WO2006059121A2 - Modulators of spindle checkpoint kinases and a taxol - Google Patents

Abstract

The invention disclosed relates to spindle checkpoint kinase modulators, especially modulators of Mps 1 kinase which may be useful in the treatment and diagnosis of disease, such as cancer. Pharmaceutical compositions and treatments are disclosed comprising a modulator of Mps l kinase activity and a taxol .

Description

Materials and Methods Relating to Modulators of Spindle

Checkpoint Kinases

Field of the Invention

The present invention relates to spindle checkpoint kinase modulators useful in the treatment and diagnosis of disease and particularly although not exclusively to modulators of Mpsl kinase activity, such as anthrapyrazolone compounds, optionally in combination with a taxol, in such treatment and diagnosis .

Background to the Invention

Taxol and taxol derivatives, e.g. Paclitaxel™ and Docetaxel™ / Taxotere, are widely employed drugs in chemotherapeutic cancer treatment. Besides reported anti-angiogenic and pro-apoptotic functions, the major benefits of these drugs appears to rely on the cytostatic action resulting from their stabilizing effect on microtubule dynamics¹. The microtubule-stabilizing activity of taxol disturbs mitotic spindle functions and prevents establishment of proper bipolar chromosome attachment resulting in a mitotic arrest. This arrest is enforced by the spindle assembly checkpoint that is active as long as chromosomes are still present that are not correctly attached to the spindle. Prolonged treatment with spindle poisons such as taxol ultimately forces cells into a mitotic catastrophe or causes mitotic exit without cytokinesis, both contributing to the lethal effects of taxol.

The chemotherapeutic benefits provided by taxol are dose limited in that high doses of taxol consistently lead to toxicity and unwanted side-effects including severe abdominal pain, neuro- and liver toxicity. The spindle assembly checkpoint is a quality control mechanism, which ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are bipolarly attached to the mitotic spindle.

The core sensory machinery constituting the spindle assembly checkpoint is a multi-protein complex assembled at the kinetochore, a proteinaceous structure embracing the centromeric region of each chromatid that organizes the complex microtubule-chromosome interactions during mitosis. Intensive research has provided compelling evidence that especially two checkpoint proteins of this sensor complex, Mad2 and BubRl, fulfil a crucial function in spindle assembly checkpoint signalling². In the absence of bipolar attachment both proteins dynamically interact with kinetochores^3"6 where they are presumably converted into an active form that can bind and inhibit Cdc20^7"17, a regulatory subunit of an E3 ubiquitin ligase essential for anaphase progression called the anaphase-promoting complex (APC)¹³. Recruitment and activation of Mad2 requires Madl^14"17, while BubRl activation requires

CENP-E¹⁸'¹⁹, underscoring that checkpoint function is a result of complex interplay between the different checkpoint components. Upon successful achievement of bipolar attachment and/or generation of tension, Mad2 and BubRl are displaced from the kinetochore leading to the release of Cdc20 which ultimately triggers APC-mediated degradation of essential mitotic targets such as Securin and Cyclin B allowing anaphase and mitotic exit to occur²⁰.

Most spindle checkpoint components are phosphorylated during mitosis and several of them are kinases²'²¹. An important kinase regulating checkpoint function is the tyrosine and serine/threonine dual-specifity kinase monopolar spindle 1 (Mpsl)²². Mutation of Mpsl in S. cerevisiae causes a monopolar spindle resulting from defective duplication of the yeast equivalent of the centrosome, called the spindle pole body (SPB) . In various species depletion or inactivation of Mpsl results in a failure of cells to arrest in mitosis in response to microtubule-depolymerizing drugs such as nocodazole^23"25. Conversely, overexpression of wild type Mpsl induces a mitotic arrest without obvious signs of spindle damage²⁶. This arrest could be abrogated by disruption of various checkpoint genes, among others also Madl, Mad2 and the yeast equivalent of BubRl, Mad3²⁶, indicating that Mpsl acts at or near the top of the pathway and fulfils a regulatory function in spindle assembly checkpoint signalling.

The amino acid and nucleotide sequence for human Mpsl is available from the NCBI database (http: //www.ncbi .nlm.nih.gov) under accession number NM_003318 (GI : 34303964) .

SP600125 (anthrafl, 9-cd]pyrazol-6 (2H) -one) is an anthrapyrazolone compound reported to be a specific and reversible inhibitor for stress activated MAP Kinases of the JNK family²⁷'²⁸.

Summary of the Invention

The inventors have found that SP600125 potently inhibits Mpsl activity in vitro and effectively ablates spindle checkpoint function in a JNK-independent manner. SP600125 has been shown to efficiently override both taxol- and nocodazole- induced mitotic blocks indicating that it targets an important spindle checkpoint component. The inventors have shown Mpsl kinase inhibition by SP600125 to be sufficient to inactivate the spindle assembly checkpoint.

Cell toxicity studies described here reveal a strong synergistic effect of SP600125 on (taxol) -mediated apoptosis of human cancer cells that was not evident with normal somatic cells. In light of the serious side-effects associated with high-dose taxol administration in cancer therapy the data presented here provides for a more acceptable anti-cancer strategy that is based on combined application of spindle- disrupting and checkpoint-silencing drugs enabling the forced progression of cancer cells into a mitotic catastrophe.

The inventors have shown that Mpsl inhibition strongly potentiates the known chemotherapeutic taxol. Given the present dose limitations for taxol in chemotherapeutic applications, this strong potentiation enables the chemotherapeutic properties of taxol to be enhanced without the need to increase the dose of taxol administered to the patient, hence avoiding the unwanted side-effects.

The synergistic effect demonstrated is tumour cell selective - the effect is not observed in normal, non-tumour cells. Thus the novel chemotherapeutic strategy provided is self-targeting to tumour cells.

The inventors have further demonstrated the ability of Mpsl- inactive cells to break taxol-induced mitotic arrest and drive cells into a lethal mitosis. Low doses of SP600125, including doses too low to inhibit JNK or BubRl, have been demonstrated by the inventors to result in a 10-fold increase in taxol sensitivity in UTA-6 osteocarcinoma cells. Similar results were observed with HBLlOO 'and T47D breast carcinoma cells as well as DLDl-colon carcinoma cells. Apoptosis was clearly and very strongly observed in JNK1/2 double negative cells in response to treatment with SP600125 and taxol compared to taxol treatment alone, demonstrating that JNK inhibition did not contribute to this process.

Increasing the toxicity of taxol using a small-compound inhibitor that ablates spindle assembly checkpoint function, SP600125 has been shown to exert a synergistic effect on drug- induced cell death of cancer cells by the chemotherapeutic drug taxol. A similar synergistic toxicity was achieved by combining taxol-treatment with RNAi-mediated depletion of Mpsl. Surprisingly, primary cells prove remarkably resistant to SP600125-mediated checkpoint override and apoptosis. SP600125 and/or similar spindle checkpoint-silencing compounds in combinational chemotherapy with checkpoint-sustaining drugs of the taxotere family are provided.

At its most general the present invention relates to spindle checkpoint kinase modulators which may be useful in the treatment and diagnosis of disease, such as cancer.

Modulators of Mpsl kinase activity are provided, which may include anthrapyrazolone compounds, e.g. the small molecule SP600125. Pharmaceutical compositions comprising such compounds, optionally in combination with a taxol, are provided together with their use in methods of medical treatment and in the manufacture of medicaments for such use.

Mpsl kinase modulators may directly or indirectly inhibit Mpsl kinase activity. Mpsl inhibitors may comprise small molecule inhibitors, e.g. SP600125 or other anthrapyrazolones. Preferred Mpsl inhibitors demonstrate specific Mpsl kinase inhibition and are selective for Mpsl over other spindle checkpoint kinases.

Screening methods and assays for identifying modulators of Mpsl kinase activity are provided. Methods are also provided for identifying the ability of test compounds to improve the ability of Mpsl kinase modulators or taxols to cause cell death when administered to cells in combination with the test compound.

According to a first aspect of the present invention there is provided a pharmaceutical composition comprising an anthrapyrazolone and a taxol. According to a second aspect of the present invention there is provided a pharmaceutical composition comprising an anthrapyrazolone and a taxol for use in a method of medical treatment .

According to a third aspect of the present invention there is provided the use of an anthrapyrazolone and a taxol in the manufacture of a medicament for the treatment of disease.

According to a fourth aspect of the present invention there is provided the use of:

(a) an anthrapyrazolone; in the manufacture of a first medicament for co-administration with a second medicament comprising: (b) a taxol; in the treatment of disease.

According to a fifth aspect of the present invention there is provided the use of: (a) a taxol; in the manufacture of a first medicament for co-administration with a second medicament comprising:

(b) an anthrapyrazolone; in the treatment of disease.

According to a sixth aspect of the present invention there is provided a method of treating a disease comprising coadministering to an individual in need of such treatment a therapeutically effective amount of an anthrapyrazolone and a therapeutically effective amount of a taxol.

Alternatively, Mpsl kinase modulators may comprise nucleic acids, such as siRNA (short interfering RNA) . Suitable siRNAs may comprise any 17-25mer, e.g. and 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24- or 25-mer, and most preferably any 19mer, sequence identical to a selected contiguous portion of the Mpsl cDNA. Suitable siRNA sequences may be complementary to the selected sequence or comprise a sequence with at least 90%, more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the selected sequence or such complementary sequence.

As examples, suitable siRNA may comprise an RNA molecule having a sequence selected from:

5'-CCCAGAGGACTGGTTGAGT-S' (SEQ ID NO. 1); 5'-CCAGATACAACAAGTGTTG-S' (SEQ ID NO. 2);

5'-TCACTGGCAGATTCCGGAG-B' (SEQ ID NO. 3);

5'-GACACCAAGCAGCAATACC-B' (SEQ ID NO. 4);

5'-GATTCTCAGGTTGGCACAG-B' (SEQ ID NO. 5) .

SEQ ID NO. s 1-4 preferably specifically modulate human Mpsl kinase. SEQ ID NO.5 preferably modulates human or mouse Mpsl kinase. siRNA complementary to any of SEQ ID No.s 1-5, or in which base T (Thymine) is replaced by base U (Uracil), form other examples of suitable siRNA.

DNA molecules encoding such siRNA' s also form aspects of the invention. Nucleic acids or nucleic acid derivatives, e.g. containing base or sugar modifications, which hybridise to the RNA or DNA molecules described above under high or very high stringency conditions also form further aspects of the invention. In another aspect of the invention a nucleic acid modulator molecule may comprise an antisense Mpsl kinase nucleic acid. Such DNA or RNA molecules may be provided as Mpsl kinase modulator compositions, medicaments or formulations .

Accordingly, in another aspect of the invention an in vitro method is provided for reducing the level of functional Mpsl kinase protein expressed in a selected cell or cells, the method comprising transforming in vitro one or more cells with one or more nucleic acid modulator molecules selected from one or more of the nucleic acid modulators described above.

The transformed cells cultured in vitro form another aspect of the invention. Methods for introducing/transplanting said transformed cells to an individual in need of treatment may form a further aspect of the invention and a method of treating a disease in the individual to whom said transformed cells have been introduced by administration of a therapeutically effective amount of taxol forms yet another aspect of the invention. Said therapeutically effective amount of taxol may provide a synergistically improved treatment in said patient, enabling lower dosages of taxol to be administered to the patient to achieve a similar or greater therapeutic effect than in a patient to whom said transformed cells have not been introduced/transplanted.

A taxol may be administered to the transformed cells when cultured in vitro to determine the effect of said taxol on the transformed cultured cells. Such method forms a further aspect of the invention.

Diseases of the invention to be treated may comprise, and methods of medical treatment may be for, diseases involving cell death or cell proliferation. More preferably treatments and diagnoses may be provided for cancer, inflammatory diseases and hyperproliferative diseases such as psoriasis.

Cancer may comprise a tumour or neoplasm. Compositions, uses and methods of the invention are provided which are suitable for use in the treatment of cancers of any kind in an individual in need of such treatment which may be any animal or human. Administration of chemotherapeutic compositions and medicaments of the invention may suitably be combined with other chemotherapeutic treatments, e.g. combination therapy with cisplatin, vincristine or vinblastine, and/or with radio- therapeutic treatments.

Co-administration may comprise simultaneous administration of two or more compounds, for example by combining the two compounds in a single composition, e.g. tablet. Coadministration may also comprise sequential administration of compounds, the time period between administration of the first and second compounds being predetermined such that the two compounds are present in active form in the patients body at the same time in order that they may directly or indirectly interact and optionally produce a synergistic effect.

In a further aspect of the present invention a method is provided for identifying compounds which modulate the kinase activity of Mpsl kinase comprising:

(i) in vitro contacting Mpsl kinase with a test compound; and (ii) determining the activity of said Mps 1 kinase.

Wherein said contacting step may comprise in vitro contacting cells expressing Mpsl kinase with said test compound. The expressed Mpsl kinase may be recombinant and may be human wild type Mpsl.

The step of determining Mpsl kinase activity may comprise detecting, optionally quantitatively detecting, autophosphorylation of Mpsl. This detection may comprise immunoprecipitation, e.g. by western blot, with an Mpsl- specific antiserum or antibody and autoradiographic detection of incorporated ³²P. Alternatively the step of determining the activity of Mpsl kinase may comprise detecting phosphorylation of an Mpsl kinase substrate, e.g. the artificial Mpsl substrate Myelin basic protein (MBP) . The test compound may interfere with or disrupt the activity of Mpsl by preventing normal interaction of Mpsl with a substrate or binding partner. The determination of Mpsl activity may involve determining the extent of this interaction, e.g. by measuring the formation of bound complex of Mpsl and binding partner or by measuring the product formed by the Mpsl-substrate interaction.

In a further aspect of the present invention a method is provided for identifying a compound which modulates the activity of Mpsl kinase comprising: (i) providing a test compound; (ii) providing a first component comprising an Mpsl kinase polypeptide, homologue, mutant, derivative or fragment;

(iii) in vitro contacting the test compound and first component under conditions in which the test compound and first component may interact; (iv) detecting a modulation of the first component.

The modulation detected in step (iv) may be the kinase activity of the first component, which activity may be the phosphorylation or autophosphorylation of the first component.

Alternatively, the modulation may be a change in the ability of the first component to bind another molecule, e.g. a substrate of the first component.

The method may comprise a further step wherein after step (iii), the first component, having been contacted with the test compound, is contacted in vitro with a second component. In step (iv) modulation of the second component may be detected in addition to or instead of detection of modulation of the first component. In this alternative arrangement the second component is preferably an in vivo substrate of the first component, i.e. a cellular substrate of Mpsl kinase. The substrate may be a protein, polypeptide or peptide fragment thereof or may be a small molecule, e.g. organic substrate. The modulation detected may comprise the phosphorylation or dephosphorylation of the second component.

The test compound may modulate the activity of Mpsl kinase towards the second component. Contact of the first component and second component may be in the presence of the test compound or follow partitioning of the first component from unbound test compound after step (iii) .

To determine the degree of modulation of the second component the result of the detection step may be compared with the result of contacting the first and second components in the absence of the prior interaction (iii) of the first component and test compound.

One or more of the first and second components or test compound may be immobilised, e.g. bound to a solid support such as a column.

In another aspect of the present invention an assay kit is provided comprising a first container having a quantity of an Mpsl kinase polypeptide, homologue, mutant, derivative or fragment therein and a second container having a quantity of an antiserum or antibody capable of binding a modulated form of said Mpsl kinase polypeptide, homologue, mutant, derivative or fragment.

The modulated form may be a phosphorylated or de- phosphorylated form of said Mpsl kinase polypeptide, homologue, mutant, derivative or fragment.

Instructions for performing a screening method for identifying a compound which modulates the activity of Mpsl kinase may also be provided with said assay kit. In a further aspect of the present invention there is provided a method for identifying compounds which improve the ability of a taxol to cause cell death comprising: (i) in vitro contacting one or more cells with a taxol;

(ii) contacting said cells with a test compound; (iii) determining the amount or extent of cell death; and (iv) comparing said amount in (iii) to the amount of cell death caused when step (ii) is omitted.

The method may be a method of identifying particular anthrapyrazolone compounds which improve the ability of a taxol to cause cell death. For example, the test compound may be an anthrapyrazolone compound or anthrapyrazolone derivative, e.g. a derivative of SP600125.

In another aspect of the present invention there is a method for identifying compounds which improve the ability of a modulator of Mpsl kinase to cause cell death comprising:

(i) in vitro contacting one or more cells with a modulator of Mpsl kinase activity;

(ii) contacting said cells with a test compound; (iii) determining the amount or extent of cell death; and

(iv) comparing said amount in (iii) to the amount of cell death caused when step (ii) is omitted.

The modulator of Mpsl kinase activity is preferably an Mpsl kinase inhibitor, more preferably an anthrapyrazolone, still more preferably SP600125.

The method may be a method of identifying particular taxol compounds which improve the ability of an Mpsl modulator to cause cell death. The test compound may be a taxol or taxol derivative .

Improved ability of a compound to cause cell death may be a synergistic improvement, i.e. wherein a normal level of cell death is obtainable by contacting the cells with a given amount of taxol/Mpsl kinase modulator, the synergy comprising the ability to obtain the normal or a greater level of cell death by contacting a smaller amount (lower dose) of taxol/ Mpsl kinase modulator together with an amount of the test compound. A high level of synergy can be said to exist where the dose of test compound is also low.

The described methods for identifying compounds may be in vitro high throughput screening assays. Test compounds may be obtained from a synthetic library of compounds.

Kinase components of the methods of the present invention may be obtained from mammalian extracts, produced recombinantly from bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines, or synthesised de novo using commercially available synthesisers.

In a further aspect of the present invention there is provided a method of assaying for compounds capable of overriding a spindle assembly checkpoint, comprising the steps of:

(a) providing one or more cells arrested in mitosis;

(b) treating said cell(s) with one or more test compounds; and (c) observing the effect of said compound (s) on said cell (s) .

Preferably arresting cells in mitosis involves treatment with taxol or nocadazole as an arresting agent. Mitotic cells may then be detached from a substrate and collected. Further treatment with an arresting agent may be used to increase the yield of mitotic cells followed by further collection of mitotic cells. The mitotic cells collected may then be treated with one or more test compounds to test for the ability of a given test compound, or combination of compounds, to override the mitotic arrest induced by the arresting agent. This may be determined by observing changes in cell morphology. The method provides a means to rapidly identify compounds capable of overriding the spindle assembly checkpoint. Compounds or specific combinations of compounds, identified, may be further analysed to investigate the molecular basis of the override. The cells tested may be mammalian or human cells.

Pharmaceutical compositions according to the invention may further comprise a pharmaceutically acceptable carrier, adjuvant or diluent. They may be formulated for oral administration, e.g. in tablet form, or for administration by other routes such as injection, e.g. in combination with a suitable fluid carrier. Injection may be parenteral or may comprise direct injection to a tissue in need of treatment, e.g. a tumour. Other possible administration routes include nasal administration.

In this specification 'an anthrapyrazolone' may be any compound of the general molecular formula shown in Figure 8A or any anthrapyrazolone derivative, having the general structure shown in Figure 8A, but being further derivatised. Possible derivatives include N-alkyl (e.g. Ci-Ci₂) substituents or placement of a halogen (Cl, F, Br or I) at the 8- or 6- position. Preferred anthrapyrazolones include the compound SP600125, anthra[l, 9-cd]pyrazol-6 (2H) -one (Figure 8A) and/or any one of the compounds shown in Figures 8B-8F. The anthrapyrazolone preferably ablates spindle checkpoint function in human and/or mouse cells. The anthrapyrazolone may be a modulator of the activity of Mpsl and may be an inhibitor of Mpsl kinase activity. In this specification ^λa taxol' may be the tricyclic diterpene taxol (Paclitaxel™) isolatable from Taxus brevifolia, the Pacific yew, and having the molecular structure shown in Figure 7A, or it may be taxotere (Docetaxel™) having the molecular structure shown in Figure 7B, or it may comprise a molecular derivative of one of these structures. The taxol preferably has chemotherapeutic properties. The taxol preferably exhibits a microtubule stabilising activity in mouse and/or human cells and may prevent establishment of proper bipolar chromosome attachment such that the cells are urged to arrest in mitosis.

Compositions, uses and methods of the invention may also comprise or use anthrapyrazolone or taxol mimetics. Suitable mimetics are organic compounds modelled to resemble the three dimensional structure of a selected anthrapyrazolone or taxol or a pharmacophore of said anthrapyrazolone or taxol.

Anthrapyrazolones or taxols forming part of the present invention may be provided in the form of pharmaceutically acceptable salts or prodrugs known to the skilled person. Such salts or prodrugs may be prepared in order to deliver a desired concentration of anthrapyrazolone or taxol in the patients blood or tissue (s) following administration.

One or more anthrapyrazolone compounds may be provided in a given composition or for a given use of the present invention. Similarly, one or more taxol compounds may also be provided. For example a composition or medicament according to the present invention may comprise two distinct anthrapyrazolone compounds and a single taxol compound.

Inhibition of Mpsl kinase may also be provided by use of an anthrapyrazolone compound and a synergistic partner.

Synergistic partners may comprise drugs (or compounds) that inhibit microtubule polymerisation (microtubule-destabilising compounds) at high concentrations such as vinca alkaloids (e.g. vincristine, vinblastine), cryptophycine, halichondrine, estramustine and colchicines. Alternatively, synergistic partners may comprise drugs (or compounds) that stimulate microtubule polymerisation (microtubule-stabilising) at high drug concentrations. In addition to taxol and taxotere described above, these may further comprise eleutherobins, epothilones, laulimalide, sarcodictyins and discodermolide . Pharmaceutical compositions, their uses and methods corresponding to the aspects and preferred features described are provided which may involve the combination of an anthrapyrazolone with one or more of the synergistic partners recited above.

Modulation

Modulation describes the ability of a compound to vary the result of an interaction between interacting substances or molecules. Thus, modulation may be detectable by a change (increase or decrease) in the level of an activity, e.g. kinase activity or inability to bind to an interacting partner molecule. Modulating compounds may have an enhancing effect or an inhibiting effect on the relevant activity or binding.

Activity

The activity of a given substance or molecule may be measured by assaying for the activity, e.g. kinase activity can be measured by assaying for phosphorylation of a known substrate, which may be the kinase itself. An activity may be a function of the interaction or binding of the given substance, e.g. Mpsl kinase, with another molecule.

Polypeptide components

Mpsl kinase used in the screening methods of the present invention may comprise the full-length protein. However, this is not always necessary. As an alternative, homologues, mutants, derivatives or fragments of the full-length polypeptide may be used, provided an Mpsl Kinase activity of such alternative form is present.

Derivatives include variants of a given full length protein sequence and include naturally occurring allelic variants and synthetic variants which have substantial amino acid sequence identity to the full length protein.

Protein fragments may be up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 150 amino acid residues long. Minimum fragment length may be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 30 amino acids or a number of amino acids between 3 and 30.

Mutants may comprise one or more addition, substitution, inversion and/or deletion compared to the corresponding wild- type polypeptide. The mutant may display an altered activity or property, e.g. binding.

Derivatives may also comprise natural variations or polymorphisms which may exist between individuals or between members of a family. All such derivatives are included within the scope of the invention. Purely as examples, conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups: (i) alanine, serine, threonine; (ii) glutamic acid and aspartic acid; (iii) arginine and leucine; (iv) asparagine and glutamine;

(v) isoleucine, leucine and valine;

(vi) phenylalanine, tyrosine and tryptophan.

Derivatives may also be in the form of a fusion protein where the protein, fragment, homologue or mutant is fused to another polypeptide, usually by standard cloning techniques, which may contain a DNA-binding domain, transcriptional activation domain or a ligand suitable for affinity purification (e.g. glutathione-S-transferase or six consecutive histidine residues) .

Test compounds

Candidate test compounds may comprise small molecules, may be synthetic or naturally occurring and may comprise organic or inorganic compounds. They may comprise known enzyme active site inhibitors, either competitive or non-competitive. A candidate test compound may comprise a peptide or an organic (mimetic) compound mimicking a selected peptide structure.

Other examples of candidate test compounds include nonfunctional homologues of Mpsl kinase target molecules and antibodies and antibody products, e.g. monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grated antibodies, which recognise the Mpsl kinase or one of its cellular substrates.

Suitable compounds may result in a change of sub-cellular localisation of the target protein which may result in sequestering of the protein thus preventing normally interacting molecules from contacting each other within the cell.

The modulating or interfering effect of a test compound may be assayed for by measuring an ability to regulate the cell cycle or to precipitate growth arrest or apoptosis. Such an assay may comprise (a) administering the candidate substance to a test cell, preferably a mammalian cell, the administration may optionally also involve administration of another compound proposed to have a synergistic effect with said test compound; and (b) determining the effect of the test compound

(optionally in combination with said another compound) on the cell cycle, e.g. by measuring induction of cell cycle arrest or cell death by apoptosis. Cell death or apoptosis can be determined by one of a number of techniques known to the person skilled in the art, e.g. the observing of morphological changes such as cytoplasmic blebbing, cell shrinkage, internucleosomal fragmentation, chromatin condensation and annexin-V staining. DNA cleavage typical of the apoptotic process may be demonstrated using TUNEL and DNA ladder assays.

Formulating pharmaceutically useful compositions and medicaments

In accordance with the present invention methods are also provided for the production of pharmaceutically useful compositions, which may be based on a substance or test compound so identified. In addition to the steps of the methods described herein, such methods of production may further comprise one or more steps selected from:

(a) identifying and/or characterising the structure of a selected substance or test compound;

(b) obtaining the substance or compound;

(c) mixing the selected substance or compound with a pharmaceutically acceptable carrier, adjuvant or diluent.

For example, a further aspect of the present invention relates to a method of formulating or producing a pharmaceutical composition for use in the treatment of a cancer, the method comprising identifying a compound in accordance with one or more of the methods described herein, and further comprising one or more of the steps of:

(i) identifying the compound; and/or

(ii) formulating a pharmaceutical composition by mixing the selected compound, or a prodrug thereof, with a pharmaceutically acceptable carrier, adjuvant or diluent . Certain pharmaceutical compositions formulated by such methods may comprise a prodrug of the selected compound wherein the prodrug is convertible in the human or animal body to the desired active agent. In other cases the active agent may be present in the pharmaceutical composition so produced and may be present in the form of a physiologically acceptable salt.

Therapeutic applications

Compounds of the present invention or identified by screening methods of the present invention may be used in the treatment of tumours and cancer in animals in need of treatment thereof. Preferably, the animal undergoing treatment is a human patient in need of such treatment. The compounds may be used in stimulating cell death.

Compounds of the invention may be formulated as pharmaceutical compositions for clinical use and may comprise a pharmaceutically acceptable carrier, diluent or adjuvant. The composition may be formulated for topical, parenteral, intravenous, intramuscular, intrathecal, intraocular, subcutaneous, oral or transdermal routes of administration which may include injection. Injectable formulations may comprise the selected compound in a sterile or isotonic medium.

Antisense nucleic acid

By antisense nucleic acid is meant a nucleic acid having substantial sequence identity to the nucleic acid formed by the sequence of complementary bases to the single strand of a target nucleic acid. The target nucleic acid may be a nucleic acid sequence encoding Mpsl kinase or a fragment thereof. Thus, the antisense nucleic acid is useful in binding the target nucleic acid and may be used as an inhibitor to prevent or disrupt the normal activity, folding or binding of the target nucleic acid or normal expression of the encoded protein.

The substantial sequence identity is preferably at least 60% sequence identity, more preferably at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100 identity.

Sequence Identity

Certain aspects of the invention concern isolated nucleic acids having a given sequence identity.

Percentage (%) sequence identity is defined as the percentage of nucleotides in a candidate sequence that are identical with nucleotides in the given listed sequence (referred to by the SEQ ID No.) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity.

Sequence identity is preferably calculated over the entire length of the respective sequences.

Where the aligned sequences are of different length, sequence identity of the shorter sequence is determined over the entire length of the longer sequence. For example, where a given sequence comprises 100 nucleotides and the candidate sequence comprises 10 nucleotides, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following examples :

(A)

Given seq: XXXXXXXXXXXXXXX (15 nucleotides) Comparison seq: XXXXXYYYYYYY (12 nucleotides) % sequence identity = the number of identically matching nucleotides after alignment divided by the total number of nucleotides m the given sequence, i.e. (5 divided by 15) x 100 = 33.3%

(B)

Given seq: XXXXXXXXXX (10 nucleotides)

Comparison seq: XXXXXYYYYYYZZYZ (15 nucleotides)

% sequence identity = number of identical nucleotides after alignment divided by total number of nucleotides in the given sequence, i.e. (5 divided by 10) x 100 = 50%.

Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art e.g. using the BLAST software available at http://www.ncbi.nlm.nih.gov/BLAST/.

Hybridisation stringency

In accordance with the present invention, nucleic acids having an appropriate level of sequence identity may be identified by using hybridisation and washing conditions of appropriate stringency.

For example, RNA-RNA hybridisations may be performed according to hybridisation methods well known to a person of skill in the art, e.g. the method of Sambrook et al., ("Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001) .

Calculation of the melting temperature (T_m) at a given salt concentration is one method of determining hybridisation stringency. Nucleic acid duplexes of low sequence identity will have a lower T_n, than nucleic acid duplexes of higher sequence identity. One of the most accurate derivations of the melting temperature is the nearest-neighbour method. This method is well known to persons of skill in the art, is suitable for calculating the T_n, of short nucleic acids and takes into account the actual sequence of the oligonucleotides as well as salt concentration and nucleic acid concentration.

The nearest-neighbour equation for both DNA and RNA based oligonucleotides is:

]

where ΔH° (Kcal/mol) is the sum of the nearest-neighbour enthalpy changes for hybrids, A is a constant (-10.8) correcting for helix initiation, ΔS° is the sum of the nearest neighbour entropy changes, R is the Gas Constant (1.99 cal K^{" 1}ITiOl^"1) and C_t is the molar concentration of the oligonucleotide. ΔH° and ΔS° values for both DNA and RNA nearest neighbour bases are publicly available (e.g. from Genosys Biotechnologies Inc.).

In general for RNA-RNA hybridisations under very high stringency conditions, the melting temperature of RNA duplexes of 100% sequence identity would be expected to be approximately greater than or equal to 60⁰C, although the actual T_m for any given duplex requires empirical calculation.

Accordingly, nucleotide sequences can be categorised by an ability to hybridise under different hybridisation and washing stringency conditions which can be appropriately selected using the above equation or by other similar methods known to persons skilled in the art. Sequences exhibiting 95-100% sequence identity are considered to hybridise under very high stringency conditions, sequences exhibiting 85-95%. identity are considered to hybridise under high stringency conditions, sequences exhibiting 70-85% identity are considered to hybridise under intermediate stringency conditions, sequences exhibiting 60-70% identity are considered to hybridise under low stringency conditions and sequences exhibiting 50-60% identity are considered to hybridise under very low stringency conditions.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Aspects .and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures

Figure 1. SP600125 promotes mitotic errors and ablates spindle checkpoint function in a JNK-independent manner.

(A) JNKl/Σ^"''^" 3T3 fibroblasts were asynchronously treated with SP600125 for 10 h and DNA profiles were determined by flow cytometry. Percentages of total 4N cells are plotted as numbers above the DNA profiles;

(B) Comparison of phospho-Histone H3 positivity (Y-axis) versus propidium-iodine staining (X-axis) of JNKl/2^"/" cells treated for 10 h with nocodazole alone or nocodazole in combination with SP600125. Mitotic cells (MI) are represented by the phospho-Histone H3 positive 4N population³¹ (upper right square) , while figures in the lower right panels denote the percentage of non-mitotic 4N cells;

(C, D) SP600125 overcomes a nocoazole-mediated mitotic arrest. JNK \/2^~'^~ cells were pretreated with nocodazole for 5h and mitotic cells were selectively collected. Cell were then reseeded into medium containing nocodazole, nocodazole/SP600125 or nocodazole/SP600125/MG132 (timepoint 0), respectively, and harvested after 2 hours. Mitotic progression was examined by FACS-mediated quantification of p- Histone H3 positivity (pHist H3, C) , determination of Cyclin B-immunoprecipitation kinase activity using Histone Hl as a substrate (D, upper panel), or Western-analysis of Cyclin B levels in total lysate (D, lower panel), respectively.

Figure 2. SP600125 abrogates spindle assembly checkpoint function in human cells.

(A) Asynchronously growing U2OS cells were incubated for 24 h with lOμM SP600125 alone, nocodazole or SP600125 in combination with nocodazole, respectively. Upper panel Light microscopic image showing morphologies of nocodazole-treated cells versus cultures incubated with nocodazole/SP600125. Lower panel DNA profiles of the differently treated U2OS cells. Figures above the profiles denote the percentage of mitotic cells (MI) as determined by p-Histone H3 positivity;

(B) U2OS cells were treated overnight with nocodazole. Mitotic cells were collected by mitotic shake-off and reseeded into fresh medium containing nocodazole alone or in combination with the specified drugs. At the indicated time points cells were harvested and mitotic progression was examined analysing p-Histone H3 positivity by flow cytometry, bar diagram shows the relative mitotic index at 3 hr after reseeding in the different combinations of drugs. For comparison a fraction of the cells was reseeded in medium without nocodazole or SP600125 and stained in parallel (release);

(C, D) U20S cells were treated as in B, but now using either nocodazole or taxol to obtain mitotic cells. After mitotic shake-off cells were reseeded in nocodazole or taxol, respectively. (C) Cyclin B-associated kinase activity Cyclin B protein levels were determined at the indicated time points in the specified drug combination. CDK4 was used as loading control. (D) Mitotic index, as determined by p-Histone H3 positivity at different time points after reseeding in the specified drug combination;

(E) Titration of the SP600125 concentrations required to override a taxol-induced arrest. Cells were treated as in (B- D) and p-Histone H3 positivity (bars) and Cyclin B-associated kinase activity were determined after 3h of co-incubation with the indicated SP600125 concentrations or an equivalent amount of solvent (mock) .

Figure 3. SP600125 treatment results in displacement of BubRl from kinetochores.

U2OS cells were transfected with Histone 2B fused to GFP (H2B- GFP) to visualize nucleus and chromosomes ³⁹ of fixed cells. Cells were synchronized in S-phase by thymidine treatment and released into normal medium or medium containing taxol or nocodazole, respectively. Cells were either grown for 15 h without further drug addition or were co-treated for the last 3h with SP600125, a combination of SP600125/MG132, or MG132 alone and immunostained with specific antibodies for BubRl or Madl.

(A) Confocal image of representative BubRl staining patterns obtained upon treatment with the indicated drug combinations; (B) Quantification of BubRl kinetochore localisation of prometaphase cells for the different combinations. An average of two independent experiments +SE is shown. For each combination 36-572 cells were counted;

(C) As in (B) but for Madl . Each 43-159 cells were scored. In none of the combinations with taxol significant kinetochore staining of Madl was detected (not shown) .

Figure 4. SP600125 inhibits hMpsl activity in a concentration- dependent manner in vitro.

(A, B) U2OS cells were treated with anisomycin to activate JNKl (A) , or nocodazole to obtain active hMpsl, BubRl and Cyclin B/Cdc2 (B), respectively. Total lysates from anisomycin-treated cells or selectively collected mitotic cells were pooled and subjected to immuncomplex kinase assays in presence of 1% DMSO (mock) or 1% DMSO and the indicated SP600125 concentrations. For comparison, activities of the respective kinases were also determined in lysates from untreated cells (A, lane 1) or thymidine-arrested cells (B, lane 1) ;

(C) Total lysates of uninfected SF9 insect cells (lane 1) or baculovirus-infected SF9 cells expressing recombinant human wild type Mpsl (Mpsl, lanes 2-8) or kinase inactive Mpsl

(Mpslkd, lane 9) were subjected to immunoprecipitation with a Mpsl-specific antiserum and autophosphorylation of immunoprecipitated Mpsl was determined in presence of 1% DMSO (mock) or 1% DMSO containing the indicated SP600125 concentrations.

Similar loading of the immunoprecipitated kinases was confirmed by Western blot. Figure 5. SP600125 co-treatment augments taxol-induced cell toxicity of U2OS osteosarcoma cells.

(A) U2OS osteosarcoma cells were treated with the indicated taxol concentrations alone, or in combination with 10 μM

SP600125. The ratio of apoptotic cells was determined 7 days after treatment, quantifying cells with sub-diploid DNA content by flow cytometry;

(B) Representative DNA profiles of U2OS cells treated for 7 days with a sub-lethal taxol dose alone (2.5 ng/ml) (see panel A) or in combination with 5 μM SP600125;

(C-E) U2OS cells were co-transfected with spectrin-GFP and either empty pRS-puro vector (pRS) or pRS-puro expressing small interfering RNA (siRNA) against hMpsl (pRS-Mpsl), respectively. After 48h cells equal numbers of GFP-positive cells were reseeded for further analysis. (C) Top panel: Mpsl protein knockdown at 72 hr post-transfection with pRS-Mpsl, bottom panel: Comparison of p-Histone H3 positivity• of pRS- and pRS-Mpsl-transfected cells upon overnight treatment with 1 μM taxol. (D) Cell death of hMpsl-depleted cells upon co- treatment with the indicated taxol concentrations for 4 days. (E) Clonogenic outgrowth of Mpsl-depleted vs. pRS-transfected cultures at a sub-lethal dose of taxol (2.5 ng/ml) .

Figure 6. Primary BJ-tert cells are refractory to SP600125- mediated spindle checkpoint override.

(A) Impact of SP600125-co-treatment on taxol-induced apoptosis of primary BJ-tert cells. BJ-tert cells were co-treated with

10 μM SP600125 and the indicated taxol concentrations.

Absolute percentages of apoptotic cells were assessed after 7 days, quantifying cells with a sub-diploid DNA content; (B, C) Different sensitivities of taxol-arrested U2OS osteosarcoma cells and primary BJ-tert cells to SP600125- mediated spindle checkpoint override. Taxol-arrested, mitotic BJ-tert cells or U2OS cells were selectively collected by mitotic shake-off, reseeded and exposed to taxol alone, taxol and SP600125, or both drugs in combination of the proteasome inhibitor MG132. Cells were harvested at the indicated time points and analyzed for p-Histone H3 positivity (B) or Cyclin B-associated kinase activity (C) , respectively. Cyclin B- associated kinase activity was compared to thymidine-arrested (S-phase) cells (Thy) as control;

(D) BJ-tert and U2OS cells were treated overnight with nocodazole or taxol in the absence or presence of SP600125 and BubRl phosphorylation was analysed by electromobility shift on Western blots.

Figure 7. Molecular structure of (A) taxol (Paclitaxel™) ; and (B) taxotere (Docetaxeϊ™) .

Figure 8. Molecular structure of (A) SP600125; and (B-F) selected anthrapyrazolone derivatives.

Figure 9. Apoptosis following treatment of JNKl/2 double negative cells with taxol or taxol and SP600125.

Figure 10 (A) Amino acid sequence for, and (B) nucleotide sequence encoding, human Mpsl protein kinase. The sequences are available from the NCBI database under accession number NM_003318 (GI :34303964) .

Detailed Description of the Best Mode of the Invention

Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

Experimental Procedures

Reagents Antisera against CDK4 (sc-260), JNKl (sc-474), TTK/hMpsl (sc- 540, used for immunoprecipitations) , and Cyclin Bl (sc-245) were from Santa Cruz Biotechnology. Antibodies against p- Histone H3 and hMPSl (used for Western blots)²⁵ were from Upstate Biotechnology; anti-p-Jun (S73) antiserum was purchased from Signaling Technology. S.taylor kindly provided sheep antiserum against human BubRl³⁵; and anti-Madl was a generous gift from A. Musacchio, who also provided baculoviruses containing His-tagged wildtype or kinase-dead hMPSl. Myelin basic protein (MBP) was from Sigma. GST-c-Jun (1-135) as substrate for JNKl was purified according to standard procedures. Histone Hl was obtained from Roche Molecular Biochemicals . SP600125 was from Biomol and used at lOμM unless stated differently. MG132, thymidine, Paclitaxel (taxol) and nocodazole were all from Sigma and employed at concentrations of 5μM, 2.5HiM, lμM, or 250ng/ml, respectively.

Expression vectors for H2B-GFP³⁸, Spectrin-GFP ^⁰ and the retrovirus-based pRetro Super (pRS) ⁴¹ have all been described. pRS vectors targeting hMpsl (pRS-Mpsl) were obtained from a siRNA kinase knockdown library ⁴². A pool of three pRS-Mpsl constructs (individual 19-mer targeting sequences: 5'- CCCAGAGGACTGGTTGAGT-3' (SEQ ID No. 1); 5'-CCAGATACAACAAGTGTTG- 3' (SEQ ID NO. 2); and 5'-TCACTGGCAGATTCCGGAG-S' (SEQ ID NO.3)) was used to knockdown expression of hMpsl.

Cell culture and transfections

Human BJ-tert foreskin fibroblasts stably transduced with human telomerase catalytic component³⁶ were grown in a 1:4 mixture of Medium 199/DMEM (Invitrogen) supplemented with 15% Fetal Calf Serum (FCS) and Penicillin/Streptomycin. Human U2OS osteosarcoma cells and murine NIH3T3 JNK l/2^"y" double deficient cells²⁹ (kind gift of E.Wagner) were cultured in DMEM supplemented with 8% FCS and Penicillin/Streptomycin. SF9 insect cells were maintained in Grace's Insect Medium (Bio Whittaker) containing 10% FCS and Penicillin/Streptomycin at 28°C. Transfections were performed according to the standard calcium phosphate protocol.

Enrichment of mitotic cells (Mitotic shake-off)

For enrichment of mitotic cells, cells were first arrested in mitosis by treatment with lμM taxol or 250ng/ml nocodazole for 18 h respectively. Mitotic cells were collected by shake-off involving vigorously rinsing the dishes with culture supernatant. Detached mitotic cells were collected and dishes were additionally washed twice with PBS containing the respective spindle poisons to increase the yield. Fractions were pooled and cells were pelleted by gentle centrifugation at 900 rpm. Resuspended cells were then reseeded into medium containing taxol or nocodazole alone, in combination with SP600125, or together with SP600125 and 5 μM MG132, respectively. Alternatively, cells were released from nocodazole by two washes with PBS and subsequent resuspension into drug-free medium. Changes in morphology were detected by phase contrast microscopy about 2-3 h after reseeding.

Immunoblots, immunoprecipitations and kinase assays Immunoblots were performed as described⁴². To activate JNKl, cells were treated with anisomycin (lOμg/ml) for 20min, to obtain active hMPSl, Cyclin B/Cdc2, or BubRl cells were incubated overnight with nocodazole (250ng/ml) . JNKl, Cyclin Bl/Cdc2, BubRl and Mpsl kinase assays were performed as described¹⁸'²⁵'⁴² but using a standardised kinase buffer⁴², equal substrate concentrations (0.25 mg/ml), and equal amounts of ATP (50μM cold ATP, 2.5μCi [γ-³²P]-ATP (Amersham) ) to ensure compatibility of the different reactions. Autophosphorylation reactions with recombinantly expressed Mpsl-His proteins were performed according to the same protocol²⁵, but in the absence of exogenous substrate. Phosphorylated substrates were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and analysed by autoradiography. Equal precipitation of the kinases was confirmed by probing the blots with an appropriate antibody.

Expression of recombinant Mpsl proteins

Recombinant wt-hMPSl-His and kd-Mpsl-His was expressed in SF9 insect cells by baculovirus-mediated gene expression according to standard procedures. For kinase assays recombinantly expressed proteins were immunoprecipitated and autokinase activity was determined as described above.

Cell cycle analysis and immunofluorescence

Cell cycle distribution and mitotic indices were determined by combined propidium iodide and p-Histone H3 staining as described⁴³. Kinetochore localisation of BubRl and Madl was judged by analysis of individual prometaphase cells by immunofluorescence microscopy and confocal microscopy as described ⁴V

Cell death assays and analysis of colony formation

2.5 xlO⁵ U2OS cells or BJ-tert cells were seeded in duplo on 10 cm dishes. Cells were treated with the different taxol concentrations and lOμM SP600125, or different SP600125 doses and 2.5ng/ml taxol. Cells were maintained in the presence of drugs until controls reached full density (~1 week) and were then harvested and fixed in 70% ethanol . Cells were stained with lOμg/ml propidium iodide and apoptosis rates were determined by quantification of cells with sub-diploid DNA content on FACS using Cell Quest software (Becton Dickinson) .

To analyse the effect of hMpsl depletion on taxol-induced cell death and colony formation, U2OS cells were transfected with pRS-Mpsl RNAi expression constructs or empty pRS and limiting amounts of Spectrin-GFP. After 48h, equal numbers of transfected cells (as determined by FACS) were re-seeded into fresh medium containing the respective taxol concentrations. For the colony formation assays transfected cells were selected by puromycin treatment (2μg/ml) and colonies were stained after 7 days with crystal violet⁴⁴. For determination of cell death, cells were harvested 4 days after treatment and resuspended in lμg/ml propidium iodide to stain membrane- damaged cells. Percentages of dead, transfected cells were assessed by quantification of PI/GFP double positive versus GFP single-positive cells by FACS.

Results

SP600125 abrogates spindle checkpoint function in a JNK- mdependent manner

SP600125 is a novel anthrapyrazolone compound previously reported as a specific and reve-rsible ATP-competitive inhibitor for stress-activated MAPKs of the JNK family²⁷'²⁸. Interestingly, in their first account Bennett and co-workers reported accumulation of 4N cells upon treatment of human naive T-cells with SP600125²⁷, which suggested a putative interference of this compound with mitotic checkpoint signalling.

To study if these effects are mediated through JNK, JNKl/2^^" double deficient fibroblasts were treated with lOμM SP600125 and DNA-profiles of SP600125-treated cells and untreated control cells were compared by flow cytometry. Interestingly, a dramatic accumulation of 4N cells and a small but significant increase in polyploidy was also observed in JNK1/2^{" ;~} deficient fibroblasts treated with SP600125 (Figure IA) . These cells are completely devoid of JNK-activity, as they lack functional genes for the two ubiquitously expressed JNKl and JNK2 isoforms and do not express the neuronal-specific JNK3 isoform²⁹. Thus, these data indicate that SP600125 interferes with cell cycle progression at some point in G2/M, but in a JNK-mdependent manner. JNK1/2^"'^" cells were then treated with nocodazole, a spindle poison that triggers microtubule depolymerization and mitotic arrest³⁰ due to the failure to satisfy the spindle assembly checkpoint under these circumstances ². As expected, in the presence of nocodazole JNKl/2^"/" cells accumulated in mitosis, as was evident by elevated phospho-Histone H3 positivity (Figure IB) that characterizes mitotic cell cultures³¹. Surprisingly, combined treatment with nocodazole and SP600125 prevented the accumulation of mitotic cells, although the total percentage of cells with a 4N DNA content was very similar between these two treatments (Figure IB) . This indicates that in the presence of SP600125, cells are either blocked in G2, or can escape a nocodazole-induced cell cycle block.

To distinguish between these two possibilities cells were first arrested in mitosis and tested if addition of SP600125 could force an escape from such an established mitotic arrest. To this end, JNKl/2^"'^" cells arrested in mitosis by nocodazole- treatment were selectively collected by mitotic shake-off and reseeded into nocodazole-containing medium in the presence or absence of SP600125. Interestingly, the percentage of p- Histone H3 positive cells decreased dramatically upon SP600125 co-treatment of mitotic JNKl/2^"/" cells, while p-Histone H3 positivity remained high in the non-co-treated samples (Figure 1C) . Likewise, Cyclin B protein and Cyclin B-associated kinase activity, which rise in late G2 and are usually sustained in spindle checkpoint-activated cells²¹, sharply dropped upon SP600125 co-treatment (Figure ID) . This indicates that these cells progress past the spindle assembly checkpoint and activate the anaphase-promoting complex (APC) , leading to degradation of Cyclin B by the proteasome. Indeed, co- treatment with the proteasome-inhibitor MG132 largely reversed the effect of SP600125 on mitotic progression as judged by restoration of p-Histone H3 positivity (Figure 1C) , Cyclin Bl protein levels, and Cyclin B-associated kinase activity (Figure ID), respectively.

Apoptosis in JNKl/2^"/" cells in response to treatment with taxol alone or taxol plus SP600125 was also measured. Figure 9 shows apoptosis in JNKl/2^'/" cells to be clearly reactive and significantly increased in response to treatment with the combination of taxol and SP600125.

SP600125 ablates spindle checkpoint function in human cells The effect of SP600125 on mitotic progression of human cells was investigated.

For this purpose, U2OS osteosarcoma cells were treated with nocodazole in the presence or absence of SP600125. Again, SP600125 almost completely inhibited the accumulation of mitotic cells that normally results from nocodazole treatment (Figure 2A) . Unlike the observation in the mouse cell lines, SP600125-induced accumulation of 4N cells was not evident at 1OyM in the U2OS cells (Figure 2A) , but was seen at higher doses of SP600125 (data not shown) . It was confirmed that SP600125 could override an established nocodazole-induced arrest in a similar experimental set-up as described above. Moreover, in the continued presence of nocodazole, addition of SP600125 to enriched mitotic cell populations induced a rapid loss of p-Histone H3 positivity (Figure 2B) and Cyclin B- associated kinase activity (Figure 2C), and both effects could again be blocked by co-treatment with MG132 (Figure 2B) . In fact, mitotic exit proceeded with similar kinetics to those observed when nocodazole was removed from the culture medium (Figure 2B) . When morphologies of the different cultures were analyzed by phase contrast microscopy, a clear flattening of SP600125-treated cells with comparable kinetics to cells that exited mitosis upon release from nocodazole (data not shown) . SP600125 prevents spindle checkpoint response to lack of attachment and tension

The experiments so far demonstrated that SP600125 treatment abrogates proper function of the spindle assembly checkpoint in absence of microtubule attachment. To explore whether

SP600125 also affects checkpoint maintenance in response to lack of tension we tested SP600125 on taxol-arrested cells. Taxol sustains attachment by stabilisation of microtubules³² but relieves tension from the attached kinetochores³³, which likewise sustains spindle assembly checkpoint activation and induces a prometaphase arrest. Comparison of SP600125/taxol versus SP600125/nocodazole co-incubated cells in a shake-off setting revealed that SP600125 could also override a taxol block very efficiently (Figure 2D) . This was also evident by a rapid drop in Cyclin B-associated kinase activity and Cyclin B protein levels observed after SP600125-treatment of taxol- arrested cells (Figure 2C), demonstrating that SP600125 can likewise overcome a mitotic-arrest induced by lack of tension. Using different concentrations of SP600125, the minimal concentration of SP600125 for efficient checkpoint-override ranged from about 2.5μM (Figure 2E), a concentration insufficient for significant inhibition of JNK-activity

SP600125 treatment leads to premature loss of BubRl from kinetochores of mitotic cells

In order to understand how SP600125 causes inactivation of the spindle checkpoint, kinetochore recruitment of two well- established spindle checkpoint proteins, Madl and BubRl, was analysed.

To this end, cells were released from a S-phase block into nocodazole- or taxol-containing medium in presence or absence of SP600125. To minimize possible effects on non-mitotic targets, SP600125 was only added for the last 3h of the observation period when cells had already progressed to G2 (12h after release) . Moreover, the proteasome inhibitor MG132 was added for this last time period to exclude indirect effects of SP600125 on BubRl or Madl localisation due to APC- activation and mitotic exit. Consistent with previous reports, BubRl was recruited to kinetochores in nocodazole- and in taxol-treated cells³⁴'³⁵ (Figure 3A) . MG132 co-incubation did not alter kinetochore localisation of BubRl, whereas additional treatment with SP600125 clearly reduced BubRl staining at the kinetochores (Figure 3A) . This was confirmed by quantification of prometaphase cells with strong staining versus weak or absent BubRl localisation at the kinetochores. About 50% of prometaphase cells in nocodazole or taxol lost BubRl from the kinetochores upon SP600125 treatment (Figure 3B) . In contrast, Madl localisation was only slightly affected and only minor differences were seen when SP600125 was co- incubated with nocodazole (Figure 3C) .

SP600125 directly inhibits kinase activity of cellular and recombinant human Mpsl

Activities of various kinases, including Mpsl ²², regulate spindle assembly checkpoint function²'²¹'²⁴'²⁵. Moreover, comparison of the ATP-binding domains of Mpsl and JNK showed a remarkable degree of similarity on the amino acid level (data not shown) . To test if SP600125 can inhibit Mpsl activity cellular Mpsl was immunoprecipitated and in vitro kinase assays were performed in the presence or absence of SP600125. In parallel BubRl kinase activity was analyzed, which recently was reported to contribute to its checkpoint function¹⁸'¹⁹. SP600125 significantly inhibited JNKl in vitro at concentrations of 0.5-lμM (Figure 4A) . Surprisingly, hMpsl activity was completely abolished at 0.5μM SP600125 (Figure 4B) , indicating that hMpsl was even more effectively inhibited in vitro than JNK. In contrast, SP600125-treatment did not affect Cyclin B/Cdc2 activity even at the highest employed drug concentration while BubRl activity was partially inhibited at the highest dose of SP600125 (Figure 4B) . To exclude that the decreased substrate phosphorylation observed in the hMpsl kinase assays resulted from inhibition of a co-iminunoprecipitated kinase interacting with Mpsl, the impact of SP600125 on autophosphorylation of recombinantly expressed hMpsl was further tested. Figure 4C illustrates that SP600125 also strongly inhibited recombinant hMpsl activity, confirming that SP600125 acts directly on hMpsl.

SP600125 augments taxol-mediated apoptosis of human cancer cells in a synergistic fashion Since the data showed that SP600125 impaired the spindle checkpoint, the inventors wondered if SP600125 could promote progression of checkpoint-challenged cells into mitotic catastrophe. Therefore, it was investigated whether SP600125 exerted a synergizing effect on taxol-mediated apoptosis of cancer cells. It was found that the effective taxol concentration capable to induce half-maximal apoptosis rates of U2OS osteosarcoma cells was about 10-fold reduced in presence of lOμM SP600125 and declined from 5ng/ml to roughly 0.5ng/ml taxol (Figure 5A). Similar results were obtained with DLD-I colon carcinoma cells (data not shown) indicating that this synergy was not restricted to the U2OS cancer cell line.

To exclude a potential influence of JNK, that is still slightly inhibited at lOμM SP600125 in vivo (data not shown) , we also analysed synthetic lethality at a concentration of SP600125 (5 μM) at which virtually no inhibition of JNK- activity in intact cells is detected (data not shown) . Also at this dose a very clear synergy between taxol and SP600125 was found, as about 60% of the cells underwent apoptosis upon co- treatment with the drugs, while only very little apoptosis was observed when each compound was added alone (Figure 5B) .

SP600125 effectively inhibited hMpsl in the kinase assays performed. It was next tested whether RNAi-mediated depletion of hMpsl would also augment taxol-induced apoptosis of human U2OS osteosarcoma cells. RNAi against hMpsl reduced total protein levels of hMpsl to about 20-30% of its initial level and this resulted in about a three-fold decrease of p-Histone H3 positivity upon taxol incubation (Figure 5C) , demonstrating that the achieved hMpsl protein depletion was sufficient to partially overcome a taxol-induced mitotic arrest in U2OS cells. When hMpsl-RNAi-transfected cells were analysed for cell death in response to increasing taxol concentrations enhanced cell death at suboptimal taxol concentrations was observed (Figure 5D) . Moreover, clonogenic outgrowth at sub- optimal taxol concentrations was strongly reduced by Mpsl depletion (Figure 5E) . This indicates that, in analogy to SP600125, Mpsl-mediated spindle checkpoint override is capable to augment taxol induced cell death of U2OS cancer cells, supporting the view that at least partially the synergistic effect of SP600125 on taxol-induced cell death relies on Mpsl inhibition.

Primary human BJ-tert fibroblasts are refractory to SP600125- mediated spindle checkpoint override The effect of SP600125 on taxol-mediated apoptosis of normal somatic cells was explored. For this BJ-tert cells were analysed, which represent primary human fibroblasts immortalised through stable expression of the catalytic component of human telomerase³⁶. Interestingly, SP600125 co- treatment of these primary cells showed only a slight additive effect on taxol-mediated apopotosis (Figure 6A), and taxol- induced cell death was much lower throughout the range of concentrations used when compared to the U2OS cells. This raises the interesting prospect that normal somatic cells retain a more stringent spindle checkpoint than transformed cells. To test this hypothesis, BJ-tert cells and U2OS cells were examined side by side for their capacity to maintain a taxol-mediated mitotic arrest in presence of SP600125. Surprisingly, even lOμM of SP600125 was not sufficient to effectively override the taxol-mediated arrest in these cells (Figure 6B and Figure 6C). Although prolonged SP600125 incubation resulted in a partial loss of p-Histone H3 positivity (Figure 6B) , still a considerable number of cells remained arrested in mitosis, which was also evident from analysis of Cyclin B-associated kinase activity (Figure 6C) . While U2OS cells rapidly lost Cyclin B/Cdc2 activity upon SP600125 addition, only a marginal reduction in Cyclin B- associated kinase activity was seen with BJ-tert cells during the whole observation period. Importantly, however, when BubRl phosphorylation was analyzed, as an alternative readout for mitotic progression³⁵, SP600125 co-treatment prevented nocodazole- and taxol-induced BubRl phosphorylation in both cell types (Figure 6D) . This conclusively demonstrates that SP600125 was active in BJ-tert cells and inactivates at least one important component of the spindle checkpoint. This is not sufficient to cause a full checkpoint override in primary cells which implies the existence of a redundant pathway controlling spindle checkpoint function that is lost in transformed cells.

Discussion

The data provided herein demonstrates that SP600125 can effectively ablate spindle checkpoint function in various mouse and human cells. Experiments with JNKl/2^"/~ cells lacking functional JNK proteins²⁹ revealed that this property of

SP600125 was JNK independent. Kinase assays implicate that the checkpoint regulator hMpsl represents an unexpected mitotic target for SP600125. The fact that SP600125 was capable of overriding both a mitotic arrest induced by lack of attachment and tension furthermore predicts the mitotic effector of

SP600125 to be an integral component or regulator of the core sensor machinery of the spindle assembly checkpoint, which certainly applies for Mpsl. Data from various organisms have provided evidence that Mpsl kinase activity is required for checkpoint function upon microtubule depolymerisation²⁴'²⁵. The experiments presented here now additionally show that RNAi against Mpsl can likewise abrogate checkpoint function upon lack of tension.

Co-treatment experiments with taxol revealed a strong synergistic effect of SP600125 on taxol-induced apoptosis of human U2OS cancer cells. The SP600125 compound has a dramatic impact on spindle checkpoint integrity, and it may be that its checkpoint-ablating action contributes to this synergism. This view is particularly substantiated by the RNAi experiments, which showed that RNAi-mediated depletion of hMpsl could largely mimic the synergizing effect of SP600125 at low taxol concentrations.

Surprisingly, similar experiments with BJ-tert cells revealed that SP600125 exerted only a marginal effect on taxol-induced cell death of normal somatic cells. The lower susceptibility of primary cells to drug-induced cell death by combined taxol and SP600125 treatment is not currently understood, although it is plausible to assume that this is a direct consequence of their remarkable insensitivity to SP600125-mediated checkpoint override. It may be that primary cells retain components or pathways, contributing to full spindle assembly checkpoint function, which are lost or compromised in cancer cells and make these more susceptible to SP600125-mediated checkpoint override. Multiple lines of evidence indicate that cancer cells indeed frequently show perturbed spindle assembly checkpoint signalling³⁷. While these mutations originally may foster tumorgenesis by promoting aneuploidy, chromosomal instability may also render cancer cells generally more susceptible to forced checkpoint override by spindle checkpoint inactivating compounds such as SP600125. In this respect a putative presence of redundant pathways in primary cells could be clinically relevant since it could facilitate the development of more effective and acceptable chemotherapeutic treatments that reliably target cancer cells without the common deleterious effects on normal cells.

The data underscores that combined administration of spindle checkpoint challenging and inactivating drugs may be a promising approach in designing novel anti-cancer strategies that attempt to take advantage of genomic instability as a classical trait of tumour cells.

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Claims

Claims :

1. A pharmaceutical composition comprising a modulator of Mpsl kinase activity and a taxol .

2. A pharmaceutical composition according to claim 1 wherein the pharmaceutical composition additionally comprises a pharmaceutically acceptable carrier, adjuvant or diluent.

3. Products containing a modulator of Mpsl kinase activity and a taxol as a combined preparation for simultaneous, separate, or sequential use in the treatment of disease.

4. Use of a modulator of Mpsl kinase activity and a taxol in the manufacture of a medicament for the treatment of disease.

5. Use of a modulator of Mpsl kinase activity in the manufacture of a first medicament for treatment of a disease by co-administration of said first medicament with a second medicament, the second medicament comprising a taxol .

6. Use of a taxol in the manufacture of a first medicament for treatment of a disease by coadministration of said first medicament with a second medicament, the second medicament comprising a modulator of Mpsl kinase activity.

7. The use of claim 5 or claim 6 wherein the coadministration is simultaneous administration, separate administration, or sequential administration.

8. A method of treating a disease comprising coadministering to an individual in need of treatment a therapeutically effective amount of a modulator of Mpsl kinase activity and a taxol.

9. The method of claim 8 wherein the modulator of Mpsl kinase activity and the taxol are administered separately, sequentially or simultaneously.

10. A kit of parts for use in the treatment of a disease, the kit comprising a first container comprising a modulator of Mpsl kinase activity and a second container comprising a taxol.

11. The kit of claim 10 wherein the kit further comprises instructions for the separate, sequential or simultaneous co-administration of said modulator of Mpsl kinase activity and said taxol.

12. The kit of claim 10 or 11 wherein the Mpsl kinase modulator and/or taxol are formulated as pharmaceutically acceptable compositions.

13. A composition, products, use, method or kit according to any one of the preceding claims wherein the modulator of Mpsl kinase activity is chosen from one or more of an anthrapyrazolone, an anthrapyrazolone derivative, a pharmaceutically acceptable anthrapyrazolone salt, a pharmaceutically acceptable anthrapyrazolone prodrug.

14. A composition, products, use, method or kit according to any one the preceding claims wherein the modulator of Mpsl kinase activity is chosen from one or more of the anthrapyrazolone SP600125, a pharmaceutically acceptable salt of SP600125, a pharmaceutically acceptable prodrug of SP600125.

15. A composition, products, use, method or kit according to any one of claims 1 to 12 wherein the modulator of Mpsl kinase activity is a short interfering RNA molecule having a length of from 17 to 25 nucleotides comprising a nucleotide sequence having at least 90% sequence identity, or complementarity, to a selected contiguous portion of the Mpsl kinase cDNA of the same length .

16. A composition, products, use, method or kit according to claim 15 wherein the RNA is chosen from one or more of SEQ ID No.1; SEQ ID No.2; SEQ ID No.3; SEQ ID No.4; SEQ ID No. 5; a sequence complementary to one of SEQ ID No. s 1-5; one of SEQ ID No. s 1-5 or its complement in which base T is replaced by base U.

17. A composition, products, use, method or kit according to any one of the preceding claims wherein the taxol is provided in the form of a pharmaceutically acceptable salt or prodrug thereof.

18. A composition, products, use, method or kit according to any one of the preceding claims wherein the modulator of Mpsl kinase activity is an inhibitor of Mpsl kinase activity.

19. A composition, products, use, method or kit according to any one of the preceding claims wherein the modulator of Mpsl kinase activity is selective for Mpsl over other spindle checkpoint kinases.

20. A composition, products, use, method or kit according to any one of the preceding claims for use in the treatment of a disease.

21. A composition, products, use, method or kit according to claim 20 wherein the disease is a disease involving cell death or cell proliferation.

22. A composition, products, use, method or kit according to claim 20 wherein the disease is cancer.

23. A method for identifying compounds which improve the ability of a microtubule-depolymerising compound or a microtubule stabilising compound to cause cell death, wherein the microtubule-depolymerising compound or the microtubule stabilising compound is capable of arresting mitosis, comprising:

(i) in vitro contacting one or more cells with a said microtubule-depolymerising compound or microtubule stabilising compound;

(ii) contacting the cells with a test compound; (iii) determining the amount or extent of cell death; and (iv) comparing said extent or amount in (iii) to the extent or amount of cell death caused when step (ii) is omitted.

24. A method according to claim 23 wherein the test compound is a modulator of Mpsl kinase activity.

25. A method according to claim 23 wherein the test compound is chosen from- one or more of an anthrapyrazolone, an anthrapyrazolone derivative, a pharmaceutically acceptable anthrapyrazolone salt, a pharmaceutically acceptable anthrapyrazolone prodrug.

26. A method for identifying a compound which modulates the activity of Mpsl kinase comprising:

(i) providing a test compound;

(ii) providing a first component having Mpsl kinase activity and comprising an Mpsl kinase polypeptide, homologue, mutant, derivative or fragment;

(iii)in vitro contacting the test compound and first component under conditions in which the test compound and first component may interact; and

(iv) detecting a modulation of the Mpsl kinase activity of the first component.

27. A method according to claim 26 wherein said kinase activity is autophosphorylation or phosphorylation activity.

28. A method according to claim 26 or claim 27 wherein after step (iii), the first component is contacted in vitro with a second component, and in step (iv) the modulation of the second component is detected in addition to, or instead of, modulation of the Mpsl kinase activity of the first component.

29. A method according to claim 28 wherein detecting the modulation of the second component comprises detecting autophosphorylation or phosphorylation of the second component .

30. A method according to claim 28 or claim 29 wherein the second component is a substrate of Mpsl kinase.

31. A method according to any one of claims 26 to 30 wherein step (ii) comprises providing a cell expressing the first component, and step (iii) comprises in vitro contacting the cell with the test compound.

32. A method according to any one of claims 26 to 31 wherein the first component is recombinant Mpsl kinase or human wild type Mpsl kinase.

33. A method according to any one of claims 26 to 32 wherein the method additionally comprises step (v) of determining whether the test compound improves the ability of a microtubule-depolymerising compound or a microtubule-stabilising compound to cause cell death, wherein the microtubule-depolymerising compound or the microtubule-stabilising compound is capable of arresting mitosis .

34. A method according to claim 33 wherein the improvement is determined by the method of claim 23.

35. An assay kit comprising a first container having a quantity of an Mpsl kinase polypeptide, homologue, mutant, derivative or fragment having Mpsl kinase activity and a second container having a quantity of an antiserum or antibody capable of binding a modulated form of said Mpsl kinase polypeptide, homologue, mutant, derivative or fragment.

36. The assay kit of claim 35 wherein the modulated form of the Mpsl kinase polypeptide, homologue, mutant, derivative or fragment is a phosphorylated or dephosphorylated form of the Mpsl kinase polypeptide, homologue, mutant, derivative or fragment.

37. The assay kit of claim 35 or claim 36 wherein the kit further comprises instructions for performing a screening method for identifying a compound which modulates the Mpsl kinase activity.

38. A method for identifying compounds which improve the ability of a modulator of Mpsl kinase to cause cell death comprising:

(i) in vitro contacting one or more cells having Mpsl kinase activity with a modulator of Mpsl kinase activity;

(ii) contacting said cells with a test compound;

(iii) determining the amount or extent of cell death; and

(iv) comparing said amount in (iii) to the amount of cell death caused when step (ii) is omitted.

39. A method according to claim 38 wherein the modulator of Mpsl kinase activity is chosen from one or more of an anthrapyrazolone, an anthrapyrazolone derivative, the anthrapyrazolone SP600125, a pharmaceutically acceptable anthrapyrazolone salt, a pharmaceutically acceptable anthrapyrazolone prodrug, a pharmaceutically acceptable salt of SP600125, a pharmaceutically acceptable prodrug of SP600125.

40. A method according to claim 38 or claim 39 wherein the test compound is a microtubule-depolymerising compound capable of arresting mitosis.

41. A method according to claim 38 or claim 39 wherein the test compound is a microtubule-stabilising compound capable of arresting mitosis.

42. The method of claim 41 wherein the microtubule- stabilising compound is a taxol .,

43. A method of assaying for compounds capable of overriding a spindle assembly checkpoint, comprising the steps of:

(i) treating one or more cells with an agent that arrests the cells in mitosis; (ii) treating said arrested cell(s) with one or more test compounds; and (iii) observing the effect of said compound (s) on said cell (S) .

44. A method according to claim 43 wherein the cells are arrested in mitosis by treating with a microtubule- depolymerising compound or a microtubule-stabilising compound.

45. A method according to claim 43 or claim 44 wherein said test compound is chosen from one or more of an anthrapyrazolone, an anthrapyrazolone derivative, a pharmaceutically acceptable anthrapyrazolone salt, a pharmaceutically acceptable anthrapyrazolone prodrug.

46. An in vitro method for reducing the level of functional Mpsl kinase protein expressed in a selected cell or cells, the method comprising contacting one or more cells with, or expressing in said cells, a short interfering RNA molecule having a length of from 17 to 25 nucleotides and comprising a nucleotide sequence having at least 90% sequence identity, or complementarity, to a selected contiguous portion of the Mpsl kinase cDNA of the same length.

47. The method of claim 46 wherein the RNA is chosen from one or more of SEQ ID No.l; SEQ ID No.2; SEQ ID

No.3; SEQ ID No.4/ SEQ ID No. 5; a sequence complementary to one of SEQ ID No. s 1-5; one of SEQ ID No.s 1-5 or its complement in which base T is replaced by base U.

48. A method according to claim 46 or claim 47 wherein the method additionally comprises administering a microtubule-depolymerising compound or microtubule stabilising compound to the cell, and determining the effect of the microtubule-depolymerising or microtubule- stabilising compound on the cell.

49. A cell transformed with, or capable of expressing, a short interfering RNA molecule having a length of from 17 to 25 nucleotides and comprising a nucleotide sequence having at least 90% sequence identity, or complementarity, to a selected contiguous portion of the Mpsl kinase cDNA of the same length, wherein the cell has a reduced level of Mpsl kinase activity compared to the Mpsl kinase activity of a wild type cell.

50. The cell of claim 49 wherein the RNA is chosen from one or more of SEQ ID No.l; SEQ ID No.2; SEQ ID No.3; SEQ ID No.4; SEQ ID No. 5; a sequence complementary to one of SEQ-ID No.s 1-5; one of SEQ ID No.s 1-5 or its complement in which base T is replaced by base U.

51. The method of any one of claims 23-25, 33 or 44 wherein the microtubule-depolymerising compound is chosen from one or more of nocodazole, a vinca alkaloid, a cryptophycine, a halichondrine, an estramυstine, a colchicine.

52. The method of any one of claims 23-25, 33 or 44 wherein the microtubule-stabilising compound is chosen from one or more of a taxol, an eleutherobin, an epithilone, a laulimalide, a sarcodictyin, a discothermolide .