecting groups according to the invention may be found in Greene, T.W. and Wuts, P.G.M., "Protective
  • the bridged indenopyrrolocarbazole compounds have evidenced important functional pharmacological activities which find utility in a variety of settings, including both research and therapeutic arenas. These derivatives are useful as therapeutic agents.
  • the activities of the compounds show positive effects on the function and/or survival of trophic factor responsive cells. Effect on the function and/or survival of trophic factor responsive cells, e.g., cells of a neuronal lineage, has been demonstrated using any of the following assays: (1) cultured spinal cord choline acetylfransferase (“ChAT”) assay; or (2) cultured basal forebrain neuron ChAT activity assay.
  • ChAT cultured spinal cord choline acetylfransferase
  • effect when used to modify the terms "function" and
  • survival means a positive or negative alteration or change.
  • An effect which is positive can be referred to herein as an “enhancement” or “enhancing” and an effect which is negative can be referred to herein as “inhibition” or “inhibiting.”
  • the terms “enhance” or “enhancing” when used to modify the terms “function” or “survival” means that the presence of a bridged indenopyrrolocarbazole compound has a positive effect on the function and/or survival of a trophic factor responsive cell compared with a cell in the absence ofthe compound.
  • the compound would evidence enhancement of survival of a cholinergic neuronal population at risk of dying (due to, e.g., injury, a disease condition, a degenerative condition or natural progression) when compared to a cholinergic neuronal population not presented with such compound, if the treated population has a comparatively greater period of functionality than the non-treated population.
  • inhibitor and “inhibition” mean that a specified response of a designated material (e.g., enzymatic activity) is comparatively decreased hi the presence of a bridged indenopyrrolocarbazole compound.
  • tr ⁇ : refers to the family of high affinity neurotrophin receptors presently comprising trkA, trkB, and trkC, and other membrane associated proteins to which a neurotrophin can bind.
  • inhibition of VEGFR implies utility in, for example, diseases where angiogenesis plays important roles, such as cancer of solid tumors, endometriosis, diabetic retinopathy, psoriasis, hemangioblastoma, as well as other ocular diseases and cancers.
  • Inhibition of trk implies utility in, for example, diseases ofthe prostate such as prostate cancer and benign prostate hype ⁇ lasia, and treatment of inflammatory pain.
  • PDGFR Platelet Derived Growth Factor Receptor
  • cancer and “cancerous” refer to any malignant proliferation of cells in a mammal. Examples include prostate, benign prostate hype ⁇ lasia, ovarian, breast, brain, lung, pancreatic, colorectal, gastric, stomach, solid tumors, head and neck, neuroblastoma, renal cell carcinoma, lymphoma, leukemia, other recognized malignancies of the hematopoietic systems, and other recognized cancers.
  • neuron As used herein the terms “neuron,” “cell of neuronal lineage” and “neuronal cell” include, but are not limited to, a heterogeneous population of neuronal types having singular or multiple transmitters and/or singular or multiple functions; preferably, these are cholinergic and sensory neurons.
  • cholinergic neuron means neurons ofthe Central Nervous System (CNS) and Peripheral Nervous System (PNS) whose neurotransmitter is acetylcholine; exemplary are basal forebrain, striatal, and spinal cord neurons.
  • nerve neuron includes neurons responsive to environmental cues (e.g., temperature, movement) from, e.g., skin, muscle and joints; exemplary is a neuron from the dorsal root ganglion.
  • a “trophic factor-responsive cell,” as defined herein, is a cell which includes a receptor to which a trophic factor can specifically bind; examples include neurons (e.g., cholinergic and sensory neurons) and non-neuronal cells (e.g., monocytes and neoplastic cells).
  • neurons e.g., cholinergic and sensory neurons
  • non-neuronal cells e.g., monocytes and neoplastic cells.
  • the bridged indenopyrrolocarbazole compounds described herein find utility in both research and therapeutic settings in, for example, inhibition of enzymatic activity.
  • the compounds can be used in the development of assays and models for further enhancement of the understanding of the roles that inhibition of serine/threonine or tyrosine protein kinase (e.g., PKC, trk tyrosine kinase) play in the mechanistic aspects of the associated disorders and diseases.
  • tyrosine protein kinase e.g., PKC, trk tyrosine kinase
  • the compounds which inhibit these enzymatic activities can be used to inhibit the deleterious consequences of these enzymes with respect to disorders such as cancer.
  • inhibition of enzymatic activity using the bridged indenopyrrolocarbazole compounds can be determined using, for example, the following assays: 1. trkA Tyrosine Kinase Activity inhibition assay;
  • Vascular Endothelial Growth Factor Receptor fVEGFR Vascular Endothelial Growth Factor Receptor fVEGFR
  • the disclosed bridged indenopyrrolocarbazole compounds can be used to enhance the function and/or survival of cells of neuronal lineage in a mammal, e.g., a human.
  • the compounds can be utilized individually or with other fused pyrrolocarbazoles and/or indolocarbazoles, or in combination with other beneficial molecules which also evidence the ability to effect the function and/or survival of a designated cell.
  • a variety of neurological disorders are characterized by neuronal cells which are dying, injured, functionally compromised, undergoing axonal degeneration, at risk of dying, etc. These disorders include, but are not limited to: Alzheimer's disease; motor neuron disorders (e.g. amyotrophic lateral sclerosis); Parkinson's disease; cerebrovascular disorders (e.g., stroke, ischaemia); Huntington's disease; AIDS dementia; epilepsy; multiple sclerosis; peripheral neuropathies (e.g., those affecting DRG neurons in chemotherapy-associated peripheral neuropathy) including diabetic neuropathy and AIDS peripheral neuropathy; disorders induced by excitatory amino acids; and disorders associated with concussive or penetrating injuries ofthe brain or spinal cord.
  • motor neuron disorders e.g. amyotrophic lateral sclerosis
  • Parkinson's disease cerebrovascular disorders (e.g., stroke, ischaemia); Huntington's disease
  • AIDS dementia e.g., epilepsy
  • multiple sclerosis e.g
  • ChAT catalyzes the synthesis ofthe neurotransmitter acetylcholine, and it is considered an enzymatic marker for a functional cholinergic neuron.
  • a functional neuron is also capable of survival. Neuron survival is assayed by quantitation ofthe specific uptake and enzymatic conversion of a dye (e.g., calcein AM) by living neurons.
  • a dye e.g., calcein AM
  • the compounds described herein find utility in a variety of settings.
  • the compounds can be used in the development of in vitro models of neuronal cell survival, function, identification, or for the screening of other synthetic compounds which have activities similar to that ofthe compounds described herein, or compounds identified by the methods described herein.
  • the compounds described herein, as well as those identified using the methods described herein, can be utilized in a research environment to investigate, define and determine molecular targets associated with functional responses.
  • the target entity to which the derivative binds can be identified, isolated, and purified for characterization.
  • the compounds, those described herein as well as those identified by using the methods described herein, are useful, inter alia, not only for enhancing trophic factor-induced activities of trophic responsive cells, e.g., cholinergic neurons, but also may function as survival promoting agents for other neuronal cell types, e.g., dopaminergic or glutamatergic.
  • Growth factor may regulate survival of neurons by signaling cascades downstream of the small GTP binding proteins that include, but are not limited to, ras, rac, and cdc42 (Denhardt,
  • ERK extracellular receptor-activated kinase
  • Stimulation of rac/cdc42 leads to an increase in activation of JNK and p38, responses that are associated with stress, apoptosis, and inflammation.
  • growth factor responses are primarily via the ERK pathway, affecting these latter processes may lead to alternative mechanisms of neuronal survival which may mimic growth factor enhancing survival properties (Xia et al, Science, 1995, 270, 1326).
  • the compounds may also function as survival promoting agents for neuronal and non- neuronal cells by mechanisms related to, but also distinct from, growth factor mediated survival, for example, inhibition ofthe JNK and p38 pathways which may lead to survival by inhibition of apoptotic cell death processes.
  • the present compounds are useful in the treatment of disorders associated with decreased ChAT activity or the death, injury to spinal cord motoneurons, and also have utility in, for example, diseases associated with apoptotic cell death of the central and peripheral nervous system, immune system and in inflammatory diseases.
  • the compounds described herein may also find utility in the treatment of disease states involving malignant cell proliferation, such as many cancers.
  • the pharmaceutically acceptable salts ofthe compounds described herein, as well as those compounds identified by the present methods include pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts.
  • the acid addition salts are inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as acetate, maleate, fumarate, tartrate, citrate and lactate
  • examples ofthe metal salts are alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt
  • examples of the ammonium salts are ammonium salt and tetramethylammonium salt
  • examples ofthe organic amine addition salts are salts with mo ⁇ holine and piperidine
  • examples ofthe amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine.
  • compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers.
  • Such compositions can be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, trans-dermal patches.
  • the composition can be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980).
  • Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like.
  • polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release ofthe active compounds.
  • Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration.
  • Formulations for trans-dermal patches are preferably lipophilic emulsions.
  • the compounds of this invention can be employed as the sole active agent in a pharmaceutical composition. Alternatively, they can be used in combination with other active ingredients, e.g., other growth factors which facilitate neuronal survival or axonal regeneration in diseases or disorders.
  • the compounds ofthe invention and pharmaceutically acceptable salts thereof can be administered orally or non-orally, e.g., as an ointment or an injection.
  • concentrations of the compounds of this invention in a therapeutic composition can vary. The concentration will depend upon factors such as the total dosage ofthe drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, the route of administration, the age, body weight and symptoms of a patient, etc..
  • the compounds of this invention typically are provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration.
  • Typical dose ranges are from about 1 ⁇ g/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day, and preferably about 0.1 to 20 mg/kg once to four times per day.
  • a preferred dosage of drug to be administered is likely to depend on variables such as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, and formulation ofthe compound excipient, and its route of administration.
  • the compounds ofthe invention including test compound and compounds identified by the methods ofthe present invention, and pharmaceutically acceptable salts thereof can be administered alone, or in the form of various pharmaceutical compositions, according to the pharmacological activity andthepu ⁇ ose of administration.
  • the pharmaceutical compositions in accordance with the present invention can be prepared by uniformly mixing an effective amount of a compound or a pharmaceutically acceptable salt thereof, as an active ingredient, with a pharmaceutically acceptable carrier.
  • the carrier may take a wide range of forms according to the forms of composition suitable for administration. It is desired that such pharmaceutical compositions are prepared in a unit dose form suitable for oral or non-oral administration.
  • the forms for non-oral administration include ointment and injection.
  • Tablets can be prepared using excipients such as lactose, glucose, sucrose, mannitol and methyl cellulose, disintegrating agents such as starch, sodium alginate, calcium carboxymethyl cellulose and crystalline cellulose, lubricants such as magnesium stearate and talc, binders such as gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose and methyl cellulose, surfactants such as sucrose fatty acid ester and sorbitol fatty acid ester, and the like in a conventional manner. It is preferred that each tablet contains 15- 300 mg ofthe active ingredient.
  • disintegrating agents such as starch, sodium alginate, calcium carboxymethyl cellulose and crystalline cellulose
  • lubricants such as magnesium stearate and talc
  • binders such as gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose and methyl cellulose
  • surfactants such as sucrose fatty
  • Granules can be prepared using excipients such as lactose and sucrose, disintegrating agents such as starch, binders such as gelatin, and the like in a conventional manner. Powders can be prepared using excipients such as lactose and mannitol, and the like in a conventional manner.
  • Capsules can be prepared using gelatin, water, sucrose, gum arabic, sorbitol, glycerin, crystalline cellulose, magnesium stearate, talc, and the like in a conventional manner. It is preferred that each capsule contains 15-300 mg ofthe active ingredient.
  • Syrup preparations can be prepared using sugars such as sucrose, water, ethanol, and the like in a conventional manner.
  • Ointment can be prepared using ointment bases such as vaseline, liquid paraffin, lanolin and macrogol, emulsifiers such as sodium lauryl lactate, benzalkonium chloride, sorbitan mono-fatty acid ester, sodium carboxymethyl cellulose and gum arabic, and the like in a conventional manner.
  • ointment bases such as vaseline, liquid paraffin, lanolin and macrogol
  • emulsifiers such as sodium lauryl lactate, benzalkonium chloride, sorbitan mono-fatty acid ester, sodium carboxymethyl cellulose and gum arabic, and the like in a conventional manner.
  • Injectable preparations can be prepared using solvents such as water, physiological saline, vegetable oils (e.g., olive oil and peanut oil), ethyl oleate and propylene glycol, solubilizing agents such as sodium benzoate, sodium salicylate and urethane, isotonicity agents such as sodium chloride and glucose, preservatives such as phenol, cresol, p- hydroxybenzoic ester and chlorobutanol, antioxidants such as ascorbic acid and sodium pyrosulfite, and the like in a conventional manner.
  • solvents such as water, physiological saline, vegetable oils (e.g., olive oil and peanut oil), ethyl oleate and propylene glycol, solubilizing agents such as sodium benzoate, sodium salicylate and urethane, isotonicity agents such as sodium chloride and glucose, preservatives such as phenol, cresol, p- hydroxybenzoic ester and chlorobutanol,
  • the lactam nitrogen protection strategy (shown in Figures 3 and 4) can be carried out by either an acid or a base-catalyzed process.
  • the acid-catalyzed reaction can be carried out with a resin-bound reagent allowing immobilization ofthe indenopyrrolocarbazole (III)/(VIII) to a polymeric support, such as a polystyrene-based, Rink acid resin (XII) ( Figure 3), providing (XIII).
  • the acid-catalyzed reaction can be carried out with a soluble reagent to yield a compound (XIV) ( Figure 4).
  • the silyl-protected compound (XV) is produced under base catalysis ( Figure 4).
  • Figure 5 describes several methods for preparing intermediate (V).
  • R 3 , R 4 , R 5 and R 6 can be carried out as described in US Patents Nos. 5,705,511 and 4,923,986, the disclosures of which are inco ⁇ orated by reference in their entirety.
  • An R 3 substituent can otherwise be introduced after the construction of the bridged indenopyrrolocarbazoles, as shown in Figure 8.
  • the 3 position of the B ring is brominated with NBS providing compound (XXXI).
  • a carbon fragment is subsequently introduced by employing palladium-catalyzed Stille, Suzuki, Heck, Kumada or Castro-
  • compound (XXXI) can provide access to compounds where the bromine group is displaced with a heteroatom, e.g. an amine-based group by utilization of Buchwald's palladium catalyzed animation chemistry.
  • a heteroatom e.g. an amine-based group
  • Buchwald's palladium catalyzed animation chemistry By an oxidative process, an oxygen linked group can be introduced at the indene carbon ofthe E ring, as shown in Figure 9, compound (XXXIV).
  • This chemistry also results in oxidation ofthe methylene group ofthe lactam (A ring) providing an imide derivative, as shown.
  • Example 3 Preparation of Compound (II-l), Compound (II-2), Compound (II-3), Compound (II-4a),-Compound (II-4b), Compound (II-6) and Compound (II-8) (Method A, Figure 6)
  • Example 3-B In a similar manner, as described above for compound II-l, resin (XIII-A) (70.3 mg) was treated with 1 , 1 -diethoxy-2-pentanone (0.75 mL) ) (which was prepared according to the literature procedure of Sworin, et al. , J. Org.
  • Example 7 Preparation of Compound II-l via Method B ( Figure 7)
  • a solution of Triton B in pyridine (0.45 M) was prepared by dissolving a 40% solution of Triton B in methanol (10 mL) in pyridine (10 mL). Solvent was removed under reduced pressure (20 mm Hg) to a final volume of ⁇ 8 mL. The residue was diluted with pyridine to 50 mL, filtered and stored under nitrogen.
  • a solution of XV (20.3 mg) in pyridine (2.0 mL) was flushed with argon and treated with 300 ⁇ L of Triton B (0.45 M in pyridine) and diethoxybutyraldehyde (50 ⁇ L).
  • resin (XHIa) (150.2 mg) was reacted with EtMgBr (1.0 mL) followed by ethyl 2,5-dioxopentanoate (Schmidt, et al. , Synthesis, 1993, 809) (1.5 mL). After workup and cleavage from the resin, the crude reaction product mixture was taken up in methylene chloride (20 mL) and treated with BF 3 etherate (20 ⁇ L). After stirring for 2.5 hours, the solution was washed with saturated aqueous NaHCO 3 and brine prior to drying over
  • Example 12 Enhancement of Spinal Cord ChAT Activity
  • Cholinergic neurons represent a major cholinergic input into the hippocampal formation, olfactory nucleus, inte ⁇ eduncular nucleus, cortex, amygdala, and parts ofthe thalamus.
  • the motoneurons are cholinergic neurons which contain ChAT (Phelps, et al. , J. Comp. Neurol, 1988, 273, 459-472).
  • ChAT activity has been used to study the effects of neurotrophins (e.g., NGF or NT-3) on the survival and/or function of cholinergic neurons.
  • neurotrophins e.g., NGF or NT-3
  • the ChAT assay also serves as an indication of the regulation of ChAT levels within cholinergic neurons.
  • Fetal rat spinal cord cells were dissociated, and experiments were performed as described (Smith, et al, J. Cell Biology, 1985, 101, 1608-1621; Glicksman, et al, J. Neurochem. , 1993, 61, 210-221). Dissociated cells were prepared from spinal cords dissected from rats (embryonic day 14-15) by standard trypsin dissociation tecliniques (Smith et al., supra.). Cells were plated at 6 x 10$ cells/cm ⁇ on poly-1-omithine coated plastic tissue culture wells in serum-free N2 medium supplemented with 0.05% bovine serum albumin (BSA) (Bottenstein, et al, Proc. Natl. Acad.
  • BSA bovine serum albumin
  • MLK3 was cloned as described (Lee, et al., Oncogene, 1993, 8, 3403-3410; Ezoe, et al, Oncogene, 1994, 9, 935-938).
  • cDNA was prepared from 200 ng polyadenylated melanocyte mRNA and 5% ofthe reaction was used as template to amplify a repertoire of PTK cDNAs using mixtures of either two or four highly degenerate oligonucleotide primers derived from the consensus sequences ofthe conserved VIb and IX subdomains of known PTKs: PTKl, 5'-CGGATCCACMGIGAYYT-3' (SEQ ID NO:l); PTK2, 5'- GGAATTCCAWAGGACCASACRTC-3 ' (SEQ ID NO:2); PTK3, 5 '- CGGATCCRTICAYMGIGAYYTIGCIGCIMGIAA-3' (SEQ ID NO:3); PTK4, 5'- GGAATTIAY
  • part ofthe PCR amplified cDNA was not cleaved, but instead was cloned blunt into M13mpl8 cleaved with Smal .
  • Nucleotide sequences were determined by chain-termination sequencing method.
  • PTKl One cDNA, identified as PTKl, was used as a probe to screen human melanoma and melanocyte cDNA libraries.
  • This cDNA was cut with Ncol, blunted with DNA polymerase (Klenow), cut again with EcoRl and ligated into the vector pCDNA3-EE cut with BamHl, blunted and then cut with EcoRl.
  • the vector pCDNA3-EE was constructed by inserting into the Hindlll/BamHl site an oligo that codes for a start codon followed by the EE epitope, MEEEEYMPME (SEQ ID NO:5) (Grassenmeyer, et al, Proc. Natl. Acad. Sci. USA, 1985, 82, 7952-7954).
  • the kinase-dead version of MLK3 was made by making the mutation K144R using PCR employing a previously published technique (Chen, et al, Biotechniques, 1994, 77, 657-659).
  • the first, mutagenic oligo was 5'- GTGGCTGTGCGGGCAGCTCGCCAG-3' (SEQ ID NO:6) and the second oligo was 5'- GAGACCCTGGATCTCGCGCTT-3' (SEQ ID NO:7).
  • MLK3 as a template, these oligos were used in PCR to generate a fragment of 806 bp and employed in a second PCR reaction using a T7 primer as the other amplimer and MLK3 as the template to generate a fragment of 1285 bp.
  • the fragment was separated by agarose gel electrophoresis, isolated, cloned into pGEM-5 (Promega) and sequenced.
  • the fragment was excised with Hindlll and Hpal, and inserted into pCDNA3-EE-MLK3 cut with Hindlll and Hpal. An additional point mutation was detected at nucleotide 1342. To correct this, a PflMl fragment (nt 1093-1418) was excised from the wild-type MLK3 and used to replace the identical fragment in the K144R mutated MLK3.
  • MLK3 protein was cloned into the baculo viral expression vector pFB-FL AG.
  • MLK3 was excised from PTKl -3.2 by digestion with Nco 1 , blunted with DNA polymerase (Klenow), cut again with Notl and ligated into pFB-FLAG digested with Stul and Notl.
  • pFB-FLAG is derived from pFB (Life Teclmologies) and has the coding sequence for the FLAG epitope (Hopp, et al, Biotechnology, 1988, 6, 1205-1210) with a start codon, MDYKDDDDK (SEQ ID NO: 8), added to the polylinker in the BamHl site.
  • Baculoviral expression ofthe kinase domain of MLK3 was achieved by excising the MLK3 fragment from the pGEXKG-MLK3(KD) using EcoRl and Xhol and ligating it into a pFB vector cut with EcoRl and Xhol in which the coding sequence for glutathione S- transferase (GST) had been cloned upstream. This was achieved by obtaining the GST coding sequence and polylinker from the pGEXKG vector by PCR using the vector as a template
  • Example 16 pGEXKG-MLK3(KD) A cDNA fragment that included both the MLK3 kinase domain and a portion ofthe leucine zipper (nt 736-1791) was obtained by PCR using the PTKl cDNA. The isolated fragment was digested with the restriction enzymes EcoR 1 and Xho 1 , sites that were included in the PCR oligos, and cloned into pGEX-KG digested with EcoRl and Xho 1. This fragment in pGEX-KG was then shortened by PCR to include only the kinase domain (nt 736-1638).
  • MLK2 was cloned using degenerate PCR (Dorow, et al. , Eur. J. Biochem. , 1993, 73, 701-710; Dorow, etal, Eur. J. Biochem., 1995, 234, 492-500).
  • Segments of cDNAs encoding catalytic subdomains of protein kinases expressed in the epithelial tumor cell line Colo 16 were amplified from RNAby reverse transcriptase PCR.
  • Degenerate PCR primers were based in sequences encoding conserved motifs in subdomains Vib and VIII ofthe epidermal-growth- factor receptor family kinase catalytic domains.
  • Sequences ofthe primers were as follows: forward primer, 5'-CGGATCCGTG(A)CACC(A)GT(CG)G(A)ACC(T)T-3' (SEQ ID NO:9), reverseprimer, 5'-GGAATTCACCA(G)TAA(G)CTCCAG(C)ACATC-3' (SEQIDNO:10).
  • Several PCR products were cloned into Ml 3 and sequenced using a T7 Super-Base sequencing kit (Bresatec).
  • One 216-bp PCR product was used as a probe to screen a human colon ⁇ gtl 1 cDNA library (Clontech, catalog #HL 10346)).
  • the fragment was random-primed labeled, hybridization was performed at 65°C and the filters washed to a stringency of 0.2X NaCl/Citrate (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.5% SDS at
  • MLK2 was cloned, from the ATG to the polyA tail, into the vector pKH3 between the BamHl and EcoRl sites in two steps as there is a BamHl site in the middle of the MLK2 sequence.
  • the vector pKH3 was constructed by inserting three copies ofthe HA epitope tag followed by a BamHl site between the Xbal and EcoRl sites ofthe pRK7 polylinker.
  • K125A the MLK2 5' BamHl fragment was cloned into the Promega p Alter vector and mutated as recommended by the manufacturer. The fragment was then cloned back into the MLK2 pKH3 vector.
  • Example 18 pcDNA3-HA-JNKl JNK1 cDNA was obtained as described (Coso, et al. , Cell, 1995, 57,1137-1146).
  • the cDNA was obtained by PCR using as a template human skeletal muscle cDNA (Invitrogen) and was cloned into the Bgl2 / Sail sites of pcDNA3-HA, a modified pcDNA3 expression plasmid encoding the HA epitope (Wilson, ⁇ t al, Cell, 1984, 37, 767-778). This was then excised from pcDNA3, including the HA epitope, and ligated into pGEX-4T3 (Pharmacia).
  • the JNK1 cDNA was excised from the pGEX-4T3 construct as a Bgl2 / Sail fragment and ligated into pcDNA3-HA, a vector with the HA epitope added in the HinD3/BamHl site of pcDNA3.
  • DLK was cloned into the expression vector pcDNA3 with the FLAG epitope added as described (Holzman, etal , J Biol. Chem., 1994, 269, 30808-30817).
  • a fragment ofthe cDNA for DLK was isolated by degenerate oligonucleotide-based PCR cloning.
  • Total RNA was extracted from embryonic day 13.5 kidneys (32 organs) and embryonic day 17.5 kidneys (16 organs) using a commercially prepared phenol/guanidine isothiocyanate reagent method according to the directions of the manufacturer (TRIzol Reagent, Life Technologies, Inc.).
  • RNA was reverse transcribed with RNase H-reverse transcriptase (Superscript, Life Technologies, Inc.) from an oligo(dT) synthetic oligonucleotide primer to single-stranded cDNA.
  • RNase H-reverse transcriptase Superscript, Life Technologies, Inc.
  • oligonucleotide primers corresponding to the protein tyrosine kinase catalytic subdomains Vib and IX originally designed by Wilks (Wilks, Proc Natl Acad Sci USA. , 1989, 86, 1603-1607) were modified to
  • the resultant 200-210-bp DNA amplification product was gel isolated, subcloned into a prepared pG ⁇ M7zf(+) plasmid (Promega), and transformed into Escherichia coli.
  • Miniprep plasmid DNA was prepared from transformed bacteria and a portion digested with EcoRl and Hindlll restriction endonucleases; clones containing inserts were sequenced.
  • the 195-bp DLK cDNA fragment obtained from the degenerate PCR was radiolabeled and used to screen approximately 1 x 10 6 recombinants of a Uni-ZAP II (Stratagene, La Jolla, CA), oligo(dT)-primed adult mouse brain cDNA library (Holzman, et al. , Mol Cell Biol, 1990, 10, 5830-5838). Filters were hybridized in abuffer consisting of 50% formamide, 5 x SSC, 3 x Denhardt's solution, 0.25% SDS, 1 mg/ml polyadenylic acid, and 200 mg/ml salmon sperm DNA at 42°C.
  • the full-length Notl-Xhol DLK cDNA fragment (3401 bp) was subcloned into the cytomegalovirus promoter based eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA) (construct designated pcDNA3-DLK).
  • pcDNA3-DLK cytomegalovirus promoter based eukaryotic expression vector
  • pcDNA3-DLK construct designated pcDNA3-DLK
  • a NH 4 -Met FLAG epitope (DYKDDDDK) (SEQ ID NO: 13) tagged constract (pFLAG-DLK) was made.
  • the PCR was used to amplify cDNA fragments which encoded a 5 ' Hindlll site, DLK' s Kozak' s consensus sequence including the initiation ATG, the FLAG epitope, and DLK cDNA open reading frame sequence extending from nucleotide 88 to an internal EcoRl site at nucleotide 758.
  • Example 20 pcDNA3-MLKl The 5' portion of MLK1 was obtained from the EST database (accession #
  • This clone was a fusion between MLK1 and another cDNA of unknown identity. It contained previously unpublished 5' sequence of MLK1 along with part ofthe previously published kinase domain of MLK1 (Dorow, etal. , Eur. J. Biochem., 1993, 213, 701 -710).
  • the MLK1 cDNA sequence from the EST clone is as follows : G A A T T C G G C A C G A G A G G A C T C G C A G G T G T C C G G C GA C G A G
  • the 3' portion of MLK1 was initially cloned by degenerate PCR as previously published (Dorow, etal, Eur. J. Biochem., 1993, 213, 701-710).
  • the protocol for cloning the 3 ' portion of MLK1 was as described above for MLK2 with the following exceptions.
  • MLK1 MLK1
  • Phage from 1 ⁇ l aliquots of amplified libraries (normal human colonic epithelia and human T84 colonic carcinoma cell line cDNA in ⁇ Uni-ZAPXR (Stratagene, cat #937204) were lysed by suspending in 20 ⁇ l water and snap freezing. A 5 ⁇ l sample ofthe lysed phage was used as a PCR template in two reactions for each library. Primers representing the vectors were taken from nucleotide sequences flanking the cloning sites. In the case of the T84 colonic cell line library, the T3 and T7 sequencing primers (Promega) were used.
  • PCR reactions contained IX PCR buffer, 2.5 mM magnesium chloride, 1U Taq polymerase (all from Bresatec), 0.2 mM dNTP and 0.4 mM each primer in a total of 50 ⁇ L. Reaction conditions were 60s at 95°C, 90s at 52°C, 90s at 72°C for 30 cycles with a 15 min extension time in the final cycle. PCR products were cloned and sequenced as a described above. The longest clone from the library screen and a PCR fragment that included additional MLK1 sequence were ligated together to create a 1.08 kb MLK1 cDNA in pUC18.
  • the MLK1 clone from the EST database was provided in the vector pBluescript (Stratagene).
  • the MLK1 cDNA from the colonic library was ligated into the EST clone by digestion ofthe former with EcoRl, blunted with Klenow, then cut with Aflll.
  • This isolated fragment was cloned into the MLK1 cDNA from the EST database cut with Xhol, blunted with Klenow, and cut with Aflll.
  • This new constract was then excised from pBluescript by digestion with Notl and Apal and ligated into pcDNA3-EE also cut with Notl and Apal . All cloning junctions were sequence verified.
  • Example 21 E.coli expression of GST-ML ⁇ pGEXKG-MLK3(KD) was transformed into E. coli strain BL21 by electroporation. Bacteria containing the plasmid were inoculated into a 15 liter Applikon fennenter in 10 liter volume ofthe following rich medium: 1.95 g/L K 2 HPO 4 , 0.9 g/L KH 2 PO 4 , 0.1 g/L ampicillin, 0.3 g/L (NH 4 ) 2 SO 4 , 0.92 g/L MgSO 4 7H 2 0, 42.7 mg/L Na citrate, 21.8 mg/L FeSO 4 7H 2 0, 0.5 mL Pichia trace metals (Higgins, et al , Methods Molecular Biology, 1998, 103, 149- 177), 20 g/L casamino acids, 40 g/L glycerol, 25.5 mg/L CaCl 2 .
  • Example 22 Purification of bacterial GST-MLK3 KD Partially-purified GST-ML ⁇ was prepared by sonicating 100 gm of bacterial cell paste in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol (DTT), pH 7.5 (buffer A). The solution was made 1% with Triton X-100, then stirred on ice for 1 hr. Supernatant solution after centrifugation for 45 min at 20,000 x g was mixed for 1 hr on ice with 10 mL glutathione Sepharose 4B resin (Pharmacia) equilibrated in buffer A. Pelleted resin was washed twice with 12.5x volume buffer A, then eluted with 20 mL 100 mM Tris-
  • Example 23 Baculoviral expression of FLAG-MLK3 and GST-MLK3 KD
  • Recombinant baculoviruses expressing the FLAG-MLK3 and GST-ML ⁇ were produced from their respective transfer vectors, pFB-FLAG-MLK3 and pFE-GST- L ⁇ using the BAC-TO-BAC system (Life Technologies) according to the instruction manual.
  • Suspension cultures of Sf21 cells were grown at 27°C/120 rpm in supplemented Grace's medium (Hink, Nature, 1970, 226, 466-467) with 10% heat-inactivated fetal bovine serum (FBS).
  • FBS heat-inactivated fetal bovine serum
  • Sf21 cells at a density of 1.5 x 10 6 cells/mL supplemented Grace's medium containing 5% FBS were infected with a multiplicity of infection (MOI) of 3.1 and harvested at 39 hr after infection.
  • MOI multiplicity of infection
  • GST-M EG KD Sf21 cells at a density of 1.5 x 10 6 cells/mL supplemented Grace's medium containing 5% FBS were infected with an MOI of
  • pelleted cells were resuspended in buffer comprised of 10 mM HEPES, 50 mMNaCl, 0.5 mM Pefabloc SC, 5 ⁇ M pepstatin, 10 ⁇ g/mL aprotinin, 10 ⁇ g/mL leupeptin, pH 7.4.
  • buffer comprised of 10 mM HEPES, 50 mMNaCl, 0.5 mM Pefabloc SC, 5 ⁇ M pepstatin, 10 ⁇ g/mL aprotinin, 10 ⁇ g/mL leupeptin, pH 7.4.
  • Supernatant solution after centrifugation for 1 hr at 147,000 xg was readjusted to pH 7.4 with 3 M Tris base and then stored at -70°C prior to purification.
  • Partially-purified baculoviral GST-ML ⁇ was prepared by glutathione affinity chromatography. For 10 mL of cell extract (26.6 mg total protein), 1 mL of glutathione Sepharose 4B resin (Pharmacia) equilibrated in 10 mM HEPES, 150 mMNaCl, pH 7.4 (buffer C) was added and protein was allowed to bind for 45 min at 4°C. Resin was then washed in column format with 30 column volumes of buffer C, then eluted with 5 column volumes of buffer C containing 20 mM glutathione. Pooled final product was dialyzed overnight against buffer C and stored in aliquots at -70°C.
  • Example 25 Purification of baculoviral FLAG-MLK3 Partially-purified baculoviral FLAG-MLK3 was prepared by antibody affinity chromatography. Protein from 15 mL of extract (19.5 mg total protein) with an additional 0.1M NaCl was bound onto a 0.25 mL column of M2 monoclonal FLAG peptide antibody coupled to agarose resin (Sigma) by repeated loading (three times total). Resin had been equilibrated with a 5 column volume wash of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS), a 3 column volume wash of 0.1M glycine, pH 3.5, followed by another 5 column volume wash with TBS, prior to chromatography.
  • Recombinant protein was primarily eluted by 5 column volumes of 0.2 mM FLAG peptide (N-Asp-Tyr-Lys-Asp-Asp-Asp-Lys-C) (SEQ ID NO:18) in TBS. Protein was stored in aliquots at -80°C prior to assay.
  • Dominant Negative Mutant A dominant negative mutant of the MLK family blocks death in differentiated PC12 cells following removal of Nerve Growth Factor
  • the PC- 12 cell line derived from a rat pheochromocytoma tumor has been used extensively as a neuronal cell model for examining the molecular events leading to neuronal death (for review, see Troy, et al, Adv. Neurology, 1997, 103-111).
  • Nerve Growth Factor induces PC- 12 cells to differentiate into a sympathetic neuronal phenotype (Greene, Cell Biol, 1978, 78, 747-755).
  • NGF differentiated PC-12 cells are dependent on NGF for survival and undergo a mo ⁇ hol ⁇ gically described apoptotic death upon removal of NGF from the culture medium.
  • a cell system was developed to determine the effect of members ofthe mixed lineage kinase family on PC-12 cell death following NGF withdrawal.
  • PC-12 cells were transfected with cDNA coding for a dominant negative (DN) mutant of MLK-3 using Pfx lipid transfer system as recommended by the manufacturer (Invitrogen, Carlsbad, CA).
  • DN dominant negative
  • a stable pool of transfectant expressing DN-MLK-3 was selected using G418 sulfate (Mediatech Inc., Hemdon, VA). Approximately 30% of cells in these pools express DN
  • MLK3 as determined by immunohistochemistry. Pools of cells stably expressing the mutant kinase were plated on polyornithine/laminin (10 ug/ml each in phosphate buffered saline) coated tissue culture 96-well format plates at a density of 2 x 10 4 cells/well and treated with 100 ng/ml of NGF for 7 days. Medium containing the NGF was removed, the cell monolayer washed with phosphate buffered saline and medium containing neutralizing NGF antibody
  • Example 27 Assay for enzymatic activity of recombinant MLK protein
  • the MLK protein expressed in either the baculovirus or bacterial expression system is enzymatically active, several assay formats may be utilized.
  • the MLK protein may be a full-length constract or a kinase domain expressed in either a baculovirus or bacterial expression system.
  • the assay may be antibody-based such as enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence (TRF), or fluorescence polarization (FP) .
  • ELISA enzyme-linked immunosorbent assay
  • TRF time-resolved fluorescence
  • FP fluorescence polarization
  • the antibody may be monoclonal or polyclonal with reactivity towards phosphoserine, phosphothreonine, or phospho-specific substrate.
  • anon- antibody-based method may be used such as radioactive gel-based assay (see Figure 11), multiscreen trichloroacetic acid (TCA) precipitation assay (Figure 13), scintillation proximity assay (SPA), flashplate method, or phosphocellulose filter assay format ( Figure 13).
  • the assay may be designed to monitor direct phosphorylation of a substrate or a coupled assay system utilizing the downstream kinases in the signaling pathway.
  • the substrate may be a specific substrate such as SEK-1 or a relatively non-specific substrate such as myelin basic protein (MBP).
  • the kinase activity of MLK-3 was assayed by monitoring the inco ⁇ oration of 32 P from [ ⁇ - 32 P]-ATP into a substrate of MLK (e.g. kinase-dead SEK- 1 ; myelin basic protein).
  • a substrate of MLK e.g. kinase-dead SEK- 1 ; myelin basic protein.
  • the 50- ⁇ l assay mixture contained Buffer A (20 mM MOPS, pH 7.2, 25 mM ⁇ -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol), 15 mM MgCl 2 , 100 ⁇ M ATP, 10 ⁇ Ci [ ⁇ - 32 P]-ATP, and 0.1 ⁇ g kinase-dead SEK-1 substrate (Stressgen, Inc; bound glutathione S-transferase-SEK-1 (GST-SEK-1) was released from glutathione-agarose beads with 10 mM glutathione, pH 8.0) or 25 ⁇ g MBP (Sigma Chemical Co.).
  • Buffer A (20 mM MOPS, pH 7.2, 25 mM ⁇ -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol), 15
  • Reaction was initiated by adding MLK protein (kinase domain or preparation containing both full-length and kinase domain) or control protein. The mixture was incubated for 30 min at 30°C. At the end ofthe reaction 2x reducing sample buffer was added. The mixture was boiled for 5 min, loaded onto either a 12% SDS-PAGE gel (using MBP as substrate) or 8% gel (SEK-1 as substrate). After electrophoresis, the gel was dried. Quantitation of phosphate inco ⁇ oration into substrate, SEK- 1 , was performed on a Molecular Dynamics Phosphorimager (Sunnyvale, CA).
  • kinase activity of bacterially-expressed GST-MLK-3 kinase domain was assessed using the Millipore Multiscreen trichloroacetic (TCA) "in-plate” assay as described by Pitt, etal,J. Biomol Screening, 1996, 7, 47-51). Assays were performed in 96-well Multiscreen
  • Each 50- ⁇ l assay mixture contained 20 mM Hepes, pH 7.4, 20 mM MgCl 2 , 20 mM MnCl 2 , 2 mM DTT, 0.1 mMNa 3 VO 4 , 1 ⁇ Ci [ ⁇ -P 32 ] ATP and 30 ⁇ g MBP substrate.
  • the reaction was initiated by adding MLK protein and allowed to proceed for 15 min at 37°C. The reaction was stopped with 25 ⁇ l of 50% TCA. The plates were allowed to equilibrate for 30 min. at 4°C, then washed with ice cold 25% TCA. Scintillation cocktail was added to the plates, and the radioactivity was determined using Wallac MicroBeta 1450 PLUS scintillation counter.
  • the protein dose response versus formation of 32 P-labeled MBP is shown in Figure 13.
  • the kinase assay was performed in a 50- ⁇ l reaction mixture containing 20 mM Hepes, pH 7.4, 20 mM MgCl 2 , 20 mM MnCl 2 , 2 mM DTT, 0.1 mM Na 3 VO 4 , 1 ⁇ Ci [ ⁇ -P 32 ] ATP and 30 ⁇ g MBP.
  • the reaction was initiated by adding MLK protein and allowed to proceed for 15 min. at 37°C.
  • the reaction was stopped with 75 ⁇ l of 75 mM phosphoric acid. An aliquot of the quenched solution was loaded directly on the phosphocellulose membrane (Pierce).
  • the 96-well phosphocellulose multiscreen plate (Millipore) may be used.
  • the membranes were washed with 75 mM H 3 PO 4 .
  • the bound 32 P-labeled phosphorylated MBP was eluted in collection tubes by adding 1 M sodium hydroxide.
  • the radioactivity was determined by Cerenkov counting in a Beckman scintillation counter (Somerset, NJ). The formation of phosphorylated MBP with increasing concentration of bacterially-expressed GST-MLK-3 kinase domain is shown in Figure 13.
  • Example 29 Assay to determine binding of compounds to recombinant MLK Family
  • K-252a (Compound III-3; see, Table 4), an indolocarbazole metabolite of Nocardia species, binds to a variety of serine/threonine and tyrosine kinases (Angeles, et al., Anal. Biochem ,1996, 236, 49-55; Knight, etal, Anal. Biochem., 1997, 247, 376-381).
  • a tritiated K-252a ligand was used to assess binding to human recombinant full length MLK-3 from a crude preparation of baculovirus infected cells.
  • [ 3 H]K-252a was specifically labelled with tritium in the 3 and 9 positions through a contract with NEN Research products (Billerica, MA) and had a specific activity of 40 Ci/mmol. Binding reactions were perfomied in 1 ml in a 96-well plate. The reaction mixture contained 50 mM MOPS buffer, pH 7, 150mM NaCl, 5 mM MnCl 2 , 1 mg/ml BSA, 1 % DMSO and 0.25 nM [ 3 H]K252a. The samples were carried out in triplicate with a concentration of 5 ug/ml of crude baculovirus derived MLK-3.
  • Nonspecific binding was defined as binding in presence of unlabeled 1.2 uM K252a and was subtracted from total binding to give specific binding. At this dilution 12-15 % ofthe total counts were non-specificaUy bound to protein and 75-85 % of these counts were specifically bound to MLK-3 ( Figure 14). All experiments were performed for 2 hrs at 4°C. [ 3 H]K252a/MLK-3 complexes were collected on GF/C Whatman filters using a Brandel harvester, washed with cold MOPS NaCl buffer and counted on a Wallac Micro Beta counter. A saturation binding experiment was performed to obtain a K d for K252a. An example ofthe results from one of these experiments is shown ( Figure 14). A K d of 0.89 nM (Confidence Limits: 0.2 to 1.5 nM) was obtained.
  • K-252a and derivatives of this compound were provided by Kyowa-Hakko Kogyo Co. Ltd. (Tokyo, Japan) (Kaneko et al, 1997).
  • Compounds were dissolved in cell culture grade dimethyl sulfoxide (DMSO) and stored in the dark at 4°C.
  • DMEM Dulbecco's modified Eagle's medium
  • HA Hemagluttinin
  • B AbCO RedCO
  • AP- 1 (c-jun) substrate was purchased from Promega (Madison, WI).
  • [ ⁇ -32p]ATP (6000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL).
  • Green Monkey Kidney Cos7 cells were obtained from ATCC, Rockville, Maryland (CRL 1651) and maintained in DMEM containing 10 % bovine serum, 2 mM glutamine, 1 mM pyruvate, 50 U/ml penicillin/streptomycin at 37°C in 10% CO 2 , 90 % air atmosphere. Cos7 cells were detached for passaging by adding 0.25 % trypsin.
  • Cos7 cells were plated at 80% confluency and transfected with 2 ug each of cDNA constructs using lipofectamine as recommended by the provider (Gibco BRL, Gaithersburg, MD).
  • the cells were treated with 0.025% DMSO or 500 nM ofthe indicated compounds for 2 hr followed by lysis in 0.4 ml Triton buffer (1 % Triton X-l 00, 50 mM sodium chloride, 10 mM Tris (pH 7.6), 0.1 % bovine serum albumin, 30 uM sodium pyrophosphate, 50 mM sodium fluoride, 20 ug/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate). JNK activity from the lysate was assayed by an immunoprecipitation/kinase assay as described below.
  • Lysate from Cos 7 cells was measured for protein concentration using the Micro BCA kit from Pierce (Rockford, IL) and equal amounts of protein were immunoprecipitated with the HA antibody for 1 hr at 4°C. Immunoprecipitates were pelleted by centrifugation in a microfuge centrifuge for 20 sec, resuspended in Triton buffer, washed by centrifugation 2 more times, followed by a final wash in Kinase buffer (20 mM Hepes pH 7.4, 20 M MgCl 2 , 2 mM dithiothreitol, 0.1 mM sodium vanadate). The immunoprecipitate was resuspended in kinase buffer containing 1 ⁇ M ATP and 5 ⁇ Ci [ ⁇ -32p]ATP and substrate (1 ⁇ g/sample of AP-
  • a cell line containing a genetically engineered inducible expression system ofthe kinase of interest may be required. For example, a PC-12 cell line transfected with a tetracycline-controlled transactivator.
  • the tetracycline-inducible cell line is fransfected with two plasmids.
  • One plasmid constitutively expresses a fusion of the cJun NH 2 -terminal transactivating domain with the yeast GAL4 DNA binding domain (cJun-DBD fusion protein).
  • the other plasmid carries the coding sequences for firefly luciferase driven by five tandem repeats ofthe GAL4 binding site.
  • MLK the downstream substrate of JNK, cJun-DBD fusion protein, is phosphorylated, binds to the GAL4 binding sites, and induces luciferase gene transcription. Luciferase is easily assayed in cell lysates by addition of its substrate (Promega, Madison, WI) and measurement of chemiluminescence.
  • E embryonic age 14.5-15.
  • Cells from only the ventral portion of the spinal cord were dissociated, and further enriched for motoneurons by centrifugation on a 6.5% step metrizamide gradient, as described previously (Henderson, et al, 1993), and were analyzed for purity by staining with low affinity neurotrophin receptor antibody (IgG- 192, Boehringer-Mannheim) (data not shown).
  • Cells were seeded onto 96-well plates previously coated with poly-1-ornithine and laminin (5 ug/ml each) at a density of 6 x 10 4 cells/cm 2 in chemically defined serum-free N2 medium (Bottenstein, etal, 1979, supra).
  • Cerebral cortices were dissected from embryonic day 18 rat fetuses and enzymatically digested to obtain a single cell suspension.
  • Cells were seeded at a density of 1.56 x 10 5 /cm 2 onto poly-omithine/laminin coated 96 well tissue culture plates in serum-free Neural Basal Medium containing B27 supplements. Plates were coated with a solution of poly- ornithine/laminin (8 ⁇ g/ml each) made in PBS for at least two hours at 37C.
  • cortical neurons were exposed to Ab25-35 (20 ⁇ M) either in the presence or absence of test compounds.
  • Ab25-35 Sigma, St.
  • Neurons cells cells cells
  • III 1 46, 56 300 65 63, 73 98, 99 89, 67 97, 96
  • Compound has formula III where Z l5 Z 2 , R l5 and R 2 are H; X is CO 2 CH 3 ; and R is OH.
  • R 2 is CH 2 CH 2 OAc
  • R 4 is CH 2 CH 2 (2-Pyridyl)
  • X is CH 2 .
  • Example 32 Immunoprecipitation of Endogenous JNK Activity from motoneuron cultures in the Absence or Presence of Indolocarbazoles or Fused Pyrrolocarbazoles
  • Purified motorneurons were plated at a density of 6 x 10 4 cells/cm 2 in 16 mm diameter wells. Cells were allowed to attach for 2 hours prior to treatment. Cells were treated with either 0.0125 % DMSO or 500 nM compound for 2 hrs in N2 defined medium. Cells were then rinsed with ice cold phosphate buffered saline followed by lysis in 0.4 ml Triton buffer as described above in example 30. Lysate from motoneuron cultures was normalized to cell number and immunoprecipitated with a JNKl antibody (cat. # sc-474) purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • JNK activity from the immunoprecipitates was assayed in the presence of 32 P-ATP and c-jun substrate as described above.
  • the profile of inhibitory activity ofthe 4 test compounds was compared in motoneurons and in Cos7 cells overexpressing either DLK, MLK-1, MLK-2 or MLK-3 (Table 3).
  • Example 33 Correlation between inhibition of MLK3-induced JNK activity in Cos7 cells and cholineacetyl transferase activity in primary embryonic cultures
  • R 2 is CH 2 CH 2 (2-Pyridyl); and R is OH.
  • Example 34 Gel shift assay for MLK activation: Activation of MLKs can lead in induction of c-jun transcription, resulting in increased c-Jun protein.
  • the increased amount of c-Jun protein can be measured by a standard assay, identified as a gel shift assay.
  • Gamer, et al Nucleic Acids Res., 1981, 9, 3047-3060, which is inco ⁇ orated herein by reference in its entirety.
  • Radiolabeled double-stranded DNA oligomers, that code for a c-Jun DNA-binding site are incubated with a nuclear cell extract followed by acrylamide gel electrophoresis and quantitation ofthe radiolabeled DNA shifted to a slower mobility. This represents the portion of DNA that is bound to the c-Jun protein and is directly proportional to the amount of c-Jun protein in the extract.
  • Activation of MLKs can also induce c-Jun phosphorylation. This can be detected using antibodies which specifically recognize the phosphorylated form of the protein in detection systems such ⁇ as, for example, Western blots or ELISAs.
  • Leibovitz' s L 15 media, glucose, sodium bicarbonate, trypsin and antibiotics were from Gibco. Muscle extract was prepared as described (Henderson, et al. , Nature, 1983, 302, 609- 611, which is inco ⁇ orated herein by reference in its entirety). AU other reagents were from
  • Motoneurons (embryonic day 5.5) were isolated with an immunological method according to the procedure set forth in Bloch-Gallego, et al, Development, 1991, 777, 221- 232, which is inco ⁇ orated herein by reference in its entirety, with modifications as described in Weng, et al. , NeuroReport, 1996, 7, 1077- 1081 , which is inco ⁇ orated herein by reference in its entirety.
  • Purified motoneurons were seeded onto 35 mm tissue culture dishes (Nunc) pre-coated with poly-DL-ornithine and laminin (1 ⁇ g/ml, Upstate Biotech). The culture medium was L 15 with sodium bicarbonate (22.5 mM), glucose (20 mM), progesterone (2x 10"
  • SG embryonal day 12 (El 2)
  • DRG dorsal root ganglia
  • CG ciliary ganglia
  • NGF Nerve growth factor
  • CNTF ciliary vomrophic factor
  • Neurons were plated in 35 mm culture dishes with grids (Nunc). Selected areas of each dish comprising together about 10% ofthe surface scanned for the presence of phase bright cells immediately after plating and again after 48 h to assess survival percentages. Cell survival was confirmed by vital staining with trypan blue (not shown). Intact DRG
  • Ganglia were placed in 96 well plates previously coated with poly-1-omithine and laminin (5 ⁇ g each/ml phosphate buffered saline) in serum-free N2 medium (Bottenstein, et al, Proc. Natl. Acad. Sci. USA, 1979, 76, 514-517, which is inco ⁇ orated herein by reference in its entirety) containing 0.05% bovine serum albumin (BSA) and maintained for 48 h at 37° and 5% CO 2 in ahumidified environment.
  • BSA bovine serum albumin
  • Compound III-3 supports the survival of chick embryonic peripheral neurons in a concentration-dependent fashion
  • motoneurons rescued with Compound III-3 exhibited phase bright cell bodies and were able to extend long neurites, which appeared slightly thicker than those induced by muscle extract (Fig. 17) . After four days in culture, 56% ofthe motoneurons were alive with Compound III-, compared with
  • Compound III-3 promotes neurite outgrowth from intact dorsal root ganglia
  • Embryos were sacrificed at E10 and their spinal cords were removed, fixed in Carnoy ' s solution ( 10% glacial acetic acid, 60% absolute ethanol, 30% chloroform), processed for serial paraffin sections, and stained with thionin. Every 20th section of lumbar segments 1-8 was counted according to previously described criteria (Clarke, et al, Methods In Cell Biology. Cell Death, 1995, Schwart & Osborne, Eds.,
  • Untimed pregnant Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). Female rat pups were injected daily, subcutaneously (SC), over the target perineal muscles, with Compound III-3 in 5% Solutol TM HS 15 or vehicle along starting on the day of birth (PI) and continuing for 5 days (P5).
  • Motor neurons were counted at X500 in serial sections from the lumbar 5 to the sacral 1 region ofthe spinal cord as described previously (Nordeen, et al, supra). The microscopic enumeration was made on coded sections by an observer blinded to the treatment groups. Motor neuron counts were corrected for cell size and section thickness (Konisgsmark, Contemporary Research Methods Newro « ⁇ t ⁇ my,Nauta&Ebbesson,Eds., 1970, Springer- Verlag, New York, pp.315-340, which is inco ⁇ orated herein by reference in its entirety) and statistical analysis was by one-way analysis of variance ( ANO V A) .
  • Perineal musculature was postfixed, decalcified, embedded in Paraplast, sectioned at 1 O ⁇ m and stained with Milligan' s Trichrome. Using bright-field microscopy (X250), BC and LA muscles in normal females and Compound III-3 -treated females (405 animals/group) were positively identified by both their location and the presence of striated fibers. The outline of muscle tissue was traced from alternate sections using a projection microscope (62.5), and the cross-sectional area was measured using a digitizing pad and a computer-based mo ⁇ hometry system (Sigmascan, Jandel Scientific). Muscle volume was calculated by talking the total cross-sectional area and multiplying it by the section thickness, and corrected for the percentage of the structure sampled.
  • Serum testosterone levels (6-7 animals/group) were measured by radioimmunoassay following the procedures set forth in Wingf ⁇ eld, et al , Steroids, 1975, 26, 311-327, which is inco ⁇ orated herein by reference in its entirety.
  • the left hypoglossal nerve was transected in the neck of adult female Sprague-Dawley rats (120-180 g) under Neu butol anesthesia, and 50 ⁇ l of Compound 111-3 or its vehicle (5% SolutolTM HS 15) were applied to a piece of Gelfoam TM (AJ Buck, Owings Mills, MD), then wrapped around the proximal end of the transected nerve. After 7 days, the animals were anesthetized and perfused with 4% paraformaldehyde in Sorenson's buffer, 0.07 M phosphate, pH 7.2.
  • the brain stem was removed and 40- ⁇ m-thick serial coronal sections were cut on a cryostat (Chiu, et al, NeuroReport, 1994, 5, 693-696, which is inco ⁇ orated herein by reference in its entirety). Every fifth section was processed for ChAT immunohistochemistry as previously described (Chiu, et al, J. Comp. Neurol., 1993, 328, 351-363, which is inco ⁇ orated herein by reference in its entirety) using a 1:350 dilution of an anti-ChAT monoclonal antibody obtained from Chemicon.
  • Compound III-3 rescued rat embryo motor neurons from apoptotic death in vitro and inhibited a signaling pathway resulting in JNKl activation in these cells (Maroney, et al. , J. Neurosci. , 1998, 18, 104- 111, which is inco ⁇ orated herein by reference in its entirety).
  • Compound 111-3 was assessed in two models of developmentally regulated programmed motor neuronal death and in a model of axotomy- induced dedifferentiation in adult motor neurons. In chicks, approximately 50% ofthe spinal cord motor neurons undergo PCD during E5- 10 (Hamburger, et al. , J. Neurosci. , 1982, 7, 38- 55; Purves, et al, Body and Brain: A Trophic Theory of Neural Connections, 1988, Harvard
  • Compound III-3 demonstrated neuroprotective activity: developmentally-regulated PCD of lumbar spinal cord motor neurons in embryos (Fig. 19), androgen-sensitive death of postnatal SNB motor neurons (Fig. 20), and axotomy-induced loss of a functional marker, ChAT, in adult hypoglossal motor neurons (Fig. 21 and Table 5).
  • Compound III-3 was efficacious when administered peripherally by sc inj ection, applied locally to the cut end of a nerve, or directly overlaid on the chick embryo chorioallantoic membrane.
  • An Inhibitor ofthe MLKS pathway demonstrates in vivo efficacy and blocks phosphorylation events downstream of MLK-3 in the MPTP model
  • MPTP was administered at a dose (40 mg/kg) that produces loss of striatal dopaminergic terminals and cell bodies in the substantia nigra.
  • Tyrosine hydroxylase was used as a marker for dopaminergic nerve terminals in the substantia nigra.
  • Systemically administered Compound 111-3 attenuated the loss of substantia nigra tyrosine hydroxylase immunoreactive neurons after MPTP lesion (Fig. 22A; Saporito et al, 1999). Since
  • Compound III-3 is a known inhibitor of MLK-3, activation of a downstream substrate of MLK-3 was measured in MPTP-treated mice.
  • Levels of phosphorylated MKK4 were measured using a phospho-MKK4 specific antibody (New England Biolabs, Beverly, MA) that recognizes the monophosphorylated form of MKK4 by either immunoblot (Fig. 22B) or ELISA (Fig. 22C).
  • MPTP administration elevated levels of phosphorylated MKK4 in the substantia nigra by up to 5 fold over control levels (Fig. 22B). Peak elevations occurred 4 hrs after administration of MPTP and coincided with peak CNS levels of MPP + .
  • MKK4 phosphorylation was attenuated by pretreatment with 1 -deprenyl, indicating that these phosphorylation events were mediated by MPP + (Fig. 22C). Moreover, MKK4 phosphorylation was partially inhibited with Compound III-3 pretreatment at a dose (1 mg/kg) that produces protection against MPTP-induced nigrostriatal dopaminergic loss (Fig. 22C). These data demonstrate that MPTP ( MPP + ) activates MKK4, a downstream substrate of MLK-3. Moreover, these data demonstrate that a known inhibitor of MLK-3 inhibits activation of this kinase pathway in vivo.
  • Inhibition of production of these two cytokines may be a useful treatment of certain inflammatory disorders of the immune system.
  • These cytokines can be easily measured by commercial ELISA kits.
  • THP-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.
  • LPS E.coli serotype 0111.B4, TCA extracted
  • ELISA kits for assaying IL- 1 and TNF- ⁇ were purchased from Boerhinger- Mannheim and assays on THP-1 culture medium were performed as directed by the manufacturer. Standard curves according to directions were obtained with each assay.
  • Assays for JNK activity were performed after immunoprecipitating the JNK protein from an extract of lysed THP-1 cells.
  • Pelleted THP-1 cells were lysed on ice for 15 min in 500 ⁇ l of Frac buffer (10 mM Tris-Hcl, pH 7.5, 50 mMNaCl, 30 ⁇ M sodium pyrophosphate, 1 mg/ml BSA, 1% Triton-X-100). The extract was centrifuged for 10 min at 14K and 5 ⁇ l of Frac buffer (10 mM Tris-Hcl, pH 7.5, 50 mMNaCl, 30 ⁇ M sodium pyrophosphate, 1 mg/ml BSA, 1% Triton-X-100). The extract was centrifuged for 10 min at 14K and 5 ⁇ l of Frac buffer (10 mM Tris-Hcl, pH 7.5, 50 mMNaCl, 30 ⁇ M sodium pyrophosphate, 1 mg/ml BSA, 1%
  • JNK antibody (Santa Cruz) was added to the supernatant.
  • the extract was rotated for 60 min at 4°C, 75 ⁇ l of washed protein A Sepharose (20% w/v in Frac) added and the extract rotated another 30 min to bind the antibody complex to the protein A Sepharose.
  • the protein A Sepharose was washed twice with Frac buffer, once with 20 mM Hepes, pH 7.6, 20 mM MgCl 2 , 2 mM DTT, then incubated for 15 min at 30° C in 30 ⁇ l of kinase buffer (20 mM hepes, 20 mM MgCl 2 , 2 MM DTT, 1 ⁇ g recombinant c-jun, and 2 ⁇ M ATP- ⁇ - 32 P, 2 ⁇ Ci. The reaction was terminated by the addition of 10 ⁇ l of 4X SDS gel loading buffer, heated for 3 min at 80° C, and the proteins were analyzed on a 10% SDS gel.
  • Jurkat cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. TNF- ⁇ was from Promega and anti CD3 and anti CD28 antibodies were from Pharmigen.
  • Jurkat experiments were done in 200 ⁇ l in a 96 well plate.
  • IL-2 was measured with an ELISA kit purchased from Boehringer Mannheim. The antibodies to CD3 and CD28 were allowed to bind to the plastic ofthe 96 well plate (18 hr in PBS) prior to addition ofthe Jurkat Cells. Cells were treated with compounds 1 hr prior to adding to the antibody coated plate. Antibodies to CD3 and CD28 were used to activate the T cell receptor and induce IL-2.
  • IL-2 was released from the Jurkat cells between 6 hr and 24 hr after initiation of induction
  • FIG.23 A No IL-2 was made constitutively (Fig.23 A CNT).
  • a Compound 1X1-3 concentration of 500 nM inhibited IL-2 induction by greater than 80% (Fig. 23B).
  • a more extensive dose response experiment was performed with Compound III-3 and with Compound 1-4 which yielded IC 50 values of 139 nM for Compound

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Pain & Pain Management (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Cardiology (AREA)
  • Zoology (AREA)
  • Psychology (AREA)
  • Wood Science & Technology (AREA)
EP01961958A 2000-08-11 2001-08-08 Modulating multiple lineage kinase proteins Withdrawn EP1309721A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US637054 1991-01-03
US63705400A 2000-08-11 2000-08-11
PCT/US2001/024822 WO2002014536A2 (en) 2000-08-11 2001-08-08 Odulating multiple lineage kinase proteins

Publications (1)

Publication Number Publication Date
EP1309721A2 true EP1309721A2 (en) 2003-05-14

Family

ID=24554347

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01961958A Withdrawn EP1309721A2 (en) 2000-08-11 2001-08-08 Modulating multiple lineage kinase proteins

Country Status (19)

Country Link
EP (1) EP1309721A2 (zh)
JP (1) JP2005503102A (zh)
CN (1) CN1458979A (zh)
AU (2) AU2001283179B2 (zh)
BG (1) BG107623A (zh)
BR (1) BR0113266A (zh)
CA (1) CA2419985A1 (zh)
CZ (1) CZ2003680A3 (zh)
HR (1) HRP20030162A2 (zh)
HU (1) HUP0501110A3 (zh)
IL (1) IL154311A0 (zh)
IS (1) IS6711A (zh)
MX (1) MXPA03001218A (zh)
NO (1) NO20030658L (zh)
NZ (1) NZ524034A (zh)
PL (1) PL366248A1 (zh)
SK (1) SK2692003A3 (zh)
WO (1) WO2002014536A2 (zh)
ZA (1) ZA200301109B (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6811992B1 (en) 1998-05-14 2004-11-02 Ya Fang Liu Method for identifying MLK inhibitors for the treatment of neurological conditions
US20060014165A1 (en) * 2003-07-14 2006-01-19 Decode Genetics Ehf. Methods of diagnosis and treatment for asthma and other respiratory diseases based on haplotype association
AU2004257748B2 (en) * 2003-07-14 2008-10-30 Decode Genetics Ehf. Method of diagnosis and treatment for asthma based on haplotype association
US20060058250A1 (en) * 2004-09-10 2006-03-16 Cephalon, Inc. Methods of treating proliferative skin diseases using carbazole derivatives
WO2014172616A2 (en) * 2013-04-18 2014-10-23 President And Fellows Of Harvard College Methods, compositions and kits for promoting motor neuron survival and treating and diagnosing neurodegenerative disorders
NZ754020A (en) * 2016-11-25 2022-11-25 Genuv Inc Composition for promoting differentiation of and protecting neural stem cells and method for inducing neural regeneration using same

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756494A (en) * 1992-07-24 1998-05-26 Cephalon, Inc. Protein kinase inhibitors for treatment of neurological disorders
US5705511A (en) * 1994-10-14 1998-01-06 Cephalon, Inc. Fused pyrrolocarbazoles
US6811992B1 (en) * 1998-05-14 2004-11-02 Ya Fang Liu Method for identifying MLK inhibitors for the treatment of neurological conditions
ATE293254T1 (de) * 1998-08-26 2005-04-15 Cephalon Inc Modulierung von mlk- (multiple lineage kinase) proteinen
US6841567B1 (en) * 1999-02-12 2005-01-11 Cephalon, Inc. Cyclic substituted fused pyrrolocarbazoles and isoindolones

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0214536A2 *

Also Published As

Publication number Publication date
JP2005503102A (ja) 2005-02-03
PL366248A1 (en) 2005-01-24
WO2002014536A9 (en) 2003-12-18
BG107623A (bg) 2003-11-28
HUP0501110A3 (en) 2006-06-28
HUP0501110A2 (en) 2006-03-28
SK2692003A3 (en) 2003-08-05
IS6711A (is) 2003-02-10
WO2002014536A2 (en) 2002-02-21
CA2419985A1 (en) 2002-02-21
CZ2003680A3 (cs) 2003-11-12
NO20030658D0 (no) 2003-02-10
ZA200301109B (en) 2004-07-20
AU2001283179B2 (en) 2006-07-13
NZ524034A (en) 2006-11-30
NO20030658L (no) 2003-04-09
CN1458979A (zh) 2003-11-26
AU8317901A (en) 2002-02-25
IL154311A0 (en) 2003-09-17
MXPA03001218A (es) 2003-05-27
HRP20030162A2 (en) 2005-12-31
WO2002014536A3 (en) 2003-01-30
BR0113266A (pt) 2005-01-04

Similar Documents

Publication Publication Date Title
EP1105728B1 (en) Modulating multiple lineage kinase proteins
KR101964954B1 (ko) 뉴로트로핀 수용체의 효현제 및 약제로서의 이의 용도
PL212956B1 (pl) Zastosowanie zwiazku o wzorze I lub II, zwiazek o wzorze II, oraz kompozycja farmaceutyczna zawierajaca zwiazek o wzorze II
SK17992000A3 (sk) Premostené indenopyrolokarbazoly
AU2001283179B2 (en) Modulating multiple lineage kinase proteins
AU2001283179A1 (en) Modulating multiple lineage kinase proteins
Watterson et al. Ligand modulation of glial activation: cell permeable, small molecule inhibitors of serine-threonine protein kinases can block induction of interleukin 1β and nitric oxide synthase II
US9440980B2 (en) Remedy for diabetes
MXPA01002020A (en) Modulating multiple lineage kinase proteins
WO2005106473A1 (ja) 神経変性疾患治療薬のスクリーニング方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030310

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1055606

Country of ref document: HK

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20110615