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WO2009030890A1 - Utilisation de composés contre le cancer/choc septique - Google Patents

Utilisation de composés contre le cancer/choc septique Download PDF

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Publication number
WO2009030890A1
WO2009030890A1 PCT/GB2008/002967 GB2008002967W WO2009030890A1 WO 2009030890 A1 WO2009030890 A1 WO 2009030890A1 GB 2008002967 W GB2008002967 W GB 2008002967W WO 2009030890 A1 WO2009030890 A1 WO 2009030890A1
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Prior art keywords
cancer
tbkl
optn
use according
kinase
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WO2009030890A9 (fr
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Philip Cohen
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University of Dundee
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University of Dundee
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Priority claimed from GB0717069A external-priority patent/GB0717069D0/en
Priority claimed from GB0724126A external-priority patent/GB0724126D0/en
Application filed by University of Dundee filed Critical University of Dundee
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Publication of WO2009030890A9 publication Critical patent/WO2009030890A9/fr
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of certain pyrimidine compounds to inhibit, for example, TBKl and/or IKX epsilon and which may therefore find application in treating cancer septic shock and/or Primary open Angle Glaucoma (POAG).
  • certain pyrimidine compounds to inhibit, for example, TBKl and/or IKX epsilon and which may therefore find application in treating cancer septic shock and/or Primary open Angle Glaucoma (POAG).
  • POAG Primary open Angle Glaucoma
  • WO2004/048343 discloses a great number of CHK-, PDK- and AKT- inhibitory pyrimidines and suggest their use in treating various diseases. Those considered to inhibit PDKl are suggested to find application in treating cancer. Nevertheless, there is no teaching of the specificity of such compounds to only these kinases, or if they display potency to other kinases, which would lead to other potential treatments and/or a possible variance to the proposed diseases to be treated.
  • POAG Primary Open Angle Glaucoma
  • NVG Normal Tension Glaucoma
  • LPG Low Tension Glaucoma
  • tumour necrosis factor ⁇ (TNF ⁇ ) has been reported to increase the severity of damage in optic nerve heads of POAG and LTG subjects [10,11]. Moreover, exposure to TNF ⁇ [12] induces the de novo expression of optineurin.
  • n is 2-4, most preferably 3.
  • R 1 is a 5 or 6 membered cyclic alkyl wherein optionally one carbon is substituted by an N.
  • Ri is a pyrrolidine group such as 1- pyrollidinyl.
  • R 2 is a thiophene group such as 2-thienyl, or is the group
  • R 3 is halo, such as I or Br.
  • kinase inhibition activity As is demonstrated in Examples section that follows, two representative compounds of the present invention (see formulae II and III) were tested for their kinase inhibition activity and showed significant potency to ERK8, MNK, Aurora B, Aurora C, MARK3, IKKepsilon, TBKl, as well as PDKl. These compounds can therefore efficiently serve for treating diseases or disorders in which inhibiting the activity of one or more of these kinases, would be beneficial.
  • the kinase is PDKl
  • the present invention relates to the treatment of diseases where it would be desirable to inhibit PDKl and at least one other identified kinase.
  • a method of treating an ERK8, MNK2, Aurora B, Aurora C, MARK3, IKKe and/or TBKl and optionally PDKl related disease or disorder is effected by administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention, as described hereinabove, either per se 5 or, more preferably, as a part of a pharmaceutical composition, mixed with, for example, a pharmaceutically acceptable carrier, as is detailed hereinunder.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • administering refers to a method for bringing a compound of the present invention and a target kinase together in such a manner that the compound can affect the enzyme activity of the kinase either directly; i.e., by interacting with the kinase itself or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
  • administration can be accomplished either in vitro, i.e. in a test tube, or in vivo, i.e., in cells or tissues of a living organism.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease or disorder, substantially ameliorating clinical symptoms of a disease or disorder or substantially preventing the appearance of clinical symptoms of a disease or disorder.
  • preventing refers to a method for barring an organism from acquiring a disorder or disease in the first place.
  • terapéuticaally effective amount refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease or disorder being treated.
  • a therapeutically effective amount can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 or the ICioo as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data. Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans.
  • toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5O and the ED 50 .
  • the dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell cultures assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, chapter 1, page 1).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for oral administration range from about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and most preferably from about 250-500 mg/kg/day.
  • therapeutically effective serum levels will be achieved by administering multiple doses each day.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • kinase related disease or disorder refers to a disease or disorder characterized by inappropriate kinase activity or over-activity of a kinase as defined herein. Inappropriate activity refers to either; (i) kinase expression in cells which normally do not express said kinase; (ii) increased kinase expression leading to unwanted cell proliferation, differentiation and/or growth; or, (iii) decreased kinase expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
  • Over-activity of kinase refers to either amplification of the gene encoding a particular kinase or production of a level of kinase activity, which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the kinase increases, the severity of one or more of the symptoms of the cellular disorder increases).
  • Over activity can also be the result of ligand independent or constitutive activation as a result of mutations such as deletions of a fragment of a kinase responsible for ligand binding.
  • Preferred diseases or disorders that the compounds described herein may be useful in preventing, treating and/or studying are cell proliferative disorders, especially cancer such as, but not limited to, papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease and Burkitt's disease.
  • cancer such as, but not limited to, papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancre
  • Another condition to which the compounds described herein may be useful in preventing, treating and/or studying is septic shock.
  • the present inventors have observed that TBKl binds in an enhanced manner to the mutant form of optineurin which causes a form of POAG.
  • the compounds described herein may therefore find use in treating POAG and/or diseases associated with optineurin activity.
  • a compound which is capable of inhibiting the binding of TBKl to a mutant form of OPTN for the manufacture of a medicament for treating POAG and/or a disease where it would be desirable to inhibit or reduce TBKl binding to mutant form of OPTN.
  • One such mutant is the OPTN (E50K) mutant.
  • Suitable compounds may include the compounds identified herein.
  • the compounds or physiologically acceptable salt, ester or other physiologically functional derivative thereof, described herein may be presented as a pharmaceutical formulation, comprising the compounds or physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic and/or prophylactic ingredients.
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation.
  • the formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy.
  • AU methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent.
  • Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored.
  • Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner.
  • Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope.
  • An active compound may also be formulated as dispersable granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
  • Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.
  • Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.
  • Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
  • compositions suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
  • Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use.
  • an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
  • An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly.
  • Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
  • Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.
  • such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent.
  • Suitable liquid propellants include propane and the chlorofluorocarbons
  • suitable gaseous propellants include carbon dioxide.
  • Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.
  • Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
  • an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
  • Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
  • the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated. Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.
  • a method of treating a patient suffering from a disease associated with abnormal cell proliferation comprising the step of administering to the subject an effective amount of a compound according to formula I, II, or III.
  • a method of treating a patient suffering from septic shock comprising the step of administering to the subject an effective amount of a compound according to formula I, II, or III.
  • a method of treating a patient suffering from POAG comprising the step of administering to the subject an effective amount of a compound which is capable of inhibiting an interaction between TBKl and a mutant form of OPTN, associated with POAG.
  • Suitable compounds include those according to formulae I, II or III.
  • BX 795 (formula II) was not only a potent inhibitor of PDKl, but also inhibited ERK8, MNK2, Aurora B, Aurora C, MARK3 and IKKe with similar potency.
  • TBKl was inhibited even more potently than PDKl (see Table I).
  • the specificity of BX 320 (formula III) was similar to BX 795, although it was a less potent inhibitor.
  • Aurora kinase (see below) and TBKl (3,4) are also attractive targets for the development of anti-cancer drugs.
  • TBKl is activated in response to hypoxia (3) and controls the production of angiogenic factors, such as VEGF and IL-8. Its levels are elevated in malignant colon and breast cancer cells.
  • TBKl is also reported to be activated by the RalB/Sec5 effector complex, restricting the initiation of apoptotic programmes and thereby aiding tumour cell survival (4).
  • BX 320 and BX 795 are not specific inhibitors of PDKl, but might be useful for assessing the physiological roles of TBKl and the closely related IKKepsilon, as they are the most potent inhibitors of these two protein kinases to be described thus far.
  • the present invention further provides use of compounds as defined herein for the manufacture of medicaments for the treatment of diseases where it is desirable to inhibit TBKl and/or IKK epsilon.
  • diseases include colon and breast cancer, septic shock and/or POAG.
  • a number of papers (5, 6, 7) have described that TBKl and IKKepsilon modulate expression of interferon and interferon inducible genes, without affecting induction of pro-inflammatory cytokines. This serves to support the proposal that the compounds disclosed herein, may find application in treating/preventing septic shock or viral infection. Mice that do not express interferon beta or IRF3 are resistant to lipopolysaccharide induced septic shock so that inhibitors of TBKl should be expected to have a similar effect.
  • Figure 1 shows that OPTN interacts with TBKl in mammalian cells.
  • IL-IR 293 cells were co-transfected with a vector expressing GST-TBKl and either empty vector (empty), or vectors expressing HA-OPTN (OPTN), HA- OPTN[E50K] (E50K) or HA-OPTN[R545Q] (R545Q). After 24 h the cells were serum starved for 16 h, then stimulated for 10 min with (+) or without (-) IL-I ⁇ (5 ng/ml) and lysed in 0.5 ml cell lysis buffer.
  • the beads were washed twice with 1 ml of cell lysis buffer plus 0.15 M NaCl and three times with 1 ml cell lysis buffer and immunoblotted with anti-GST antibodies to detect GST-TBKl (C) IL-IR 293 cells were stimulated for the times indicated with 5 ng/ml IL- l ⁇ .
  • the extracts (2 mg protein) were incubated for Ih at 4°C with anti-OPTN (4 ⁇ g). Protein G-Sepharose (10 ⁇ l beads) was then added and incubated for 45 min at 4°C.
  • the suspension was centrifuged to pellet the protein G-Sepharose, the beads were washed three times with lysis buffer plus 150 niM NaCl, denatured in SDS and immunoblotted as in A using anti-TBKl and anti-OPTN.
  • An aliquot of the cell lysate 80 ⁇ g protein was also immunoblotted with antibodies that recognise the active phosphorylated forms of ERKl and ERK2 (ERK 1/2) to monitor stimulation by IL- l ⁇ (D).
  • the experiment was carried out as in A, except that cells were transfected using HA-tagged TKBl and GST-tagged wild- type or mutant OPTN.
  • IL-IR cells were transfected with vectors encoding WT-OPTN or OPTN[E50K]. After 24h the cells were serum starved for 20 h, then stimuklated for 15 min with 5 ng/ml IL- l ⁇ . Following cell lysis, the HA-OPTN was immunoprecipitated from cell extract (1 mg protein) with anti-HA (5 ⁇ g) coupled to protein G Sepharose. After washing three times as in C, bound proteins were released by denaturation in SDS and immuoblotted for the presence of endogenous TBKl as well as for anti-HA.
  • Figure 2 shows characterisation of the interaction between OPTN and TBKl.
  • A Sequence alignment of the TBKl interaction domains found in human (h) TANK, NAPl and SINTBAD with OPTN. Identities are shown in white lettering on a black background and similar residues in white lettering on a grey background.
  • B IL-IR cells were transfected as in Figure 1 with vectors expressing GST-TBKl and either empty vector (control), a vector expressing wild type (WT), a vector expressing Ha- OPTN[E50K] (E50K) or a vector expressing HA-OPTN[M98K] (M98K).
  • the cells were lysed and GST-TBKl pulled down from 1 mg cell lysate protein on gluthatione-Sepharose beads as in Figure 1.
  • the beads were washed, denatured in SDS, subjected to SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with an HA-antibody to detect OPTN.
  • the membrane was stripped and re-probed with an antibody that recognizes GST as shown.
  • Aliquots of the cell lystates 50 ⁇ g protein were also immunoblotted with HA-antibodies as in Figure 1.
  • the experiment was carried out as in B, except that the cells were transfected with vectors expressing GST-TBKl and HA-OPTN[I -127] in which Glu50 has been mutated to Lys (E50K) or Met98 to Lys (M98K).
  • Figure 3 shows the C-terminal region of TBKl is required for interaction with OPTN.
  • IL-IR cells were transfected with vectors expressing GST, wild type GST-TBKl (W/T) or GST-TBKl [1-687] (1-687) and vectors expression HA-tagged OPTN or HA-tagged TANK. The experiment was then earned out exactly as described in the legend to Figure 2.
  • Protein kinase inhibitors BX 795 and BX 320 were synthesised in accordance with the teaching of WO 2004/048343.
  • All protein kinases were of human origin and encoded full length proteins, unless indicated otherwise. They were either expressed as glutathione S-transferase (GST) fusion proteins in Escherichia CoIi or as hexahistidine (His6)-tagged proteins in insect Sf21 cells. GST fusion proteins were purified by affinity chromatography on glutathione- Sepharose, and His6-tagged proteins on nickel/nitriloacetate-agarose. The procedures for expressing some of the protein kinases used herein have been detailed previously (1,2). The following sections outline the DNA vectors synthesised and the procedures used to express and purifying protein kinases that have not been reported previously.
  • kinases were expressed in Sf21 cells: Aurora B and Aurora C, extra-cellular signal-regulated kinase 8 (ERK8), microtubule affinity regulating kinase 3 (MARK3), protein kinase Ba[118-480][S473D], protein kinase B ⁇ (PKB ⁇ [120-481][S474D], 3-phosphoinositide-dependent protein kinase-1 [52-556] (PDKl [52-556], IKK ⁇ , TBKl,
  • insect Sf21 cells were incubated for 1 h with the protein phosphatase inhibitor okadaic acid (50 nM).
  • JNK isoforms were activated with MKK4 and MKK7, MNK2 with p38 ⁇ MAP kinase; PKB ⁇ , PKB ⁇ , with PDKl, and ERKl with MKKl.
  • bacterial pellets expressing cyclin A2 and CDK2 were mixed together, lyse and purified on glutathione Sepharose.
  • the GST-tags were removed by cleavage with PreScission protease and the CDK2-cyclin A2 complex purified by chromatography on SP-Sepharose.
  • Protein kinases assayed at 20 ⁇ M ATP were:- JNKl, PDKl 5 CHKl, CHK2, CDK2 and Aurora B. Protein kinases assayed at 50 ⁇ M ATP were:-MNK2, IKKepsilon and TBKl.
  • the assays were initiated with MgATP, stopped by addition of 5 ⁇ l of 0.5 M orthophosphoric acid and spotted on to P81 f ⁇ lterplates using a unifilter harvester (PerkinElmer, Boston, MA 02118, USA).
  • the IC50 values of inhibitors were determined after carrying out assays at 10 different concentrations of each compound.
  • Aurora B and Aurora C were both assayed against the substrate peptide LRRLSLGLRPvLSLGLRRLSLGLRRLSLG (300 ⁇ M), ERKl and ERK8, against myelin basic protein (MBP, 0.33mg/ml).
  • MARK3 was assayed against the peptide KKKVSRSGLYRSPSMPENLNRPR (300 ⁇ M), MNK2 against the eIF4E protein (0.5mg/ml).
  • PKB ⁇ was assayed against the peptide GRPRTSSFAEGKK (30 ⁇ M).
  • TBKl were assayed against (AKPKGNKDYHLQTCCGSLAYRRR) (300 ⁇ M).
  • the substrates used for other protein kinases were described previouslyfl, 2].
  • enzymes were diluted in 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% (v/v) 2-mercaptoethanol and assayed in 50 mM
  • HA antibody and Interleukin-l ⁇ were purchased from Sigma, the OPTN antibody from Abeam, and the TBKl antibody from Cell Signalling Technologies, while the GST antibody was provided by the Division of Signal Transduction Therapy at Dundee.
  • Glutathione-sepharose was obtained from GE Healthcare, while the IL-IR cells were a gift from Tularic Inc., USA.
  • OPTN and TBKl were cloned and inserted into pEBG6P and pCMV5 to express GST and HA-tagged proteins, respectively, in the IL-R cells.
  • TANK was cloned and also expressed in pCMV5.
  • Site-sirected mutageneisis was carried out using the Quick Change method (Stratagene) but using KOD Hot Start Polymerase.
  • IL-IR cells were cultured and lysed [13] and SDS-PAGE and immunoblotting carried out as described previously [14].
  • Table 1 Inhibition of protein kinases by BX795 and BX320.
  • BX795 and related compounds are not only inhibitors of PDKl but also inhibit ERO, MNK2, Aurora B, Aurora C, MARK3 and IKK ⁇ with similar potency, leading to potential new utilities.
  • TBKl was inhibited even more potently than PDKl.
  • TBKl is elevated in malignant colon cancer and breast cancer. Further more phosphorylation of proteins by TBKl has a role in interferon production. It has been reported that overexpression of interferon may interfere with the immune system.
  • BX 320 The specificity of BX 320 was similar to BX 795, although it was a less potent inhibitor.
  • the present study indicates that BX 320 and BX 795 are not specific inhibitors of PDKl, but might be useful for assessing the physiological roles of TBKl and the closely related IKK ⁇ , as they are the most potent inhibitors of these two protein kinases. More importantly these compounds may be useful for the treatment of diseases such as cancer or septic shock that can be caused by the uncontrolled activation of TBKl and/or IKK epsilon.
  • yeast two hybrid screens were carried out using OPTN as bait. Approximately 1 x 10 6 yeast colonies were screened from a human foetal brain cDNA library and a human leukocyte cDNA library. One positive clone was detected in each library that interacted with OPTN and encoded the C-terminal region of TBKl (residues 572-729 from the brain library and residues 601- 729 from the leukocyte library) (results not shown). This result was unexpected since had been reported previously that neither of the two unidentified protein kinase activities found associated with OPTN immunoprecipitates were TBKl [12]. We therefore carried out further experiments to investigate whether these proteins were capable of interacting with one another in cells.
  • mice that do not express TBKl die just prior to birth because they become hypersensitive to TNF ⁇ -induced apoptosis of the liver and lethality can be rescued by crossing to mice that do not express TNFRl [22].
  • the adenovirus E3-14.7K protein binds to OPTN, and this interaction blocks the protective effect of the E3-14.7K protein on TNF ⁇ -induced apoptosis [24].
  • McWhirter et al (PNAS 101, 233 238, 2004) Demonstrate that induction of type I interferon and related genes depends on TBKl . They also show that IKKepsilon and TBKl directly phosphorylate serine residues that are critical for IRF3 activation. 7. Hemmi et al (J Exp Med 199, 1641-1650, 2004) indicate that TBKl and IKK are essential for the activation of IFN beta and IFN inducible genes.
  • Tumor necrosis factor-alpha a potentially neurodestructive cytokine produced by glia in the human glaucomatous optic nerve head. Glia 32, 42-50.
  • TANK a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-kappaB activation. Genes Dev 10, 963-973.
  • NAK is recruited to the TNFRl complex in a TNFalpha-dependent manner and mediates the production of RANTES: identification of endogenous TNFR-interacting proteins by a proteomic approach. J Biol Chem 279, 53266-53271.

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Abstract

Cette invention concerne l'utilisation de certains composés de pyrimidine dans le but d'inhiber, par exemple, TBK1 et/ou IKKepsilon et qui partant, peuvent s'utiliser pour le traitement du choc septique lié au cancer et/ou du glaucome primitif angle ouvert (GPAO).
PCT/GB2008/002967 2007-09-03 2008-09-02 Utilisation de composés contre le cancer/choc septique Ceased WO2009030890A1 (fr)

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Application Number Priority Date Filing Date Title
GBGB0717069.9 2007-09-03
GB0717069A GB0717069D0 (en) 2007-09-03 2007-09-03 Use of compounds for cancer/septic shock
GBGB0724126.8 2007-12-11
GB0724126A GB0724126D0 (en) 2007-12-11 2007-12-11 Use of compounds for cancer/septic shock

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Cited By (19)

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WO2011046970A1 (fr) * 2009-10-12 2011-04-21 Myrexis, Inc. Composés d'amino-pyrimidine en tant qu'inhibiteurs de tbkl et ou d'ikk epsilon
WO2012161877A1 (fr) 2011-05-23 2012-11-29 Merck Patent Gmbh Dérivés de pyridine et pyrazine
WO2013024282A2 (fr) 2011-08-15 2013-02-21 Domainex Limited Composés et leurs utilisations
DE102011112978A1 (de) 2011-09-09 2013-03-14 Merck Patent Gmbh Benzonitrilderivate
DE102011119127A1 (de) 2011-11-22 2013-05-23 Merck Patent Gmbh 3-Cyanaryl-1H-pyrrolo[2.3-b]pyridin-Derivate
WO2013117285A1 (fr) 2012-02-09 2013-08-15 Merck Patent Gmbh Dérivés de la furo[3,2-b]- et de la thiéno[3,2-b] pyridine comme inhibiteurs de tbk1 et ikk
US9249114B2 (en) 2011-05-23 2016-02-02 Merck Patent Gmbh Thiazole derivatives
KR101660863B1 (ko) 2015-04-03 2016-09-28 주식회사 녹십자 IKKε 및 TBK1 억제제로서의 7-아자인돌 또는 4,7-다이아자인돌 유도체 및 이를 포함하는 약학적 조성물
KR101663277B1 (ko) 2015-03-30 2016-10-06 주식회사 녹십자 TNIK, IKKε 및 TBK1 억제제로서의 피라졸계 유도체 및 이를 포함하는 약학적 조성물
KR20160127568A (ko) 2015-04-27 2016-11-04 주식회사 녹십자 TNIK, IKKε 및 TBK1 억제제로서의 화합물 및 이를 포함하는 약학적 조성물
US9556426B2 (en) 2009-09-16 2017-01-31 Celgene Avilomics Research, Inc. Protein kinase conjugates and inhibitors
WO2017106556A1 (fr) * 2015-12-17 2017-06-22 Gilead Sciences, Inc. Composés inhibiteurs de kinases se liant à tank
WO2018019341A1 (fr) 2016-07-26 2018-02-01 Karl Rosa Procédé de transfection avec systèmes de transfert de gènes non viraux
US10040781B2 (en) 2014-09-26 2018-08-07 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
US10072001B2 (en) 2014-06-03 2018-09-11 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
CN109718374A (zh) * 2017-10-27 2019-05-07 中国科学院上海生命科学研究院 Irf3抑制剂在制备yap过度激活的癌症的治疗或预防药物中的用途
WO2019156438A1 (fr) 2018-02-07 2019-08-15 Korea Research Institute Of Chemical Technology Composés de phényle fusionné à un hétérocycle pour l'inhibition de tnik et utilisations médicales associées
WO2019156439A1 (fr) 2018-02-07 2019-08-15 Korea Research Institute Of Chemical Technology Composés destinés à l'inhibition de tnik et utilisations médicales associées
US11542492B2 (en) 2009-12-30 2023-01-03 Celgene Car Llc Ligand-directed covalent modification of protein

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US20060212007A1 (en) * 2005-02-24 2006-09-21 Peters Donna M Method for reducing intraocular pressure using integrin-linked kinase inhibitor

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10662195B2 (en) 2009-09-16 2020-05-26 Celgene Car Llc Protein kinase conjugates and inhibitors
US9556426B2 (en) 2009-09-16 2017-01-31 Celgene Avilomics Research, Inc. Protein kinase conjugates and inhibitors
WO2011046970A1 (fr) * 2009-10-12 2011-04-21 Myrexis, Inc. Composés d'amino-pyrimidine en tant qu'inhibiteurs de tbkl et ou d'ikk epsilon
JP2013507449A (ja) * 2009-10-12 2013-03-04 ミレクシス， インコーポレイテッド ＴＢＫ１および／またはＩＫＫεの阻害剤としてのアミノ−ピリミジン化合物
US11542492B2 (en) 2009-12-30 2023-01-03 Celgene Car Llc Ligand-directed covalent modification of protein
US9249114B2 (en) 2011-05-23 2016-02-02 Merck Patent Gmbh Thiazole derivatives
WO2012161877A1 (fr) 2011-05-23 2012-11-29 Merck Patent Gmbh Dérivés de pyridine et pyrazine
US9273029B2 (en) 2011-05-23 2016-03-01 Merck Patent Gmbh Pyridine-and pyrazine derivatives
WO2013024282A3 (fr) * 2011-08-15 2013-05-16 Domainex Limited Composés et leurs utilisations
WO2013024282A2 (fr) 2011-08-15 2013-02-21 Domainex Limited Composés et leurs utilisations
WO2013034238A1 (fr) 2011-09-09 2013-03-14 Merck Patent Gmbh Dérivés de benzonitrile en tant qu'inhibiteurs de kinases
DE102011112978A1 (de) 2011-09-09 2013-03-14 Merck Patent Gmbh Benzonitrilderivate
WO2013075785A1 (fr) 2011-11-22 2013-05-30 Merck Patent Gmbh Dérivés 3-cyanaryl-1h-pyrrolo[2,3-b]pyridine
DE102011119127A1 (de) 2011-11-22 2013-05-23 Merck Patent Gmbh 3-Cyanaryl-1H-pyrrolo[2.3-b]pyridin-Derivate
WO2013117285A1 (fr) 2012-02-09 2013-08-15 Merck Patent Gmbh Dérivés de la furo[3,2-b]- et de la thiéno[3,2-b] pyridine comme inhibiteurs de tbk1 et ikk
US10072001B2 (en) 2014-06-03 2018-09-11 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
US10259811B2 (en) 2014-06-03 2019-04-16 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
US10253019B2 (en) 2014-09-26 2019-04-09 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
US10040781B2 (en) 2014-09-26 2018-08-07 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
KR101663277B1 (ko) 2015-03-30 2016-10-06 주식회사 녹십자 TNIK, IKKε 및 TBK1 억제제로서의 피라졸계 유도체 및 이를 포함하는 약학적 조성물
KR101660863B1 (ko) 2015-04-03 2016-09-28 주식회사 녹십자 IKKε 및 TBK1 억제제로서의 7-아자인돌 또는 4,7-다이아자인돌 유도체 및 이를 포함하는 약학적 조성물
KR20160127568A (ko) 2015-04-27 2016-11-04 주식회사 녹십자 TNIK, IKKε 및 TBK1 억제제로서의 화합물 및 이를 포함하는 약학적 조성물
CN108430993A (zh) * 2015-12-17 2018-08-21 吉利德科学公司 Tank-结合激酶抑制剂化合物
WO2017106556A1 (fr) * 2015-12-17 2017-06-22 Gilead Sciences, Inc. Composés inhibiteurs de kinases se liant à tank
US10316049B2 (en) 2015-12-17 2019-06-11 Gilead Sciences, Inc. Tank-binding kinase inhibitor compounds
WO2018019341A1 (fr) 2016-07-26 2018-02-01 Karl Rosa Procédé de transfection avec systèmes de transfert de gènes non viraux
DE102016113714A1 (de) 2016-07-26 2018-02-01 Rosa Karl Transfektionsverfahren mit nicht-viralen Genliefersystemen
CN109718374A (zh) * 2017-10-27 2019-05-07 中国科学院上海生命科学研究院 Irf3抑制剂在制备yap过度激活的癌症的治疗或预防药物中的用途
WO2019156439A1 (fr) 2018-02-07 2019-08-15 Korea Research Institute Of Chemical Technology Composés destinés à l'inhibition de tnik et utilisations médicales associées
WO2019156438A1 (fr) 2018-02-07 2019-08-15 Korea Research Institute Of Chemical Technology Composés de phényle fusionné à un hétérocycle pour l'inhibition de tnik et utilisations médicales associées
EP4477271A2 (fr) 2018-02-07 2024-12-18 Korea Research Institute of Chemical Technology Composés pour inhiber la tnik et utilisations médicales associées

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