WO2009030890A1 - Pyrimidine compounds for the treatment of cancer, septic shock and/or primary open angle glaucoma - Google Patents

Abstract

The present invention relates to the use of certain pyrimidine compounds to inhibit, for example, TBKl and/or IKK epsilon and which may therefore find application in treating cancer septic shock and/or Primary open Angle Glaucoma (POAG).

Description

PYRIMIDINE COMPOUNDS FOR THE TREATMENT OF CANCER, SEPTIC SHOCK AND/OR PRIMARY OPEN ANGLE GLAUCOMA

Field of the Invention

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).

Background to the Invention

Approximately 500 protein kinases are encoded by the human genome and in recent years the importance of protein kinases as drug targets has increased with substantial resource being invested by the drug discovery industry to identify suitable modulators of this enzyme class.

In addition to providing important starting points for the development of suitable new therapeutics small cell-permeant inhibitors of protein kinases have become invaluable reagents with which to investigate the physiological roles of protein kinases, because they can be used simply and rapidly to block endogenous kinase activity in normal cells and tissues, as well as transformed cell lines. In recent years a plethora of protein kinase inhibitors have become available commercially and researchers are often faced with a bewildering variety of compounds from which to choose from, each purported to be a "specific" inhibitor of a particular protein kinase. It is therefore difficult to decide which compound will switch off the activity of the protein kinase or signalling pathway under investigation, both effectively and specifically. 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.

Feldman et al (2005) J. Biol.Chem. 280 pi 9867- 19874 describes a number of small molecule inhibitors of PDKl. The compounds are described in WO2004/048343. The compounds are shown to be effective in inhibiting growth of a range of tumor cell lines.

Primary Open Angle Glaucoma (POAG) is a leading cause of irreversible blindness affecting 35 million people worldwide. The disease is genetically heterogeneous and mutations in the protein optineurin (OPTN) are associated with a form of POAG associated with normal intraocular pressure, termed Normal Tension Glaucoma (NTG) or Low Tension Glaucoma (LTG) [8,9]. A study of 54 families with autosomal dominant adult onset glaucoma, 17% had one of gour sequence mutations in OPTN, the most prevalent being the OPTN[E50K] mutant. How this mutation in OPTN might cause POAG is unknown. However, tumour necrosis factor α (TNFα) has been reported to increase the severity of damage in optic nerve heads of POAG and LTG subjects [10,11]. Moreover, exposure to TNFα [12] induces the de novo expression of optineurin. These observations suggest that some forms of POAG may be caused by an abnormal response to this cytokine. However, little is known in rrelation to what kinases may bind OPTN.

It is amongst the objects of the present invention to provide compounds which display a high degree of activity and/or specificity to particular kinases and may therefore serve as drug candidates or as starting points for further derivatisation and kinase inhibition studies.

It is a further object of the present invention to provide compounds for potential use as drug candidates for use in treating cancer septic shock and/or POAG.

It is a further object to provide compounds which display a significant inhibitory effect on one or more of the following kinases: TBKl, IKKepsilon, MARK3, AURORA B, AURORA C, MNK2, ERK8 and/or PDKl.

Summary of the Invention

In a first aspect there is provided use of a compound according to formula (I)

wherein n is from 2-6; Ri is a 5 or 6 membered cyclic alkyl ring or aromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an O, N or S; R₂ is a 5 or 6 membered cyclic alkyl ring or arromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an O, N or S, or is a linear or branched substituted or unsubstituted Ci-C₆ alkyl, C₁-C₆ alkoxy or C₂-C₆ alkenyl, wherein when substituted the substituent group may be a C(=0)NH₂;C00H; OH, NH₂, NO₂, and R3 is a halo, H, CH₃, CN, NO₂ for the manufacture of a medicament for treating a disease where it is desirable to inhibit one or more of the following kinases: TBKl, IKKepsilon, MARK3, AURORA B, AURORA C₅ MNK2, ERK8 and/or PDKl.

Preferably n is 2-4, most preferably 3.

Preferably R₁ is a 5 or 6 membered cyclic alkyl wherein optionally one carbon is substituted by an N. More preferably Ri is a pyrrolidine group such as 1- pyrollidinyl. Preferably, R₂ is a 5 or 6 membered arromatic ring wherein optionally one carbon is substituted by S, or is a branched C₂-C₄ alkyl substituted by COOH, or Q=O)NH₂. More preferably, R₂ is a thiophene group such as 2-thienyl, or is the group

Preferably R₃ is halo, such as I or Br.

Particularly preferred compounds are represented by formula (II) and (III) shown below

(II)

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. When 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.

Hence, according to another aspect of the present invention, there is provided a method of treating an ERK8, MNK2, Aurora B, Aurora C, MARK3, IKKe and/or TBKl and optionally PDKl related disease or disorder. The method according to this aspect of the present invention 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 se₅ or, more preferably, as a part of a pharmaceutical composition, mixed with, for example, a pharmaceutically acceptable carrier, as is detailed hereinunder. The term "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.

The term "administering" as used herein 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. As used herein, 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.

Herein, the term "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.

Herein, the term "preventing" refers to a method for barring an organism from acquiring a disorder or disease in the first place.

The term "therapeutically 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.

For any compound used in this invention, a therapeutically effective amount, also referred to herein as a therapeutically effective dose, can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ 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.

Moreover, 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 ED₅₀. The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD₅₀ and ED₅₀. 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 ED₅₀ 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. Preferably, therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake, 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.

As used herein, 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.

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.

Thus, in a further aspect there is provided use of 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.

For use according to the present invention, 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.

Pharmaceutical formulations 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.

Pharmaceutical formulations 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. Alternatively, 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. As one possibility 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, and 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.

As a further possibility 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.

It should be understood that in addition to the aforementioned carrier ingredients 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.

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.

In a further aspect there is provided 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.

In a further aspect there is provided 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.

In a further aspect there is provided 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.

In the present study the inventors found that 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. Interestingly, 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).

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 IKKepsilon, as they are the most potent inhibitors of these two protein kinases to be described thus far.

Thus, 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. Such 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.

Detailed Description

The present invention will now be further described by way of example and with reference to the Figures which show:

Figure 1 shows that OPTN interacts with TBKl in mammalian cells. (A) 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 β (5 ng/ml) and lysed in 0.5 ml cell lysis buffer. In order to pull down HA-OPTN bound to GST-TBKl, an aliquot of the cell extract (1 mg protein) was added to 20 μl of gluthatione-Sepharose beads, and after 1 h at 4⁰C 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. After denaturation in SDS, the supernatants were subjected to SDS-PAGE, transferred to nictrocellulose membranes and immunoblotted (IB) with anti-HA to detect the presence of HA-OPTN (B) Cell extracts from A (1 mg protein) were added to 10 μl of protein G-Sepharose beads coupled to 5 μg of HA antibody. After incubation for 2 h at 4°C 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- lβ. Following cell lysis, the extracts (2 mg protein) were incubated for Ih at 4°C with anti-OPTN (4 μg). Protein G-Sepharose (10 μl 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 μg protein) was also immunoblotted with antibodies that recognise the active phosphorylated forms of ERKl and ERK2 (ERK 1/2) to monitor stimulation by IL- lβ (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. After pulling down GST-OPTN from the cell extracts, bound proteins were released by denaturation in 1% SDS, subjected to SD-PAGE, transferred to nitrocellulose membranes and immunoblotted with antibodies that recognise HA. The membranes were stripped and re-probed with antibodies that recognise GST. (E) 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- lβ. Following cell lysis, the HA-OPTN was immunoprecipitated from cell extract (1 mg protein) with anti-HA (5 μg) 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). After 48 h, 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 μg protein) were also immunoblotted with HA-antibodies as in Figure 1. C. 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.

Materials and Methods

Protein kinase inhibitors BX 795 and BX 320 were synthesised in accordance with the teaching of WO 2004/048343.

Source and purification of kinases.

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.

Expression in E.coli

The following proteins were expressed in E.coli:- CHK2[5-543], cyclin- dependent protein kinase 2 (CDK2), MAP kinase-interacting kinase 2 (MNK2), extracellular signal-regulated kinase 1 (ERKl). Expression in SOl cells

The following 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 Bβ (PKBβ[120-481][S474D], 3-phosphoinositide-dependent protein kinase-1 [52-556] (PDKl [52-556], IKKε, TBKl,

Activation of protein kinases

In order to produce activated forms of Aurora B and Aurora C, 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 p38α MAP kinase; PKBα, PKBβ, with PDKl, and ERKl with MKKl. To activate CDK2, 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. It was then activated with CAK1/CDK7 followed by chromatography on nickel-nitrilotriacetate agarose to remove CAK1/CDK7, which binds to this column by virtue of its C-terminal His6 tag. All the other protein kinases were active as expressed.

Protein kinase assays

All assays (25.5μl) were carried out at room temperature (21⁰C) and were linear with respect to time and enzyme concentration under the conditions used. Assays were performed for 30 min using Multidrop Micro reagent dispensers (Thermo Electron Corporation, Waltham, MA 02454, USA) in a 96- well format. The concentration of magnesium acetate in the assays was 10 mM and the [γ-33P] ATP (800 cpm / pmol) was used at 5, 20 or 50 μM as indicated, in order to be at or below the Km for ATP for each enzyme. Protein kinases assayed at 5 μM ATP were:- ERKl, ERK8, PKBα, MARK3, Aurora C. Protein kinases assayed at 20 μM ATP were:- JNKl, PDKl₅CHKl, CHK2, CDK2 and Aurora B. Protein kinases assayed at 50 μM ATP were:-MNK2, IKKepsilon and TBKl.

The assays were initiated with MgATP, stopped by addition of 5 μl of 0.5 M orthophosphoric acid and spotted on to P81 fϊlterplates 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 (300μM), ERKl and ERK8, against myelin basic protein (MBP, 0.33mg/ml). MARK3 was assayed against the peptide KKKVSRSGLYRSPSMPENLNRPR (300μM), MNK2 against the eIF4E protein (0.5mg/ml). PKBβ was assayed against the peptide GRPRTSSFAEGKK (30μM). TBKl were assayed against (AKPKGNKDYHLQTCCGSLAYRRR) (300 μM). The substrates used for other protein kinases were described previouslyfl, 2].

Unless stated otherwise, 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

Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1% (v/v) 2-mercaptoethanol.

Further experimental procedures

Materials. The HA antibody and Interleukin-lβ (IL- lβ) 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.

DNA cloning. Full length 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.

Cell culture and immunoblotting. The IL-IR cells were cultured and lysed [13] and SDS-PAGE and immunoblotting carried out as described previously [14].

Results

Table 1: Inhibition of protein kinases by BX795 and BX320.

The concentrations of compounds used in the assays are indicated below each molecule and the results are presented as % activity remaining in the presence of inhibitors compared to control incubations with inhibitor omitted (averages of duplicate determinations). Each experiment was repeated two or three times with similar results. Further details of the assays are given under experimental procedures. It should be noted that a further approximately 70 different kinases were also tested and the compounds had little or poor activity towards these kinases. As such, the data shown is only in relation to the kinases which are affected to a significant degree and the data below is provided as an example.

The present work therefore shows that the compounds BX 320 and BX 795 are not specific inhibitors of PDKl, as suggested by Feldman et al (2005), referred to above, but show significant activity towards further kinases, notably TBKl, IKKepsilon, MARK3, Aurora C, Aurora B, MARK2 and ERK8. These new observations open up the possibility of treating conditions not previously suggested in the art.

In Summary

The inventors show herein that BX795 and related compounds are not only inhibitors of PDKl but also inhibit ERO, MNK2, Aurora B, Aurora C, MARK3 and IKKε 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.

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 IKKε, 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.

Further experimentation in relation to binding of TBKl to OPTN

To identify proteins that interact with OPTN, yeast two hybrid screens were carried out using OPTN as bait. Approximately 1 x 10⁶ 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.

In initial experiments, we studied the association between OPTN and TBKl by co-expression of vectors encoding glutathione S-transferase (GST) fused to TBKl and haemagglutinin (HA)-tagged OPTN in human embryonic kidney (HEK) 293 cells that stably express the IL-I receptor (termed IL-IR cells). We found that the expressed HA-OPTN could be "pulled down" on glutathione-Sepharose together with GST-TBKl (Figure IA), while GST-TBKl could be immunoprecipitated with anti- HA antibodies (Figure IB). We also showed that the endogenous TBKl in IL-IR cells could be immunoprecipitated with the endogenous OPTN (Figure 1C). The interaction between OPTN and TBKl was not affected by stimulation with IL-I (Figures IB, IB and 1C).

Strikingly, the amount of TBKl bound to OPTN was enhanced when wild type (WT)-OPTN was replaced by the OPTN[E50K] mutant that is associated with LTG forms of POAG (Figures IA and IB). In contrast, the OPTN[R545Q] mutant, which has been detected in a few individuals with POAG [8,15,16], but does not show association with POAG in most populations [15,16], interacted with TBKl in a similar manner to WT-OPTN (Figures IA and IB). When the tags were reversed and vectors expressing GST-OPTN co-transfected with vectors expressing HA-TBKl, the results were even more striking. Only the GST-OPTN[E50K] mutant, but not WT- OPTN or OPTN[R545Q], interacted with TBKl (Figure ID). These experiments suggested that the large GST-tag attached to the N-terminus of OPTN may interfere with binding to wild-type TBKl, this interference being overcome, at least partially, in the OPTN[E50K] mutant, presumably as a result of a conformational change.

Interestingly, when HA-OPTN or HA-OPTN[E50K] were transfected into IL- IR cells and immunoprecipitated with anti-HA, endogenous TBKl was only associated with HA-OPTN[E50K] and not HA-OPTN (Figure IE). These observations again suggested that HA-OPTN[E50K]was much more efficient than HA-OPTN at displacing TBKl from binding partners (including endogenous OPTN) in these cells. Similar results were obtained with OPTN-HA in which the tag was attached to the C-terminus of OPTN.

Three other proteins, termed TANK [17], NAPl [18] and SINTBAD [19] have been reported to bind to TBKl. We noticed that residues 78-121 of OPTN possessed significant homology with the TBKl -binding domains of TANK, NAPl and SINTBAD that have been identified previously [19] (Figure 2A). Interestingly, in one adult with POAG there was a two base pair insertion that resulted in the expression of a truncated OPTN protein in which only the N- terminal 127 residues of the protein were expressed [8]. We found that OPTN[I -127] in which Glu50 was changed to Lys interacted with TBKl, although OPTN[I -127] without this mutation did not (Figure 2B). This finding is consistent with the notion that residues 78-121 are important for the binding of TBKl to OPTN. Interestingly, this region includes Met98 and there is a statistical association of the Met98Lys mutant with NTG forms of glaucoma in some human populations [8,15,16,20], but not others [16,21]. However, unlike the OPTN[E50K] mutant, the OPTN[M98K] mutant did not show increased binding to TBKl in co-transfection experiments (Figure 2C).

The results described above suggested that OPTN might bind to the same region of TBKl as TANK₅ NAPl and SINTBAD. It has been shown previously that deletion of the C-terminal 41 residues of TBKl (residues 688-729) prevents interaction with TANK [17] and in Figure 2D we show that, similar to TANK, wild type OPTN is also unable to bind TBKl [1-688]. This is in agreement with the yeast two-hybrid results described earlier, which showed that OPTN interacts with the C- terminal region of TBKl . Taken together, these results indicate that TBKl is likely to bind via its terminus to the homologous regions of four different proteins, namely TANK, NAPl, SINTBAD and OPTN.

Discussion

The results described in the paper have identified TBKl as a protein kinase that interacts with OPTN, and have also shown the OPTN[E50K] mutant that is associated with POAG exhibits a strikingly enhanced interaction with TBKl. These findings raise the question of whether the enhanced interaction of OPTN[E50K] with TBKl is the cause of POAG. Several lines evidence suggest that this could be the case. Firstly, TBKl is activated when cells are stimulated with TNFα [22,23] and TNFα increases the severity of damage in the optic nerve heads of POAG and LTG subjects [10,11]. Secondly, mice that do not express TBKl die just prior to birth because they become hypersensitive to TNFα-induced apoptosis of the liver and lethality can be rescued by crossing to mice that do not express TNFRl [22]. Thirdly, the adenovirus E3-14.7K protein binds to OPTN, and this interaction blocks the protective effect of the E3-14.7K protein on TNFα-induced apoptosis [24]. These observations indicate that OPTN and TBKl play critical roles in the regulation of TNFα-stimulated apoptosis and suggest that mutations interfering with the normal functioning of these proteins may sensitise cells to TNFα-induced damage.

References

1. Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95-105.

2. Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. (2003) The specificities of protein kinase inhibitors: an update. Biochem J 371, 199-204.

59. Bayascas, J. R., Leslie, N. R., Parsons, R., Fleming, S. and Alessi, D. R. (2005) Hypomorphic mutation of PDKl suppresses tumorigenesis in PTEN(+/-) mice. Curr Biol 15, 1839-1846.

3. Korherr, C, Gille, H., Schafer, R., Koenig-Hoffmann, K., Dixelius, J., Egland, K.A., Pastan, I. and Brinkmann, U. (2006) Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBKl and the IRF3 pathway. Proc Natl Acad Sci U S A 103, 4240-4245

4. Chien, Y., Kim, S., Bumeister, R., Loo, Y. M., Kwon, S. W., Johnson, C. L., Balakireva, M. G., Romeo, Y., Kopelovich, L., Gale, M., Jr., Yeaman, C, Camonis, J. H., Zhao, Y. and White, M. A. (2006) RaIB GTPase-mediated activation of the IkappaB family kinase TBKl couples innate immune signaling to tumor cell survival. Cell 127, 157-170 937.

5. Perry et al (J Exp Med 199, 1651-1658, 2004) compared the role of TBKl in interferon responses induced by a number of stimuli. TBK-/- mice were deficient in their ability to up regulate IFN beta production.

6. 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.

8. Rezaie, T., Child, A., Hitchings, R., Brice, G., Miller, L., Coca-Prados, M., Heon, E., Krupin, T., Ritch, R., Kreutzer, D., Crick, R.P. and Sarfarazi, M. (2002) Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 295, 1077-1079.

9. Sarfarazi, M. and Rezaie, T. (2003) Optineurin in primary open angle glaucoma. Ophthalmol Clin North Am 16, 529-541.

10. Tezel, G. and Wax, M.B. (2000) Increased production of tumour necrosis factor-alpha by glial cells exposed to stimulated ischemia or elevated hydrostatic pressure induces apoptosis in cocultured retinal ganglion cells. J Neurosci 20, 8693-8700.

11. Yuan, L. and Neufeld, A.H. (2000) Tumor necrosis factor-alpha: a potentially neurodestructive cytokine produced by glia in the human glaucomatous optic nerve head. Glia 32, 42-50.

12. Schwamborn, K., Weil, R., Courtois, G., Whiteside, S. T. and Israel, A. (2000) Phorbol esters and cytokines regulate the expression of the NEMO-related protein, a molecule involved in a NF-kappa B-independent pathway. J Biol Chem 275, 22780-22789.

13. Stafford, MJ., Morrice, N.A., Peggie, M.W. and Cohen, P. (2006) Interleukin-l stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and SER62. FEBS Lett 580, 4010-4014.

14. Morton, S. Davis, RJ., McLaren, A. and Cohen, P. (2003) A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. Embo J 22, 3876-3886. 15. Alward, W.L., Kwon, Y.H., Kawase, K., Craig, J.E., Hayreh, S.S., Johnson, A.T., Khanna, C.L., Yamamoto, T., Mackey, D.A., Roos, B.R., Affatigato, L.M., Sheffield, V. C. and Stone, E.M. (2003) Evaluation of optineurin sequence variations in 1,048 patients with open-angle glaucoma. Am J Ophthalmol 136, 904- 910.

16. Ayala-Lugo, R.M., Pawar, H., Reed, D.M., Lichter, P.R., Moroi, S.E., Page, M., Eadie, J., Axocar, V., Maul, E., Ntim-Amponsah, C, Bromley, W., Obeng- Nyarkoh, E., Johnson, A.T., Kijek, T.G., Downs, C.A., Johnson, J. M., Perez- Grossmann, R.A., Guevara-Fujita, M.L., Fujita, R., Wallace, M.R. and Richards, J.E. (2007) Variation in optineurin (OPTN) allele frequencies between and within populations. MoI Vis 13, 151-163.

17. Cheng, G., and Baltimore, D. (1996) TANK, a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-kappaB activation. Genes Dev 10, 963-973.

18. Fujita, F., Taniguchi, Y., Kato, T., Narita, Y., Furuya, A., Ogawa, T., Sakurai, H., Joh, T., Itoh, M., Delhase, M., Karin, M. and Nakanishi, M. (2003) Identification of NAPl, a regulatory subunit of IkappaB ldnase-related kinases that potentiaties NF-kappaB signaling. MoI Cell Biol 23, 7780-7793.

19. Ryzhakov, G. and Randow, F. (2007) SINTBAD, a novel component of innate antiviral immunity, shares a TBKl -binding domain with NAPl and TANK.

20. Aung, T., Ebenezer, N.D., Brice, G., Child, A.H., Prescott, Q., Lehmann, O. J., Hitchings, R.A. and Bhattacharya, S. S. (2003) Prevalence of optineurin sequence variants in adult primary open angle glaucoma: implications for diagnostic testing. J Med Genet 40, elOl.

21. Wiggs, J.L., Auguste, J., Allingham, R.R., Flor, J.D., Pericak- Vance, M.A., Rogers, K. LaRocque, K.R., Graham, F.L., Broomer, B., Del Bono, E., Haines, J.L. and Hauser, M. (2003) Lack of association of mutations in optineurin with disease in patients with adult-onset primary open-angle glaucoma. Arch Ophthalmol 121, 1181-1183.

22. Bonnard, M., Mirtsos, C, Suzuki, S., Graham, K., Huang, J., Ng, M., Itie, A., Wakeham, A., Shahinian, A., Henzel, W.J., Elia, A.J., Shillinglaw, W., Mak, T. W., Cao, Z. and Yeh, W.C. (2000) Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription. Embo J 19, 4976-4985.

23. Kuai, J., Wooters, J., Hall, J.P., Rao, V.R., Nickbarg, E., Li, B., Chatterjee-Kishore, M., Qiu, Y. and Lin, L.L. (2004) 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.

Claims

Claims

1. Use of a compound according to formula (I)

wherein n is from 2-6; R₁ is a 5 or 6 membered cyclic alkyl ring or aromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an O, N or S; R₂ is a 5 or 6 membered cyclic alkyl ring or arromatic cyclic ring, wherein optionally one or more carbons in the ring structure is substituted by an O, N or S, or is a linear or branched substituted or unsubstituted C₁-C₆ alkyl, Ci-C₆ alkoxy or C₂-C₆ alkenyl, wherein when substituted the substituent group may be a C(=O)NH₂;COOH; OH, NH₂, NO₂, and R3 is a halo, H, CH₃, CN, NO₂ for the manufacture of a medicament for treating a disease where it is desirable to inhibit one or more of the following kinases: TBKl, IKKepsilon, MARK3, AURORA B, AURORA C, MNK2, ERK8 and/or PDKl.

2. Use according to claim 1 wherein preferably n is 2-4.

3. Use according to either of claim 1 or 2 wherein Ri is a 5 or 6 membered cyclic alkyl wherein optionally one carbon is substituted by an N.

4. Use according to claim 3 wherein R₁ is a pyrrolidine group such as 1- pyrollidinyl.

5. Use according to any preceding claim wherein R₂ is a 5 or 6 membered aromatic ring wherein optionally one carbon is substituted by S, or is a branched C₂- C₄ alkyl substituted by COOH, or C(=0)NH₂.

6. Use according to any preceding claim wherein R₂ is a thiophene group such as 2-thienyl, or is the group

7. Use according to any preceding claim wherein R₃ is halo, such as I or Br.

8. Use according to claim 1 wherein the compounds are selected from shown below

9. Use according to any preceding claim wherein 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.

10. Use according to any preceding claim wherein the kinase related disease or disorder refers to a disease or disorder charecterized by inappropriate kinase activity or over-activity of a kinase.

11. Use according to any preceding claim wherein 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.

12. Use according to any preceding claim for use in preventing, treating and/or studying cell proliferative disorders, especially cancer such as, 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.

13. Use according to any of claims 1 to 11 for use in preventing, treating and/or studying septic shock.

14. Use according to any of claims 1 to 11 for use in treating POAG and/or diseases associated with optineurin activity.

15. Use of 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.

16. Use according to claim 15 wherein the mutant is the OPTN (E50K) mutant.

17. 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.

18. 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.

19. 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, such as a compound according to formulae I, II or III.

20. Use according to any of claim 1 to 11 for the manufacture of medicaments for the treatment of diseases where it is desirable to inhibit TBKl and/or IKK epsilon.

21. Use according to claim 20 for treating colon and breast cancer, viral infection, septic shock and/or POAG.