WO2008132464A2 - Mek5 and related proteins as biomarkers of neurodegenerative diseases - Google Patents

Mek5 and related proteins as biomarkers of neurodegenerative diseases Download PDF

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WO2008132464A2
WO2008132464A2 PCT/GB2008/001469 GB2008001469W WO2008132464A2 WO 2008132464 A2 WO2008132464 A2 WO 2008132464A2 GB 2008001469 W GB2008001469 W GB 2008001469W WO 2008132464 A2 WO2008132464 A2 WO 2008132464A2
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module
mek5
erk5
mef
neurodegenerative disease
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PCT/GB2008/001469
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French (fr)
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WO2008132464A3 (en
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John R. Mayer
Weng Choon Chan
Zahra Nooshin Rezvani
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The University Of Nottingham
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to diagnostic methods and therapies for neurodegenerative illnesses characterised by the occurrence of intracellular protein aggregates, including, but not limited to, Alzheimer's Disease, Dementia with Lewy Bodies and Parkinson's Disease.
  • the invention relates to mitogen activated protein kinase kinase 5 (MEK5) and mitogen activated protein kinase kinase 1 (MEKl) as targets for developing diagnostic tests and therapies for progressive neurodegenerative illness.
  • MEK5 mitogen activated protein kinase kinase 5
  • MEKl mitogen activated protein kinase kinase 1
  • Progressive dementias which include Alzheimer's disease, Dementia with Lewy Bodies, dementia associated ' with Parkinson's disease, Huntington's Disease, prion diseases and a number of rarer conditions, cause a progressive loss of mental function resulting from the death of nerve cells.
  • Alzheimer's Disease acetyl cholinesterase inhibitors
  • Parkinson's Disease dopaminergics and dopamine agonists
  • Current therapies for Alzheimer's Disease acetyl cholinesterase inhibitors
  • Parkinson's Disease dopaminergics and dopamine agonists
  • no medications are approved specifically to treat Dementia with Lewy Bodies or most other types of progressive dementia.
  • therapies which can halt or slow disease progression.
  • diagnostic tools for progressive neurodegenerative diseases At present the only unambiguous way to diagnose neurodegenerative disease is autopsy. Diagnosis in living patients is a difficult and complex procedure based on a battery of physical, neurological and cognitive tests.
  • Alzheimer's Disease is characterised by intraneuronal neurofibrillary tangles and extracellular amyloid deposits.
  • Dementia with Lewy Bodies is characterised by intraneuronal Lewy bodies with extraneuronal amyloid plaques 1-3 . Lewy bodies also occur in Parkinson's Disease.
  • Huntington's Disease and the other polyglutamine disorders are associated with aggregation of the polyQ-expansion-containing proteins including huntingtin.
  • Prion diseases such as Creutzfeld-Jacob Disease are associated with the aggregation of the PrP So protein.
  • a major research focus into targets for novel therapies is on the proteins which aggregate in these disorders.
  • the extracellular amyloid is generated when the Alzheimer precursor protein (APP) is cleaved to produce the aggregate-prone A ⁇ peptides.
  • APP Alzheimer precursor protein
  • the aggregation of hyperphosphorylated tau and ⁇ -synuclein in neuronal inclusions are the halhnarks of Alzheimer's disease and dementia with Lewy bodies respectively.
  • There is also intense work on the kinases which phosphorylate tau primarily cyclin dependent kinase 5, glycogen synthetase kinase 3 ⁇ and the MARCK kinase 6 .
  • the aggregated proteins found in the inclusions which characterise Alzheimer's Disease and Dementia with Lewy Bodies are also ubiquitylated 7 .
  • Protein ubiquitylation is one of the two major mechanisms for protein degradation in all cells, the other being autophagy 8 .
  • the brains of patients succumbing to dementing illness show extensive loss of neurones in several regions including the hippocampus and cerebral cortex. The relationship between protein phosphorylation, protein ubiquitylation, neuronal death and chronic neurodegenerative disease is not well understood.
  • a greater understanding of the cellular response to the aggregation of proteins is key to understanding the pathogenesis of these diseases and developing novel diagnostic and therapeutic interventions.
  • a generic response to the aggregation of proteins has been studied by looking for changes in gene expression in cells expressing two aggregate-prone proteins, namely the proteasomal S 8 ATP ase and the metabotropic glutamate receptor 15 .
  • This experiment uncovered a major increase and subsequent decrease in the expression of the gene for mitogen activated protein kinase kinase 5 (MEK5) which mirrored the accumulation and elimination of the aggregate-prone proteins from cells by the protein catabolic systems.
  • MEK5 mitogen activated protein kinase kinase 5
  • MEK5 (also known as MKIC5, PRKMK5, MAPKK5 and MAP2K5) is a kinase kinase involved in the mitogen activated protein kinase (MAPK) signalling pathway.
  • MAPK mitogen activated protein kinase
  • This pathway comprises several different families of interacting kinases forming 'modules', which act in parallel pathways to regulate diverse activities including cell division, gene expression and apoptosis.
  • a kinase kinase kinase phosphorylates a kinase kinase, -which phosphorylates a kinase, which triggers regulatory processes in the cell.
  • MEK5 is phosphorylated by MEKK2,3/Tpl2 and atypical protein kinase C.
  • MEK5 has only one known kinase substrate, ERK5/BMK1.
  • EREL5 specifically phosphorylates the myocyte enhancer factor (MEF) family of transcription factors that activate expression of neuronal survival genes during development.
  • MEF myocyte enhancer factor
  • the down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/MAPK module facilitates cell death by apoptosis 9 .
  • the microarray analysis described above shows for the first time changes in gene expression, including activation of the MEK5 module and inactivation of the MEK3 module, in response to protein aggregation in cells. However these cells were not of neuronal origin, nor were they expressing proteins implicated in ⁇ neurodegeneration.
  • the aggresomes formed in this experimental system disappeared within 3 days and had no effect on the viability of the cells.
  • the changes in gene expression detected in this system appear to be a generic response to protein aggregation and would not necessarily be expected to mirror the process which causes neuronal loss in the brains of patients with neurodegenerative disease.
  • the present invention provides novel methods, assays and materials useful in the diagnosis of progressive neurodegenerative illnesses including, but not limited to, Alzheimer's Disease, Parkinson's Disease and Dementia with Lewy Bodies. It further provides novel targets for development of neuroprotective therapeutic agents and imaging agents, and novel screening methods and materials to discover such therapeutic and imaging agents.
  • the present invention is based upon the immunohistochemical (IHC) demonstration that neurones containing neurofibrillary tangles and Lewy bodies in human brain show a large increase in the expression of MEK5 ( Figures 1 to 4) and its effector protein ERK5 (see appendix for methods and data). In contrast, adjacent neurones without these inclusions do not show enhanced expression of the kinase module. This demonstrates for the first time that the MEK5/ERK5/MEF survival and cytoprotective responses take place in chronic neurodegenerative diseases, and implies that a mechanism of neuronal survival is operative at certain stages in the diseases.
  • IHC immunohistochemical
  • p62 binds to ⁇ biquitylated proteins and is present in inclusions in all the neurodegenerative disease ⁇ .
  • a recent report indicates that the p62 protein and MEK5 both have a ubiquitin-like PBl domain through which p62 and MEK5 can bind together n .
  • IHC shows that MEK5 and p62 are both present in inclusions containing ubiquitylated proteins. This is the first report of a connection between ⁇ biquitylation of aggregate-prone proteins, p62 and the MEK5/ERK5/MEF module for activation of neuronal survival genes.
  • MEK5 is also present in areas of granulovacuolar degeneration in the hippocampus which do not contain the p62 protein. These areas are thought to represent areas of intense autophagic activity to eliminate aggregate-prone proteins by the autophagosome-lysosome system. This is the first report that MEK5 is associated with the autophagolysosomal system.
  • MEKl is a dual-specificity tyrosine/threonine protein kinase that phosphorylates threonine and tyrosine in the activation loop of ERKl, thereby activating ERKl.
  • Zhu et al, (2003) J Neurochem 86, 136-142 reports that the cellular location of MEKl is altered in Alzheimer's disease but that the overall levels of MEKl are unchanged.
  • MEKl immunostaining in Alzheimer's disease We consider that there is a small subgroup of neurones that have neurofibrillary tangles that have nuclear MEKl.
  • MEKl may also be a target for diagnosis and therapy of such, disease.
  • the MEKl kinase shares 40% sequence homology with the MEK5 kinase including both enzymes sharing the same allosteric site.
  • the MEKl enzyme has been crystallised.
  • the MEKl enzyme has been studied carefully since the enzyme is expressed in tumour cells and is a good target for inhibition to prevent cancer cell division.
  • Several compounds have been made that bind to the allosteric site of MEKl, including PD 0325901, and have been shown to be well tolerated in phase II clinical trials.
  • the MEK5 enzyme is immunochemically detected in both intraneuronal neurofibrillary tangles and Lewy bodies in patients succumbing to chronic neurodegenerative diseases including Alzheimer's disease and Dementia with Lewy bodies.
  • the present invention provides accurate, sensitive methods and materials for diagnosing and monitoring progressive neurodegenerative disease including, but not limited to, Alzheimer's disease, Parkinson's Disease, Dementia with Lewy Bodies and prion diseases, in humans or animals.
  • the diagnostic methods and materials are based on the finding, reported here, that neuronal expression of the MEK5/ERK5/MEF module provides an index of protein aggregation and neurodegeneration.
  • the invention also provides a novel target for developing imaging agents and therapies against progressive neurodegenerative disease in humans or animals. Neuronal death in the ageing brain could be prevented, by up- regulating the MEK5/ERK5/MEF module which promotes cell survival, and/or down-regulating the MEK3/p38 MAPK module which promotes apoptosis. A therapy based oil this invention could potentially halt the progression of chronic neurodegenerative disease.
  • a first aspect of the invention provides a method of diagnosing a neurodegenerative disease in an individual comprising measuring the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the individual.
  • the module involving MEKl is Ras/Raf/MEKl (MEK2)/ERK1 (ERK2).
  • a further aspect of the invention provides a method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising measuring the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the individual.
  • determining whether a sample of body fluid or tissue contains a certain level of one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or the level of one or more components of the MEK3/p38 MAPK module may be diagnostic of a neurodegenerative disease or it may be used by a clinician as an aid in reaching a diagnosis.
  • the methods of the invention may be used for presymptomatic screening of a patient who is in a risk group for developing a neurodegenerative disease, e.g. a patient having a family history of such diseases. Hence the methods of the invention may also be considered as aiding in the diagnosis of neurodegenerative diseases.
  • levels of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), are elevated in neuronal cells which have high levels of protein aggregation.
  • down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/p38 MAPK module facilitates cell death by apoptosis.
  • Such components can exit the neuronal cells and enter into bodily fluids and/or accumulate in certain body tissues.
  • the level of such a component in a body fluid is an indicator of the level of that component that existed in specific neuronal cells which have lysed, and correlates with the state of the disease.
  • Body fluids isolated from an individual having, or susceptible to having, a neurodegenerative disease will have a characteristically elevated level of a component of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or a component of the MEK3/p38 MAPK module.
  • a direct link can be established between the level of protein aggregation in the neuronal cells of an individual and a sample of body fluid or tissue taken from the same individual. This is considered to be the first diagnostic test available for neurodegenerative diseases.
  • the results obtained from the methods of the invention may be used in association with other clinical indicators of neurodegenerative disease to allow the clinician to reach a diagnosis.
  • MMSE 'Mini-Mental State Examination
  • MMSE Folstein et al (1975) J Psychiatr Res 12:189-198
  • ADAC-Cog 'Alzheimer's Disease Assessment Scale Cognitive Subscale
  • the level of the component(s) of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least Wz fold higher, or it may be at least 2-fold or 3 -fold higher, in the sample than the level of the same component in a body fluid or tissue sample taken from an individual who is not suffering from a neurodegenerative disease.
  • the level of the component(s) of the MEK3/p38 MAPK module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least 1 Vz fold lower, or it may be at least 2-fold or 3-fold lower, in the body sample than the level of the same component in a body fluid or tissue sample taken from an individual who is not suffering from a neurodegenerative disease.
  • the method may further comprise the step of comparing the level of one or more component(s) of the MEK5/ERK5/MEF module, or an activator of. the MEK5/ERK5/MEF module, or a component of the MEK3/p38 MAPK module in the sample to the level of the same component(s) in a normal body fluid or tissue sample, i.e. a sample from an individual who does not have a neurodegenerative disease.
  • components of the MEK5/ERK5/MEF module we include the protein kinases MEK5, ERK5 and the transcription factor MEF.
  • the MEK5/ERK5/MEF module is well known in the art (Watson et at (2001) Nat. Neuroscience 4, 981-988) and information about the MEK5, ERK5 and transcription factor MEF genes and polypeptides is readily available to the skilled person.
  • MEK5 the human MEK5 polypeptide sequence is given in Genbank Accession Ql 3163 and presented below.
  • MEK5 as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEK5 and are included within the scope of this term.
  • MEK5 we mean the MEK5 polypeptide sequence shown below.
  • ERK5 polypeptide sequence is given in Genbank Accession Q13164 and presented below.
  • the term "ERK5" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to ERK5 and are included within the scope of this term.
  • ERK5 we mean the ERK5 polypeptide sequence shown below.
  • ERK5 polypeptide sequence fGenbank Accession 013164) •
  • MEF The human MEF polypeptide sequence is given in Genbank Accession Q06413 and presented below.
  • MEF the MEF polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEF and are included within the scope of5 this term. Preferably, by MEF we mean the MEF polypeptide sequence shown below.
  • an activator of the MEK5/ERK5/MEF module we include molecules which can interact with and activate the function of the MEK5/ERK5/MEF module, i.e. lead to the activation of the MEK5/ERK5/MEF module. Methods of measuring the activation of components of the MEK5/ERK5/MEF module are discussed below in relation to the screening methods of the invention.
  • activators include p62, the MEKK2,3/Tpl2 protein kinase and
  • aPKC "atypical protein kinase C”
  • MEKK2,3/T ⁇ l2 and aPKC are well known in the art (Segfried et at (2005) MoI Cell Biol 25, 9820-9828; Hirano et al
  • MEKK2,3/Tpl2 and aPKC genes and polypeptides is readily available to the skilled person.
  • MEKK2,3/Tpl2 is a protein kinase which interacts with and phosphorylates MEK5. On phosphorylation MEK5 becomes "active".
  • p62 The human p62 polypeptide sequence is given in " Genbank Accession Ql 3501 and presented below.
  • the term "p62” as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to p62 and are included within the scope of this term. Preferably, by p62 we mean the p62 polypeptide sequence shown below.
  • MEKK2,3/Tpl2 The human MEKK2,3/Tpl2 polypeptide sequence is given in Genbank Accession Q9Y2U5 and presented below.
  • MEKK2,3/Tpl2 as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEKK2,3/Tpl2 and are included within the scope of this term.
  • MEKK2,3/Tpl2 we mean the MEKIC2,3/Tpl2 polypeptide sequence shown below.
  • MEK3/p38 MAPK module components of the MEK3/p38 MAPK module
  • MEK3 and ⁇ 38 MAPK both of which are well known in the art. Therefore information about the MEK3 and p38 MAPK genes and polypeptides is readily available to the skilled person.
  • MEK3 the human MEK3 polypeptide sequence is given in Genbank Accession P46734 and presented below.
  • MEK3 as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEK3 and are included within the scope of this term.
  • MEK3 we mean the MEK3 polypeptide sequence shown below.
  • p38 MAPK The human p38 MAPK polypeptide sequence is given in Genbank Accession Q15759 and P53778 and presented below.
  • the term "p38 MAPK” as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to p38 MAPK and are included within the scope of this term. Preferably, by to p38 MAPK we mean the to p38 MAPK polypeptide sequence shown below.
  • MEKl The human MEKl polypeptide sequence is given in Genbank Accession Q02750 and presented below.
  • MEKl as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEKl and are included within the scope of this term. Preferably, by MEKl we mean the MEKl polypeptide sequence shown below.
  • MEKl polypeptide sequence (Genbank Accession 002750) MPKKKPTPIQ LNPAPDGSAV NGTSSAETNL EALQKKLEEL ELDEQQRKRL EAFLTQKQKV GELKDDDFEK ISELGAGNGG VVFKVSHKPS GLVMARKLIH LEIKPAIRNQ IIRELQVLHE CNSPYIVGFY GAFYSDGEIS ICMEHMDGGS
  • Ras polypeptide sequence is given in Genbank Accession AAMl 2633 for N-Ras and presented below.
  • the term "Ras” as used herein includes this polypeptide sequence as well as other members of the Ras family such as K-Ras and H-Ras and naturally occurring variants thereof. Further animal species also have equivalent polypeptides to Ras and are included within the scope of this term.
  • Ras we mean the Ras polypeptide sequence shown below.
  • N-Ras polypeptide sequence (Genbank Accession AAM12633)
  • Raf polypeptide sequence is given in Genbank Accession P04049 and presented below.
  • the term "Ras” as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to Raf and are included within the scope of this ⁇ term. Preferably, by Rafwe mean the Raf polypeptide sequence shown below. - - • --
  • the component of the MEK5/ERK5/MEF module is MEK5.
  • the activator of the MEK5/ERK5/MEF module is p62 or MEKK2,3/Tpl2.
  • the methods of the invention include the step of measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module in a sample of body fluid.
  • the component is a polypeptide.
  • Assaying protein levels in a biological sample can occur using any art-known method.
  • Preferred for assaying protein levels in a biological sample are antibody- based techniques.
  • protein expression in tissues can be studied with classical immunohistological methods.
  • the specific recognition is- provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies.
  • an immunohistological staining of tissue section for pathological examination. is obtained.
  • Tissues can also be extracted, e.g. with urea and neutral detergent, for the liberation of protein for Western-blot or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol.
  • quantitation of protein can be accomplished using isolated protein as a standard.
  • This technique can also be applied to body fluids. With these samples, a molar concentration of protein will aid to set standard values of protein content for different body fluids, like serum, plasma, urine, spinal fluid, etc. The normal appearance of protein amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject.
  • antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify the protein. The level of protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify the protein.
  • the level of protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • Such an ELISA for detecting a rumor antigen is described in Iacobelli
  • the above techniques may be conducted essentially as a “one-step” or “two-step” assay.
  • the "one-step” assay involves contacting protein with immobilized antibody and, without washing, contacting the mixture with the labelled antibody.
  • the "two-step” assay involves washing before contacting the mixture with the labelled antibody.
  • Other conventional methods may also be employed as suitable.
  • Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.
  • Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available.
  • Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction.
  • radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technecium (99mTc)
  • fluorescent labels such as • fluorescein and rhodamine, and biotin.
  • any suitable protein quantitation method for example ELISA, electrophoresis, chromatography or mass spectography.
  • RIA radioimmunoassay
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • sandwich assays using monoclonal and/or polyclonal antibodies include sandwich assays using monoclonal and/or polyclonal antibodies.
  • Antibody staining of cells on slides may be used, using antibodies to the MEK5/ERK5/MEF module in methods well known in cytology laboratory diagnostic tests, as well known to those skilled in the art.
  • the level of the polypeptide may be determined using a molecule which selectively binds to the polypeptide.
  • Antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A “ manual of techniques", H ZoIa (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference. Suitable antibodies include the anti- MEK5 antibody (KAP-MA003) made available by. Bioquote Ltd., (York, U.K.); the ERK5 antibody from Santa Cruz Biotechology Inc; an anti-p62 antibody (p62 lck ligand) from BD Biosciences (Oxford, U.K.).
  • antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.
  • synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs)
  • dAbs domain antibodies
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al; J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al; J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the body fluid may be cerebrospinal fluid (CSF) or blood or the tissue is nasal neuro-epithelial tissue.
  • CSF cerebrospinal fluid
  • Means of collecting such body samples from an individual are well known to those skilled in the art.
  • a further aspect of the invention provides a method of diagnosing or monitoring a neurodegenerative disease in an individual comprising detecting the level of one or more components of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in the brain of the individual, for example in neurons in the brain of the individual.
  • Components of the MEK5/ERK5 pathway are considered to be elevated in neurons in chronic neurodegenerative diseases.
  • a further aspect of the invention provides a method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising detecting the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in the brain of the individual, for example in neurons in the brain of the individual.
  • the components of the MEK5/ERK5 pathway are considered to be elevated in neurons. However, protein components of this pathway may be released into cerebral spinal fluid where they may be detected with suitable assays.
  • component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or one or more components of the MEK3/ ⁇ 38 MAPK module we include those components and activators listed above in relation to the earlier aspects of the invention.
  • the level of said component(s) may be measured using neuroimaging, preferably magnetic resonance imaging (MRI).
  • Other types of imaging that may be useful include positron emission scanning (PET), for example using a 124 I- or 18 F-labelled compound; single photon emission computed tomography (SPECT or SPET), for example using a 99m Tc- or 123 I-labelled compound; or nuclear magnetic resonance (NMR), for example using a 19 F-labelled compound.
  • PET positron emission scanning
  • SPECT or SPET single photon emission computed tomography
  • NMR nuclear magnetic resonance
  • Methods of neuroimaging, including magnetic resonance imaging, of proteins in the neurons in the brain of an individual are well known. Such methods include where an agent (s) which can bind to the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/p38 MAPK module (or to component(s) of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is supplied to a patient. The agent then passes the blood-brain barrier and binds to the said component. The level of said agent in the brain of a patient can then be detected, thus providing an indication of the level of said component(s) in the patient's brain. Examples of agents which can selectively bind to said component(s) are provided below in Example 3.
  • a further aspect of the invention provides a method of measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in neurons in the brain of the individual comprising measuring the level of an agent(s) which can bind to 'the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/ ⁇ 38 MAPK module (or the one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in the neurons in the brain of a patient.
  • Such a method can include where an agent(s) which can bind to the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is supplied to a patient.
  • the agent then passes the blood-brain barrier and binds to the said
  • the level of said agent(s) may be measured using neuroimaging, preferably magnetic resonance imaging or other imaging technique as indicated above.
  • the levels of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module are elevated in neuronal cells in the brain which have high levels of protein aggregation.
  • down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/ ⁇ 38 MAPK module facilitates cell death by apoptosis.
  • the level of such a component in the brain neurons of an individual can correlate with the state of the neurodegenerative disorder.
  • the level of MEKl (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is similarly considered to correlate with the state of the neurodegenerative disorder.
  • these methods of the invention may also be considered as aiding in the diagnosis of neurodegenerative diseases.
  • the level of the component(s) of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module (or of component(s) of the MEKl module (for " example MEKl) or the activator(s) of the MEKl module) which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least IH fold higher, or it may be at least 2-fold or 3-fold higher, in brain neurons than the level of the same component in brain neurons of an individual who is not suffering from a neurodegenerative disease.
  • the level of the component(s) of the MEK3/p38 MAPK module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least IVi fold lower, or it may be at least 2-fold or 3-fold lower, in brain neurons than the level of the same component in brain neurons of an individual who is not suffering from a neurodegenerative disease.
  • the method further -comprises the step of comparing the level of one or more component(s) of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or one or more component(s) of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in neuronal cells in the patient's brain to the level of the same component(s) in a normal brain, i.e. a brain of an individual who does not have a neurodegenerative disease.
  • the method may comprise comparing the level of the component with the level observed previously in the patient's brain. This may be useful in, for example, assessing progression of the patient.
  • the component of the MEK5/ERK5/MEF module is MEK5.
  • the activator of the MEK5/ERK5/MEF module is p62 or MEKK2,3/Tpl2.
  • the component of the MEKl module is MEKl.
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module and assaying for activation of the MEK5/ERK5/MEF module component(s).
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERK5/MEF module and assaying for inhibition of the MEK5/ERK5/MEF inhibitor.
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for activation of the activator.
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK3/p38 MAPK module and assaying for inhibition of the MEK3/p38 MAPK module component(s).
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of one or more components MEK5/ERK5/MEF module.
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of a MEK5/ERK5/MEF module inhibitor.
  • a further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of an activator MEK5/ERK5/MEF module.
  • a further aspect of the invention is a method for detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of one or more components of the MEK3/p38 MAPK module.
  • a further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module (or MEKl module) and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF module (or MEKl module) com ⁇ onent(s).
  • a further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERIC5/MEF module ⁇ ' and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF inhibitor.
  • a further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for binding to, activation or inhibition of the activator.
  • a further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK3/p38 MAPK module and assaying for binding to, activation or inhibition of the MEK3/p38 MAPK module component(s).
  • useful compounds are able to bind to the specified component. Activation or inhibition of that component is not considered to be essential, but ability to activate or inhibit the
  • component may serve as a useful indicator of ability to bind ' to the component.
  • the techniques that are useful in measuring binding directory are well known in the art and are, for example, based on radiolabeled ligands.
  • the level of binding affinity should be at least in the ⁇ M range.
  • the methods of the invention described above are "screening assays". Methods of detecting the activity of the protein kinases MEK5 and ERK5 (or MEKl and ERKl, for example) are well known in the art. Such methods routinely use phosphoepitope antibodies, which recognise when a substrate of the protein ldnase has been phosphorylated and hence the activity of the kinase itself. In such a
  • an experiment is performed in which a ldnase is incubated with a substrate with and without the presence of the test compound.
  • the activity of the kinase to its substrate is then measured using the particular phosphoepitope antibody. In this way the effect of the test compound on the activity of the kinase can be measured.
  • the screening methods may measure any changes in the level of one or more polypeptide component(s) of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module, one or more polypeptide component (s) of the MEK3/p38 MAPK module.
  • Methods by which polypeptide levels can be measured are provided above in relation to earlier aspects of the invention.
  • the screening methods may measure any changes in the level of one or more polynucleotides encoding component(s) of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), one or more polypeptide component (s) of the MEK3/p38 MAPK module.
  • Methods of measuring the change in the level of polynucleotides in a cell are routine in the art and include, for example, northern blotting and RT-PCT.
  • screening assays which are capable of high throughput operation will be particularly preferred.
  • Examples may include cell based assays and protein-protein binding assays.
  • An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used.
  • an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and 32 P-ATP or 33 P-ATP and with the test compound. Conveniently this is done in a 96-well format.
  • the plate is then counted using a suitable scintillation counter, using known parameters for 32 P or 33 P SPA assays. Only 32 P or 33 P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used.
  • the therapeutic screening methods of the invention do not include detecting compounds which upregulate ERK5 activity.
  • MEF is a transcription factor.
  • An assay of MEF activity may be conducted by measuring the effect of a test compound on the amount of a
  • MEF-regulated reporter gene as would be appreciated by a person skilled in the art.
  • a gene encoding the Luciferase polypeptide can be fused next to a MEF-responsive nucleic acid sequence and the resulting reporter gene construct integrated into a suitable cell. Then an experiment is set up in which a cell with this reporter gene is incubated with a test compound and the effect of the compound on Luciferase activity in the cell is assessed and compared to an equivalent cell which has not been exposed to the test compound. In this way the effect of the test compound on the activity of MEF can be measured.
  • An example of a reporter gene assay which could be used to measure MEF activity is provided in Martin et ⁇ l (1993) Proc. Natl. Acad. Sci USA 90, 5282-5286.
  • MEK3/p38 MAPK complements the activities of the MEK5/ERK5/MEF module since the MEK3/ ⁇ 38 MAPK module facilitates cell death by apoptosis.
  • compounds which inhibit the activity of the MEK3/p38 MAPK module may be useful in preventing apoptosis in neuronal cells and therefore useful for the treatment of neurodegenerative disease.
  • Representative genes and polypeptide sequences for the MEK3/p38 MAPK module polypeptide are provided herein.
  • MEK3 is also a protein kinase and hence similar protein kinase assays to those set out above can be used to assay for MEK3 activity.
  • activation of the MEK5/ERK5/MEF kinase module (or MEKl module) an increase in the activity of the MEK5/ERK5/MEF module (or MEKl module)” and “upregulation of the MEK5/ERK5/MEF module (or MEKl module” we include where the test compound increases the activity or expression of that component by 1.5x, 2x, 5x, 1Ox 5 2Ox, 5Ox, 10Ox, 20Ox, 300X, 40Ox, 50Ox, 60Ox 5 70Ox 5 80Ox, 900 or 100Ox or more compared to a component of the MEK5/ERK5/MEF module (or MEKl module, as appropriate) not exposed to the test compound.
  • an inhibitor of the MEK5/ERK5/MEF module we include 2'-amino-3'- methoxyfiavone or an O-alkyl iV-arylanthranilyl hydroxamic acid or analogues thereof which are capable of binding to MEK5 (and MEKl).
  • Example 3 presented below provides further information on these compounds.
  • inhibitors of the MEK5/ERK5/MEF module (or MEKl module) inhibitor and “inhibition of the MEK3/p38 MAPK module” we include where the test compound decreases the functional activity or expression of the inhibitor by 1.5x, 2x, 5x, 10x, 2Ox, 50x, 10Ox or more compared to an inhibitor of the MEK5/ERK5/MEF module (or MEKl module) or MEK3/ ⁇ 38 MAPK module not exposed to the test compound.
  • activation of the activator of the MEK5/ERK5/MEF module we include where the test compound increases the activity or expression of that component by 1.5x, 2x, 5x, 1Ox 5 20x, 5Ox, 10Ox 5 20Ox, 300X, 40Ox 5 50Ox 5 60Ox 5 70Ox 5 800x, 900x or 100Ox or more compared to an activator of the MEK5/ERK5/MEF module (or MEKl module) not exposed to the test compound. ⁇
  • cell we include a cell which contains a MEK5/ERK5/MEF module (or MEKl module), and/or an activator of the MEK5/ERK5/MEF module (or MEKl module), and/or an MEK5/ERK5/MEF module (or MEKl module) inhibitor, and/or a MEK3/p38 MAPK module.
  • Examples of such cells include primary neurones and neuronal cell lines.
  • Such cells are well known in the art and include, for example, mouse N 2 a and PC12 cell lines.
  • the above aspects of the invention are screening methods to identify drugs or lead compounds of use in treating, diagnosing, -monitoring or imaging neurodegenerative diseases.
  • the compound may be a drug-like compound or lead compound for the development of a drug- like compound.
  • a drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non- selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • Suitable characteristics for an imaging compound will also be known to those skilled in the art, and may include the ability to be labelled with a suitable isotope, for example a fluorine isotope.
  • the methods may be used as "library screening" methods, a term well known to those skilled in the art.
  • the methods of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of the polypeptides mentioned herein. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result.
  • the library may preferably be from the same source as the said functional equivalent that is expressed in the said host cell, ie. a human expression library may be screened for effects on host cells expressing human equivalent proteins.
  • a further aspect of the invention provides a compound detected according to any one of the screening assay aspects of the invention for use in medicine.
  • a further aspect of the invention provides a pharmaceutical composition comprising a compound as detected according to any one of the screening assay aspects of the invention and a pharmaceutically acceptable excipient.
  • the aforementioned compounds or a formulation thereof may be administered by any conventional method hicluding oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses ' over a period of time.
  • a compound Whilst it is possible for a compound to be adrninistered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • a further aspect of the invention provides the use of a material which binds to a component of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module or binds to a component of the MEF3/p38 MAPK module in the diagnosis or monitoring of a neurodegenerative disease in an individual or to determine the susceptibility of an individual to developing a neurodegenerative disease or to detect compounds useful for the treatment of neurodegenerative disease.
  • the diagnostic or imaging techniques described herein may be useful for assessing the effect of a treatment regime on a patient and may therefore be useful in evaluating that treatment regime and/or in formulating and testing alternative treatment regimes. The diagnostic or imaging techniques may therefore be useful in clinical trials and may provide a more rapid and quantifiable outcome measurement than, for example, measurement of early behavioural changes.
  • a further aspect of the invention provides a kit of parts useful for diagnosing (or monitoring, as discussed above) neurodegenerative disease comprising a material which is capable for use in determining the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEF3/p38 MAPK module.
  • the kit of parts comprises a control sample containing one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEF3/p38 MAPK module
  • the control sample may be a negative control (which contains a level of said component which is not associated with a neurodegenerative disease) or it may be a positive control (which contains a level of said component which is associated with a neurodegenerative disease).
  • the kit may contain both negative and positive controls.
  • the ldt may be usefully contain controls of said components polypeptide which correspond to different levels in a sample such that a calibration curve may be made.
  • the kit further comprises means for obtaining sample of body fluid or tissue from a patient.
  • Examples of materials which bind to components of MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module, (or MEKl module) or binds to the MEF3/p38 MAPK module are provided above.
  • the material may be an antibody.
  • the material binds to MEK5 (or MEKl), more preferably the material is an anti-MEK5 (or anti-MEKl) antibody, as described above and in the accompanying examples.
  • the material binds to MEK3; preferably the material is an anti- MEK3 antibody.
  • an embodiment of these aspects of the invention is wherein the material is a compound which binds to components of the MEK5/ERK5/MEF module and which is modified with a phase contrast agent.
  • an embodiment of these aspects of the invention is. wherein the material is 2'- amino-3'-methoxyflavone or an 0-alkyl JV-aryl anthranilyl hydroxamic acid or analogues thereof which are capable of binding to MEK5.
  • Example 3 presented below provides further information on these compounds.
  • Such compounds can be further modified to include a phase-contrast agent such as F 19 (and heteronuclei C 3 -F 19 ) for use in MRI visualisation of neurodegeneration (Higuchi supra).
  • a further aspect of the invention provides the use of an oligonucleotide encoding one or more components of the MEK5/ERK5/MEF .module (or MEKl module) or an activator the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease.
  • the members of the MEK5/ERK5/MEF module, MEKl module, activators of the MEK5/ERIC5/MEF module or MEKl module and the MEK3/p38 MAPK module are well known to those skilled in the art.
  • the skilled person could identify an appropriate oligonucleotide sequence from Genbank with no inventive contribution required.
  • a polynucleotide sequence encoding MEK5 is provided in Genbanlc accession number BC008838; ERK5 polynucleotide sequence is provided in Genbank accession number BC030134; MEF polynucleotide sequence is provided in Genbank accession number BC026341; p62 polynucleotide sequence is provided in Genbank accession number BC003139; MEKK2,3/T ⁇ l2 polynucleotide sequence is provided in Geribank accession number NM_006609; p38 MAPK polynucleotide sequence is provided in Genbank accession number BC027933 and BCOl 5742; MEK3 polynucleotide sequence is provided in Genbank accession number BC032478.
  • a further aspect of the invention provides the use of a component of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or a component of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease.
  • the component is MEK5.
  • a further aspect of the invention provides a method of treating an individual suffering from a neurodegenerative disease comprising supplying to a patient an ' appropriate quantity of a compound detected according to any one of the previous screening methods of the invention.
  • a further aspect of the invention provides the use of a compound detected according to any one of the previous therapeutic screening methods of the invention in the manufacture of a medicament for preventing or treating a neurodegenerative disease.
  • a further aspect of the invention provides the use of a compound detected according to any one of the previous imaging screening methods of the invention in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
  • a further aspect of the invention provides a method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of upregulating MEK5/ERK5/MEF module (or MEKl module) activity either directly or by downregulating the activity of one or more inhibitors of MEK5/ERK5/MEF module (or MEKl module) activity.
  • a further aspect of the invention provides the use of an agent or agents capable of upregulating MEK5/ERK5/MEF module activity (or MEKl module activity) either directly or by downregulating the activity of one or more inhibitors of MEK5/ERK5/MEF module activity (or MEKl module activity) in the manufacture of a medicament for treating a neurodegenerative illness in a human
  • a further aspect of the invention provides a method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of inhibiting MEK3/p38 MAPK module activity.
  • a further aspect of the invention provides the. use of a pharmaceutically effective amount of an agent or agents capable of inhibiting MEK3/p38 MAPK module activity in the manufacture of a medicament for treating a neurodegenerative illness in a human or animal.
  • the method or medicament of the previous aspects of the invention does not comprise an agent or a compound which upregulates ERK5 activity.
  • a further aspect of the invention provides a method of preventing neuronal cell death comprising upregulating the activity of the MEK5/ERK5/MEF module (or MEKl module) and/or downregulating the activity of the MEK3/p38 MAPK module in the neuronal cell.
  • a preferred embodiment is wherein the neurodegenerative disease or illness is progressive.
  • the progressive neurodegenerative disease is Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy, multisystem atrophy, motor neurone disease (amyotrophic lateral sclerosis) or frontotemporal dementia.
  • the individual may be a human or animal; preferably the individual is a human.
  • the compounds described in Example 3 or compounds identified by the screening • methods of the invention in relation to the MEK5 module may also be useful in treating, diagnosis, monitoring or imaging (as appropriate) of cancer, particularly prostate cancer.
  • MEK5 has been implicated in cancer, particularly prostate cancer.
  • a further aspect of the invention provides a compound which binds to a component of the MEK5/ERK5/MEF modulate or to an activator of the MEK5/ERK5/MEF module or binds to a component of the MEF3/p38PAK module, for example, for use in treating, imaging, diagnosing or monitoring cancer, for example prostate cancer.
  • the compounds useful in treating and imaging prostrate cancer are the same as for neurodegeneration.
  • a further aspect of the invention provides the anthranilyl hydroxamic acid analogue JV-(Cyclopropylmethoxy)-3 ,4-difluoro-2-(2-fluoro-4- iodophenylamino)benzamide; 2-(4-Bromo-2-fluorophenylamino)-7v r -
  • the anthranilyl hydroxamic acid analogue is 2-(4-Chloro-3- memoxyphenylarnino)-7V-ethoxy-3 ,4-difluorobenzamide; (2-(4-Bromo-3 - methoxyphenylamino)-3,4-dirluorophenyl)methanol; or (2-(4-Chloro-3- methoxyphenylammo)-3,4-difluorophenyl)methanol; or radiolabelled or fluorescently labelled derivative any thereof.
  • a further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention for use in medicine.
  • a further aspect of the invention provides a pharmaceutical composition comprising an anthranilyl hydroxamic acid analogue of the invention and a pharmaceutically acceptable excipient.
  • a further aspect of the invention provides the anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition for use in aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
  • a further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention or related pharmaceutical composition in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
  • the neurodegenerative disease may be a progressive neurodegenerative disease.
  • the progressive neurodegenerative disease may be Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy, multisystem atrophy, motor neurone disease (amyotrophic lateral sclerosis) or frontotemporal dementia.
  • the individual may be a human or animal patient.
  • a further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition for use in aiding diagnosing, imaging or monitoring of prostate cancer.
  • a further aspect of the invention provides, the use of an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of prostate cancer.
  • a furfltier aspect of the invention provides a method of aiding diagnosing, imaging or monitoring of prostate cancer, wherein the patient is administered an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition.
  • Figure 1 Immunohistochemical detection of MEK5, p62 and ERK5 in neurones containing Lewy bodies, from the temporal cortex from a case of Dementia with Lewy Bodies.
  • Figure 2 Immunohistochemical detection of MEK5, p62 and ERK5 in neurones containing neurofibrillary tangles, from the temporal cortex from a case of Dementia with Lewy Bodies.
  • FIG. 3 Imrnunohistochemical detection of MEK5, p62 and ERK5 in neurones showing granulovacuolar degeneration (GVD), from the hippocampus from a case of Dementia with Lewy Bodies.
  • VFD granulovacuolar degeneration
  • FIG. 4 A. Expression profile of the MEK5 gene. At the beginning of the analysis of gene expression protein aggregation has already started (Supplementary Methods). The expression of MEK5 exhibits a clear up-regulation that proceeds gradually and peaks at 24 h, before the peak of maximal aggregate load (at 48 h) and subsequently subsides. Data are from pooled RNA samples derived from 6 culture plates (control or co-transfected) transfected simultaneously and harvested at the designated times.
  • MEK5/ERK5 module responds to serum growth factors in the medium (11).
  • FIG. 5 Aggregate formation exhibits high specificity even during times of considerable aggregate load (here at 16 h post transfection).
  • a 3D projection of the interior of a co-transfected cell shows that S 8 ATP ase and mGluRl ⁇ receptor aggregate individually and do not intermix. Aggregates emitting one fluorescent signal were encircled on the panels on the left side, at different depths, and then projected on to the panels of the other fluorescent signal on the right side. Signals were not observed to coincide in any case. This observation was also verified by 3D rotational examination (not shown).
  • Figure 6 The schematic structure of MEKl with Mg, ATP ⁇ thin stick) and PD318088 (thick stick) bound, showing a 'closed' conformation involving the 'activation loop' (yellow) and helix C (green) (Ohren, 2004).
  • Figure 7 The ligand PD318088 bound to MEKl, in which the ligand B-ring via van der Waals interaction makes contact with the hydrophobic side-chain of Met 143 (ball-and-stick) (Ohren, 2004).
  • the bound ATP was removed in order to facilitate visualisation of the ligand interactions.
  • Figure 9 Methods for production of radioimaging ligands for SPECT and PET
  • Figure 10 A. Expression analysis of MEKl. BL21(DE3)pLys cells were lysed by sonication and 50 ⁇ l of soluble fraction was analysed on 5-20% SDS-PAGE and stained with Coomassie blue.
  • Figure 11 Analysis of the imidazole eluted His-tagged MEKl recombinant protein from the nickle-charged resin. 50 ⁇ l eluted protein was loaded on each. Lane. 1: elution using 500 mM imidazole; Lane 2: elution using 50 mM EDTA. The figure shows 500 mM imidazole is slightly more efficient than EDTA.
  • Figure 12 Fluorescence polarization assay was employed to assess the binding of chemical reagents to the MEKl recombinant protein in competition with fluorescent probe. • ⁇
  • Binding reaction was carried out for 30 minutes at room temperature in a 384- well plate (384 ShallowWell, NuncTM) in final volume of 150 ⁇ l with 2 nM fluorescent probe and increasing concentration of MEKl protein as indicated.
  • the binding affinity was also measured with 500 nM, 200 nM and 100 nM fluorescent probe (data not shown) which gave similar affinity constant (Zd) 485 nM.
  • ERK5 antibody from Santa Cruz Biotechology Inc. and is rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 516-815 of the carboxy terminus of human ERK5. .
  • p62 is a scaffolding protein that can bind multiubiquitin chains attached to misfolded and aggregated proteins and which is found in all intracellular inclusions containing ubiquitylated proteins in chronic neurodegenerative diseases.
  • the deposits of MEK5 and p62 accumulate in overlapping deposits in neurones containing Lewy bodies ( Figure 1) and neurofibrillary tangles ( Figure 2), which is expected since MEK5 binds to p62.
  • the MEK5 effector kinase ERK5 is also detected in the nuclei of afflicted cells ( Figure 1 and 2).
  • MEK5 and ERK5 are also present in areas of granulovacuolar degeneration (GVD) in the hippocampus which do not contain the p62 protein ( Figure 3). These areas are thought to represent areas of intense autophagic activity to eliminate aggregate-prone proteins by the autophagosome-lysosome system.
  • FIGS 1-3 Immunohisto chemical detection of MEIC5 and ERK5 in neurones from the temporal cortex from a case of dementia with Lewy bodies.
  • Fig 1 immunostaining of MEK5 in a neurone containing a Lewy body
  • Fig 2 MEK5 in a neurone containing a neurofibrillary tangle
  • Fig 3 MEK5 in a neurone showing granulovacuolar degeneration (GVD)
  • FIG 1 ERK5 in neurone containing a Lewy body
  • Fig 3 ERK5 in a neurone containing a neurofibrillary tangle
  • Fig 3 ERK5 in a neurone containing GVD.
  • An anti-MEK5 antibody directed to the N- terminus of the protein was used for immunocytochemical detection of the protein
  • Example 2 Gene expression changes in response to aggregate-prone proteins identifies mitogen activated protein kinase kinase 5 (MEK5) which, with ERK5, is abundant in neurones in Alzheimer-related disease.
  • MEK5 mitogen activated protein kinase kinase 5
  • Intra-neuronal protein aggregation is central to chronic neurodegenerative disease.
  • the genetic response to aggregate-prone proteins has not been previously investigated.
  • a pan-genomic screen of expression changes to protein aggregates reveals that the expression of MEK5 matches the accumulation and dissolution of protein aggregates.
  • Immunochemical extrapolation to Alzheimer-related disorders showed that MEK5 ⁇ and ERK5 are abundant in afflicted neurons in the brain. The findings have novel implications for neuronal survival.
  • Intraneuronal protein aggregates are the single most consistent molecular feature of all the chronic neurodegenerative disorders. This diverse range of neurological conditions including Alzheimer's disease (AD), Parkinson's disease (PD) and
  • AD Huntington's disease
  • a significant development in understanding neurodegeneration came with the experimental demonstration of a novel type of intracellular protein deposit termed the "aggresome".
  • the aggresome originally identified by protein microinjection studies 1 ' 2 in cultured cells, is an intracellular structure formed in response to aberrantly folded 3 or mutated protein 4 .
  • Aggresome formation may be cytotoxic and contribute to neuronal cell death.
  • reports advocating a cytoprotective rather than cytotoxic function for aggresomes have been published with increasing frequency in recent years 5 ' . Aggresomes may be an end-point in the cellular response to aggregate-prone proteins.
  • MEK5 has one known down-stream phosphorylation target, ERK5, and this kinase ' module is specifically activated by axonally-delivered neurotrophins to cause the expression of neuronal survival genes 10 .
  • the MEK5/ERK5 module activates the myocyte enhancer factor to trigger neuronal expression of the survival genes.
  • MEF2C has a major role in survival of neurons during development 11 and the MEK5/ERK5 module appears to be central in the response to cellular insults including oxidative stress 12 .
  • the findings presented here may suggest the existence of a previously unsuspected cellular mechanism for detecting aggregate-prone proteins that includes the MEK5/ERK5 signalling pathway.
  • the GFP-S 8 ATPase fusion protein cDNA was expressed ligated in the pEGFP- Cl vector (Promega, Southampton, UK).
  • the FLAG-tagged mGluRl ⁇ receptor cDNA was expressed in the pcDNA 3.1 Zeo (+/-) plasmid vector (Invitrogen Corp., Carlsbad, CA, USA).
  • Mouse monoclonal anti-FLAG (M2) was purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK) and used to detect FLAG- tagged mGluRl ⁇ . Immunostaining was performed using anti-mouse Alexafluor 594-conjugated antibody supplied by Molecular Probes (Eugene, Oregon, USA) as secondary antibody.
  • Immunohistochemical detection of p62 in paraffin sections was performed with mouse monoclonal anti-p62 (p62 lck ligand) directed to the C-terminus of the protein purchased from BD Biosciences (Oxford, U.K.). Immunohistochemical detection of ERK5 in paraffin sections was performed with rabbit polyclonal antibody (H300: directed to the carboxy-terminus of ERK5) purchased from Auto gen Bioclear, (Came, Wiltshire, U.K).
  • HEK293 cells were co-transfected with vectors expressing S 8 ATPase/mGluRl ⁇ by the calcium phosphate method.
  • Culture plates (100 mm) at 50-70% confluence were selected and media changed 3 h prior to transfection.
  • To prepare the initial CaCl 2 -DNA solution for each 100 mm plate sterile deionised H 2 O, 10 ' ⁇ g of purified DNA (each vector) and 83 ⁇ l of 1.5 M CaCl 2 were added sequentially.
  • the CaCl 2 -DNA solutions were made up to 500 ⁇ l with sterile water.
  • Experimental design included a minimum post-transfection incubation period (4 h) for DNA absorption and cell adherence to the plates. Aggresome formation was assessed in three experiments by confocal microscopy, gene expression profiling and apoptosis evaluation. Cells were harvested at O h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h and 72 h after the post-transfection incubation period.
  • FLAG-mGluRl ⁇ and GFP-S 8 ATPase were detected via immunofluorescence on fixed cell preparations.
  • Cells were fixed with paraformaldehyde (4%) followed by serial application of staining antibodies and mounting in DAPI-Vectashield immunofluorescence medium (Vector Laboratories, Burlingame, CA, USA), shortly before visualization by confocal microscopy.
  • Cells were cultured on plates containing cover slips. At each time point coverslips were removed from the plate and carefully washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.47 mM KH 2 PO 4 , pH 7.3).
  • each coverslip was fixed in ImI of fresh paraformaldehyde (4%) in PBS. Each coverslip was left for 40 min at room temperature. Subsequently, cells were washed with 10 mM Glycine in PBS and then twice with PBS. An aliquot of freshly made 0.1% (v/v) Nonidet-P40 (IGEPAL-63; ICN Biomedical Inc., Aurora, Ohio, USA) in PBS was subsequently applied to each coverslip to permeabilise the cells. Permeabilisation was allowed to proceed for 5 min at room temperature followed by washing 3 times with PBS. Cells were then incubated with 500 ⁇ l of Blocking Buffer (1% donkey serum in PBS) for 1 h before application of primary antibody.
  • Blocking Buffer 1% donkey serum in PBS
  • the anti-FLAG primary antibody was applied in 1:100/ 1 :200 dilutions in blocking buffer and the coverslips were incubated overnight in a humid chamber at 4 0 C, in the dark. Following incubation with the primary antibody, coverslips were washed 3 times with PBS and incubated with the secondary anti-mouse Alexafluor 594 antibody , prepared as a 1 :50 dilution in blocking buffer. The secondary antibody incubation was carried out for 1 h at room temperature, followed by PBS washing and mounting in DAPI-Vectashield immunofluorescence medium. All immunostaining reactions were carried out with minimum exposure of cells to light to avoid fading of the contained GFP-S8 ATPase fluorescent signal. Confocal microscopy image analyses were performed using the LSM510 Browser software supplied by Carl Zeiss company (Oberkochen, Germany).
  • RNA samples were used for cRNA preparation.
  • the first and second strand cDNA synthesis was performed using the Superscript Choice System (Life Technologies) according to the manufacturer's instructions except using an oligo(dT) primer containing a T7 RNA polymerase promoter site.
  • Labelled cRNA was prepared using the BioArrayTM RNA Transcript labelling kit (Enzo Life Sciences, Farmingdale, NY, USA) in total reaction volumes of 40 ⁇ l. Biotin- labelled CTP and UTP (Enzo) were used in the reaction together with unlabelled NTPs. Following in vitro transcription, the unincorporated nucleotides were removed using RNeasy columns (Qiagen).
  • the probe array was exposed to 10 washes in 6x SSPE-T at 25 °C followed by four washes in 0.5x SSPE-T at 50 0 C.
  • the biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, 10 mg/ml (Molecular Probes, Eugene, OR), in 6x SSPE-T for 30 min at 25 0 C followed by 10 washes in 6x SSPE-T at 25 0 C.
  • the probe arrays were scanned at 560 nm using a confocal laser- 10 scanning microscope with an argon ion laser as the excitation source (Hewlett Packard GeneArray Scanner G2500A). The readings from quantitative scanning were analysed by the Expressionist Analyst software (version 5.0.3) from Genedata AG (Basel, Switzerland), which was also used for normalization of the different arrays prior to the analysis.
  • HRP horse radish peroxidase conjugated goat anti-rabbit [1:5000 in Marvel (1% w/v) in TTBS] was then applied to the membrane for 1 hour at room • temperature followed by washing (2 x 10 minutes) in TTBS and finally in TBS for 10 minutes. Antigen was visualized by ECL with film exposure for 5 minutes.
  • Routine paraffin-embedded sections and antigen retrieval techniques were used for the immunocytochemical analyses of a laboratory control tissue block from a case of Dementia with Lewy bodies.
  • the synthetic molecules 2'-amino-3'-methoxyflavone (also known as PD98059) and 2'-amino-3'-methoxy-thiofiavone are highly selective and potent inhibitors of MEKl (Dudley, 1995; Kataoka, 2004). These flavones selectively block the activation of MEKl, without inhibitory effects on the phosphorylation activities of both upstream and downstream kinases.
  • the high selectivity for MEKl is not surprising since in contrast to many of the well established kinase inhibitors, e.g.
  • these compounds bind to inactive MEKl via a non-competitive mechanism and selectively block the activation of MEKl.
  • PD318088 binds to a unique allosteric pocket within the hinge region (i.e. between the N-terminal ⁇ -sheets and the C-terminal ⁇ -helical lobes), which is separate from but adjacent to the Mg- ATP binding cavity (Ohren, 2004).
  • the bound ligand induced significant conformational changes (see Figure 6; Ohren, 2004) of the MEKl helical 'activation loop' and helix C, as well as deformation of the catalytic site.
  • a solution of LiHIsO)S was prepared by the addition of 2.5 M butyllithium in hexanes (26.8 ml, 67 mmol) to a solution of hexamethyldisilazane (14.1 ml, 67 mmol)' in THF (50 ml) at -78 °C. This solution was warmed to ambient temperature over 1 h, which was then gradually added to a solution of 5-amino-2- chloroanisole (7.1 g, 45.0 mmol) in THF (100 ml) at -78 °C. The resulting dark suspension was then stirred for a further 1 h.
  • outlined in Figure 9 are two simple methods for the production of the radiopharmaceuticals, JV-(ethoxy)-3 ,4-difluoro-2-(2-fluoro-4-[ 123 I]iodophenyl- amino)benzamide and 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-iV-(2- [ 18 F]fluoroethoxy)benzamide, from the compounds 7 and 4, for use in SPECT and PET respectively.
  • PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270, 27489-27494.
  • Endogenous ERK5 or tagged-ERK5 was immunoprecipitated with anti-ERK5 antibody (Ab 1) or anti-tag antibody and incubated for 10 min at 3O 0 C with myelin basic protein (final concentration, 2 mg/ml) in reaction buffer (20 mM Tris-Cl (jpH 7.5], 10 mM MgCl 2 , and 100 ⁇ M ATP, or 2 ⁇ Ci of [ ⁇ - 32 P]ATP).
  • Antibodies were purchased as follows: rabbit anti-ERK5 antibody from Sigma (Ab 1) and Calbiochem (Ab 2); rabbit anti-phospho-ERK5 antibody from Sigma (Ab 1) and Calbiochem (Ab 2); rabbit anti-phospho-ERK5 antibody from Sigma (Ab 1) and Calbiochem (Ab 2); rabbit anti-phospho-ERK5 antibody from Sigma (Ab 1) and Calbiochem (Ab 2); rabbit anti-phospho-ERK5 antibody from
  • the human MEKl (Gene Bank Accession number: L11284.1) spanning 2,167 bps and contains 11 exons, was cloned by PCR utilizing cDNA derived form HeLa cells as the template.
  • the primers utilized for full length MEKl amplification were: MEKl forward :5'CGC GGA TCC ATG CCC AAG AAG AAG CCG3';
  • MEKl reversed 'GCAA GCT TCG TTA GAC GCC AGC AGC ATG3'.
  • Direct ligation of the Ml length MEKl cDNA into the ⁇ RSET-A plasmid was performed using BamHI and HindIII restriction sites introduced into the forward and reverse primers respectively.
  • MEKl recombinant protein is N-terminally tagged with 6 histidine residues for further purification. The final construct was verified by DNA sequence analysis.
  • MEKl cDNA was inserted into different plasmids, pET-41a, pET-28a (Novagen) and pGEX-4T-l (Amersham Biosciences) to produce the MEKl recombinant protein with different tags.
  • BL21(DE3)pLyse E. coli competent cells (Novagen) were utilized as a bacterial host for expression of the recombinant MEKl protein. Optimization of the expression was completed.
  • the BL21(DE3)pLyse cells were grown in the LB medium at 37 0 C and induced with 500 ⁇ M isopropyl- ⁇ -D-thiogalactopyranoside
  • the pRSET-A plasmid gave the best expression of MEKl recombinant protein.
  • the other plasmids did not yield a sufficient amount of protein in comparison to pRSET-A.
  • cell growth at 37 0 C produced the best result.
  • LB culture 1 litre LB culture was utilized. After 6 h of IPTG induction, the cell pellet was harvested (3000g, 10 rnin at room temperature) and bacterial cells lysed in ' lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40 and 0.1% SDS) and then sonicated on ice for 5 x at high output setting. The lysate was centrifuged at high speed (1000Og, 10 min at room temp), insoluble fraction was spun down and supernatant was collected.
  • ' lysis buffer 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40 and 0.1% SDS
  • nickel-charged resin was employed to purify the His-tagged protein under native conditions. Imidazole was added to the supernatant to a final concentration of 10 mM and applied to the prepared 700 ⁇ l nickel-charged resin. The column was incubated with gentle shaking overnight at 4 0 C. The resin was then washed 3x with TBS, TBS-20 mM Tris and TBS-40 mM Tris (pH 7.5) respectively. . The recombinant MEKl protein was eluted using 1000 ⁇ l of 500 mM imidazole. The purified protein was analysed on 5-20% SDS-PAGE electrophoresis (Figure 11).
  • the resin product in the reaction column was treated with 20% piperidine (2.5 ml mm “1 , 10 min), washed with DMF (2.5 ml min "1 , 15 min), excess DMF removed and treated with a pre-prepared mixture of 3,4-difluoro-2- (2-fluoro-4-iodophenylamino)benzoic acid (98 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol) and ⁇ yV-diisopropylethylamine (0.087 ml, 0.5 mmol) in DMF (1 ml) for 1O h.
  • the resin product was washed with DMF (2.5 ml min '1 , 10 min), treated with 4% hydrazine monohydrate in DMF (2.5 ml min "1 , 30 min), washed with DMF (2.5 ml min '1 , 15 min), excess DMF removed and then treated with a pre- prepared mixture of 5 -carboxy fluorescein (100 mg, 0.26 mmol), 7-aza-l- hydroxybentriazole (34 mg, 0.25 mmol) and ⁇ yV'-diisopropylcarbodiimide (0.110 ml, 0.25 mmol) in DMF (1 ml) for 14 h. The resin product was then successively washed with DMF (2.5 ml min "1 , 15 min), 20% piperidine in DMF (2.5 ml min "1 , 15 min), and DMF (2.5 ml min "1 , 15 min).
  • the coloured resin product was collected, rinsed with CH 2 Cl 2 , dried in vacuo, and then treated with the acidolytic mixture trifluoroacetic acid (6 ml) - H 2 O (0.6 ml) - iPr 3 SiH (0.2 ml) for 2 h at ambient temperature.
  • the suspension was filtered, the filtrate was evaporated to dryness in vacuo and the residual material was triturated with diethyl ether (3 x 5 ml) to afford the title compound as a bright yellow solid (48 mg).
  • Reversed-phase HPLC was carried out using WatersTM 510 pumps, WatersTM 484 detector and HypersilTM Pep 100-C 1S analytical column (150 x 4.6 mm, 5 ⁇ m) at a flow rate of 1.20 ml min "1 and the effluent was monitored at 220 nm. Gradient elution was from 50 % to 100 % B in 20 min, and the eluents used were: solvent A (0.06% v/v TFA in MiUi-Q water) and solvent B (0.06% v/v TFA in MeCN - Milli-Q water, 9:1 v/v). The solid product was established by HPLC to be >90% pure; ⁇ t . 8.48 min.
  • a fluorescence polarization assay (FP) was employed.
  • the assay is based on competition between the fluorescent compound [TV 0 - (fluorescein-5-carbonyl)-iV E -(3,4-difluoro-2-(2-fluoro-4- iodophenylamino)benzoyl)-L-lysyl amide; Compound Code: WCC030F] and increasing amounts of competing compound, determined as fluorescence polarization intensity (milli-Polarization, mP).
  • assessments that can be performed on a target . imaging compound.
  • the objective of the work is to identify compounds that bind to MEK5 better than MEKl for the neuroimaging analyses.
  • the compounds are designed to fit into the MEK1/MEK5 allosteric regulation site.
  • Zatloukal, K. et al. p62 is a common component of cytoplasmic inclusions in protein aggregation diseases.

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Abstract

A method of diagnosing, monitoring or imaging a neurodegenerative disease in an individual the method comprising measuring the levels of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the individual. A method of detecting compounds useful for the diagnosis, monitoring, imaging or treatment of neurodegenerative disease comprising contacting a test compound with a member of the MEK5/ERK5/MEF module and assaying for an increase in the activity of the MEK5/ERK5/MEF module. Use of a component of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or the MEK3/p38 MAPK module in a method of detecting compounds useful for the diagnosis, monitoring, imaging or treatment of neurodegenerative disease.

Description

METHODS
The present invention relates to diagnostic methods and therapies for neurodegenerative illnesses characterised by the occurrence of intracellular protein aggregates, including, but not limited to, Alzheimer's Disease, Dementia with Lewy Bodies and Parkinson's Disease. The invention relates to mitogen activated protein kinase kinase 5 (MEK5) and mitogen activated protein kinase kinase 1 (MEKl) as targets for developing diagnostic tests and therapies for progressive neurodegenerative illness.
Demographic predictions indicate that by 2015 approximately 40% of the population of the United States, Europe and Japan will be over the age. of 60, 15% of the population will be over the age of 70, 10% over 80 and 5% over 90. A consequence of this dramatic expansion of the ageing population will be a concomitant increase in the number of people with chronic neurodegenerative diseases causing dementia. Progressive dementias, which include Alzheimer's disease, Dementia with Lewy Bodies, dementia associated ' with Parkinson's disease, Huntington's Disease, prion diseases and a number of rarer conditions, cause a progressive loss of mental function resulting from the death of nerve cells.
Current therapies for Alzheimer's Disease (acetyl cholinesterase inhibitors) and Parkinson's Disease (dopaminergics and dopamine agonists) can provide only temporary and limited relief from symptoms. At present, no medications are approved specifically to treat Dementia with Lewy Bodies or most other types of progressive dementia. There is a clear need for therapies which can halt or slow disease progression. There is also a need for diagnostic tools for progressive neurodegenerative diseases. At present the only unambiguous way to diagnose neurodegenerative disease is autopsy. Diagnosis in living patients is a difficult and complex procedure based on a battery of physical, neurological and cognitive tests. A test which could accurately detect the early stages of disease in body fluids, or by neuroimaging techniques, would have clear utility in allowing medical intervention before irreversible brain damage has occurred. . There is an enormous research, literature on the molecular mechanisms that cause dementing illness. The literature stems from neuropathological observations, and shows that a wide range of neurodegenerative disorders share a common molecular feature: the occurrence of intracellular protein aggregates. Alzheimer's Disease is characterised by intraneuronal neurofibrillary tangles and extracellular amyloid deposits. Dementia with Lewy Bodies is characterised by intraneuronal Lewy bodies with extraneuronal amyloid plaques 1-3. Lewy bodies also occur in Parkinson's Disease. Huntington's Disease and the other polyglutamine disorders are associated with aggregation of the polyQ-expansion-containing proteins including huntingtin. Prion diseases such as Creutzfeld-Jacob Disease are associated with the aggregation of the PrPSo protein. A major research focus into targets for novel therapies is on the proteins which aggregate in these disorders.
The extracellular amyloid is generated when the Alzheimer precursor protein (APP) is cleaved to produce the aggregate-prone Aβ peptides. There are intense studies on the enzymes responsible for APP cleavage as targets for therapeutic intervention 4. The aggregation of hyperphosphorylated tau and α-synuclein in neuronal inclusions are the halhnarks of Alzheimer's disease and dementia with Lewy bodies respectively. There is also intense work on the kinases which phosphorylate tau, primarily cyclin dependent kinase 5, glycogen synthetase kinase 3β and the MARCK kinase 6. The aggregated proteins found in the inclusions which characterise Alzheimer's Disease and Dementia with Lewy Bodies are also ubiquitylated 7. Protein ubiquitylation is one of the two major mechanisms for protein degradation in all cells, the other being autophagy 8. The brains of patients succumbing to dementing illness show extensive loss of neurones in several regions including the hippocampus and cerebral cortex. The relationship between protein phosphorylation, protein ubiquitylation, neuronal death and chronic neurodegenerative disease is not well understood.
There are many investigations into protein aggregate formation and searches for compounds to prevent intracellular protein aggregation 5. However it is not yet clear whether protein aggregation is cytotoxic, contributing to neuronal cell death, or cytoprotective, promoting neuronal survival. An alternative approach, is to develop drugs which protect neurons from damage or death. Most neuroprotective drug development has focused on mimicking the effects of nerve growth factor, or on reducing oxidative stress. As no neuroprotective drug has yet reached late-stage clinical trials for treatment of neurodegenerative disease, the utility of the current drug candidates is not yet known. There is a need to identify further pathways which can be manipulated to promote neuronal survival.
A greater understanding of the cellular response to the aggregation of proteins is key to understanding the pathogenesis of these diseases and developing novel diagnostic and therapeutic interventions. A generic response to the aggregation of proteins has been studied by looking for changes in gene expression in cells expressing two aggregate-prone proteins, namely the proteasomal S 8 ATP ase and the metabotropic glutamate receptor 15. This experiment uncovered a major increase and subsequent decrease in the expression of the gene for mitogen activated protein kinase kinase 5 (MEK5) which mirrored the accumulation and elimination of the aggregate-prone proteins from cells by the protein catabolic systems. There was a concomitant down regulation of the expression of the MEK3 gene.
MEK5 (also known as MKIC5, PRKMK5, MAPKK5 and MAP2K5) is a kinase kinase involved in the mitogen activated protein kinase (MAPK) signalling pathway. This pathway comprises several different families of interacting kinases forming 'modules', which act in parallel pathways to regulate diverse activities including cell division, gene expression and apoptosis. A kinase kinase kinase phosphorylates a kinase kinase, -which phosphorylates a kinase, which triggers regulatory processes in the cell. MEK5 is phosphorylated by MEKK2,3/Tpl2 and atypical protein kinase C. MEK5 has only one known kinase substrate, ERK5/BMK1. EREL5 specifically phosphorylates the myocyte enhancer factor (MEF) family of transcription factors that activate expression of neuronal survival genes during development. The down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/MAPK module facilitates cell death by apoptosis 9. The microarray analysis described above shows for the first time changes in gene expression, including activation of the MEK5 module and inactivation of the MEK3 module, in response to protein aggregation in cells. However these cells were not of neuronal origin, nor were they expressing proteins implicated in ■ neurodegeneration. The aggresomes formed in this experimental system disappeared within 3 days and had no effect on the viability of the cells. The changes in gene expression detected in this system appear to be a generic response to protein aggregation and would not necessarily be expected to mirror the process which causes neuronal loss in the brains of patients with neurodegenerative disease.
The present invention provides novel methods, assays and materials useful in the diagnosis of progressive neurodegenerative illnesses including, but not limited to, Alzheimer's Disease, Parkinson's Disease and Dementia with Lewy Bodies. It further provides novel targets for development of neuroprotective therapeutic agents and imaging agents, and novel screening methods and materials to discover such therapeutic and imaging agents.
The present invention is based upon the immunohistochemical (IHC) demonstration that neurones containing neurofibrillary tangles and Lewy bodies in human brain show a large increase in the expression of MEK5 (Figures 1 to 4) and its effector protein ERK5 (see appendix for methods and data). In contrast, adjacent neurones without these inclusions do not show enhanced expression of the kinase module. This demonstrates for the first time that the MEK5/ERK5/MEF survival and cytoprotective responses take place in chronic neurodegenerative diseases, and implies that a mechanism of neuronal survival is operative at certain stages in the diseases. Neurones failing to activate this system or where the system becomes dysfunctional may succumb to the deleterious effects of protein aggregation and undergo programmed cell death, accounting for the enormous loss of neurones in afflicted areas of the brain in dementing illness. While MEK5 is implicated in the pathogenesis of diseases characterised by inappropriate cell survival such as cancer and cardiac hypertrophy, and downregulation of MEK5 is known in the art as a potential means of developing novel therapies for such diseases, the current disclosure provides the first indication that upregulation of MEK5 has potential therapeutic utility and diagnostic utility.
The expression of the protein p62/sequestosome was simultaneously investigated in the same histological sections. p62 binds to ύbiquitylated proteins and is present in inclusions in all the neurodegenerative disease π. A recent report indicates that the p62 protein and MEK5 both have a ubiquitin-like PBl domain through which p62 and MEK5 can bind together n. Here, IHC shows that MEK5 and p62 are both present in inclusions containing ubiquitylated proteins. This is the first report of a connection between ύbiquitylation of aggregate-prone proteins, p62 and the MEK5/ERK5/MEF module for activation of neuronal survival genes. MEK5 is also present in areas of granulovacuolar degeneration in the hippocampus which do not contain the p62 protein. These areas are thought to represent areas of intense autophagic activity to eliminate aggregate-prone proteins by the autophagosome-lysosome system. This is the first report that MEK5 is associated with the autophagolysosomal system.
This study provides the first mechanistic connection between the ubiquitylation of aggregate-prone proteins and the MEK5/ERK5 module of pro-survival signalling proteins. It also suggests for the first time a reason for the persistence of ubiquitylated proteins in inclusions in all the chronic neurodegenerative diseases.
Ubiquitylation of aggregate-prone proteins could provide the physical anchor for the linked p62/MEK5/ERK5 cluster of proteins. The unexpected observation that a specific kinase pathway known to control cell survival is activated in dementing illnesses has considerable practicable implications for the diagnosis and treatment of chronic neurodegenerative diseases. It indicates that neurones could be rescued by suitable compounds at early stages of disease when oligomeric forms of hyperphosphorylated tau, α-synuclein and prion protein are being degraded by the ubiquitin proteasome system and autophagy. MEKl is closely related to MEK5. MEKl is a dual-specificity tyrosine/threonine protein kinase that phosphorylates threonine and tyrosine in the activation loop of ERKl, thereby activating ERKl. Zhu et al, (2003) J Neurochem 86, 136-142 reports that the cellular location of MEKl is altered in Alzheimer's disease but that the overall levels of MEKl are unchanged. We have done a further analysis of MEKl immunostaining in Alzheimer's disease. We consider that there is a small subgroup of neurones that have neurofibrillary tangles that have nuclear MEKl. We consider that MEKl may also be a target for diagnosis and therapy of such, disease.
The MEKl kinase shares 40% sequence homology with the MEK5 kinase including both enzymes sharing the same allosteric site. The MEKl enzyme has been crystallised. The MEKl enzyme has been studied carefully since the enzyme is expressed in tumour cells and is a good target for inhibition to prevent cancer cell division. Several compounds have been made that bind to the allosteric site of MEKl, including PD 0325901, and have been shown to be well tolerated in phase II clinical trials. We show that the MEK5 enzyme is immunochemically detected in both intraneuronal neurofibrillary tangles and Lewy bodies in patients succumbing to chronic neurodegenerative diseases including Alzheimer's disease and Dementia with Lewy bodies. We consider that synthesis and characterisation of new compounds that bind to the MEKl allosteric site will provide the pharmacophore for a family of novel compounds that bind to the shared allosteric " site in MEK5. One of these compounds can then be developed as an imaging agent for MEK5 in inclusions in chronic neurodegenerative disease.
It is quite clear that such an imaging agent is essential for diagnosis and evaluation of the effects of drugs e.g. acetyl cholinesterase inhibitors for the treatment of these disorders. This becomes more apparent since the extraneuronal amyloid imaging agent (11 C-PIB) currently used in positron emission tomography (PET) for imaging extraneuronal amyloid in the brains of patients with these diseases shows, in a longitudinal series of imaging analyses, that amyloid deposition occurs early in the brains in patients with mild cognitive decline (MCI) but then appears to have reached stabilised constant amounts in patients with Alzheimer's disease ■ (Dr. David Brooks, MRC clinical Sciences Centre, Imperial College, London, presented at Alzheimer Research Trust annual meeting at Bristol, March 2008). Clearly, there is a need for comparative imaging of intraneuronal inclusions, which are thought to provide a much better index of the extent of disease, and therefore response to treatment. Therefore, imaging of MEK5-containiαg inclusions is considered to provide a much better measure of the extent of disease and response to existing and new treatments.
The present invention provides accurate, sensitive methods and materials for diagnosing and monitoring progressive neurodegenerative disease including, but not limited to, Alzheimer's disease, Parkinson's Disease, Dementia with Lewy Bodies and prion diseases, in humans or animals. The diagnostic methods and materials are based on the finding, reported here, that neuronal expression of the MEK5/ERK5/MEF module provides an index of protein aggregation and neurodegeneration. The invention also provides a novel target for developing imaging agents and therapies against progressive neurodegenerative disease in humans or animals. Neuronal death in the ageing brain could be prevented, by up- regulating the MEK5/ERK5/MEF module which promotes cell survival, and/or down-regulating the MEK3/p38 MAPK module which promotes apoptosis. A therapy based oil this invention could potentially halt the progression of chronic neurodegenerative disease.
A first aspect of the invention provides a method of diagnosing a neurodegenerative disease in an individual comprising measuring the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the individual. The module involving MEKl is Ras/Raf/MEKl (MEK2)/ERK1 (ERK2).
A further aspect of the invention provides a method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising measuring the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the individual.
It will be appreciated that determining whether a sample of body fluid or tissue contains a certain level of one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or the level of one or more components of the MEK3/p38 MAPK module, may be diagnostic of a neurodegenerative disease or it may be used by a clinician as an aid in reaching a diagnosis. Furthermore, the methods of the invention may be used for presymptomatic screening of a patient who is in a risk group for developing a neurodegenerative disease, e.g. a patient having a family history of such diseases. Hence the methods of the invention may also be considered as aiding in the diagnosis of neurodegenerative diseases.
This is because, as set out above, levels of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), are elevated in neuronal cells which have high levels of protein aggregation. Similarly, down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/p38 MAPK module facilitates cell death by apoptosis. Such components can exit the neuronal cells and enter into bodily fluids and/or accumulate in certain body tissues. The level of such a component in a body fluid is an indicator of the level of that component that existed in specific neuronal cells which have lysed, and correlates with the state of the disease. Body fluids isolated from an individual having, or susceptible to having, a neurodegenerative disease will have a characteristically elevated level of a component of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or a component of the MEK3/p38 MAPK module. Hence a direct link: can be established between the level of protein aggregation in the neuronal cells of an individual and a sample of body fluid or tissue taken from the same individual. This is considered to be the first diagnostic test available for neurodegenerative diseases. The results obtained from the methods of the invention may be used in association with other clinical indicators of neurodegenerative disease to allow the clinician to reach a diagnosis. Examples of other clinical indicators include 'Mini-Mental State Examination' (MMSE, Folstein et al (1975) J Psychiatr Res 12:189-198; 'Cognitive Test of Mental Decline'; 'Addenbrookes Cognitive Examination' (Cambridge); 'Alzheimer's Disease Assessment Scale Cognitive Subscale (ADAC-Cog)'.
The level of the component(s) of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least Wz fold higher, or it may be at least 2-fold or 3 -fold higher, in the sample than the level of the same component in a body fluid or tissue sample taken from an individual who is not suffering from a neurodegenerative disease.
The level of the component(s) of the MEK3/p38 MAPK module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least 1 Vz fold lower, or it may be at least 2-fold or 3-fold lower, in the body sample than the level of the same component in a body fluid or tissue sample taken from an individual who is not suffering from a neurodegenerative disease.
Thus, the method may further comprise the step of comparing the level of one or more component(s) of the MEK5/ERK5/MEF module, or an activator of. the MEK5/ERK5/MEF module, or a component of the MEK3/p38 MAPK module in the sample to the level of the same component(s) in a normal body fluid or tissue sample, i.e. a sample from an individual who does not have a neurodegenerative disease.
When used herein in all aspects- of the invention, by "components of the MEK5/ERK5/MEF module" we include the protein kinases MEK5, ERK5 and the transcription factor MEF. The MEK5/ERK5/MEF module is well known in the art (Watson et at (2001) Nat. Neuroscience 4, 981-988) and information about the MEK5, ERK5 and transcription factor MEF genes and polypeptides is readily available to the skilled person.
For example, the human MEK5 polypeptide sequence is given in Genbank Accession Ql 3163 and presented below. The term "MEK5" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEK5 and are included within the scope of this term. Preferably, by MEK5 we mean the MEK5 polypeptide sequence shown below.
MEK5 polypeptide sequence fGenbank Accession 013163)
MLWLALGPFP AMENQVLVIR IKIPNSGAVD WTVHSGPQLL FRDVLDVIGQ VLPEATTTAF EYEDEDGDRI TVRSDEEMKA MLSYYYSTVM EQQVNGQLIE
PLQIFPRACK PPGERNIHGL KVNTRAGPSQ HSSPAVSDSL PSNSLKKSSA
ELKKILANGQ MNEQDIRYRD TLGHGNGGTV YKAYHVPSGK ILAVKVILLD
ITLELQKQIM SELEILYKCD SSYIIGFYGA FFVENRISIC TEFMDGGSLD
VYRKMPEHVL GRIAVAVVKG LTYLWSLKIL HRDVKPSNML VNTRGQVKLC DFGVSTQLVN SIAKTYVGTN AYMAPERISG EQYGIHSDVW SLGISFMEIQ
KNQGSLMPLQ LLQCIVDEDS PVLPVGEFSE PFVHFITQCM RKQPKERPAP
EELMGHPFIV QFNDGNAAVV SMWVCRALEE RRSQQGPP
The human ERK5 polypeptide sequence is given in Genbank Accession Q13164 and presented below. The term "ERK5" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to ERK5 and are included within the scope of this term. Preferably, by ERK5 we mean the ERK5 polypeptide sequence shown below. ERK5 polypeptide sequence fGenbank Accession 013164) •
MAEPLKEEDG EDGSAEPPAR EGRTRPHRCL CSAKNLALLK ARSFDVTFDV
GDEYEIIETI GNGAYGVVSS ARRRLTGQQV AIKKIPNAFD VVTNAKRTLR
5 ELKILKHFKH DNIIAIKDIL RPTVPYGEFK SVYVVLDLME SDLHQIIHSS
QPLTLEHVRY FLYQLLRGLK YMHSAQVIHR DLKPSNLLVN ENCELKIGDF
GMARGLCTSP AEHQYFMTEY VATRWYRAPE LMLSLHEYTQ AIDLWSVGCI
FGEMLARRQL FPGKNYVHQL QLIMMVLGTP SPAVIQAVGA ERVRAYIQSL PPRQPVPWET VYPGADRQAL SLLGRMLRFE PSARISAAAA LRHPFLAKYH0 DPDDEPDCAP PFDFAFDREA LTRERIKEAI VAEIEDFHAR REGIRQQIRF QPSLQPVASE PGCPDVEMPS PWAPSGDCAM ESPPPAPPPC PGPAPDTIDL
TLQPPPPVSE PAPPKKDGAI SDNTKAΆLKA ALLKSLRSRL RDGPSAPLEA PEPRKPVTAQ ERQREREEKR RRRQERAKER EKRRQERERK ERGAGASGGP STDPLAGLVL SDNDRSLLER WTRMARPAΆP ALTSVPAPAP APTPTPTPVQ-5 PTSPPPGPLA QPTGPQPQSA GSTSGPVPQP ACPPPGPAPH PTGPPGPIPV
PAPPQIATST SLLAAQSLVP PPGLPGSSTP GVLPYFPPGL PPPDAGGAPQ
SSMSESPDVN LVTQQLSKSQ VEDPLPPVFS GTPKGSGAGY GVGFDLEEFL
■ NQSFDMGVAD GPQDGQADSA SLSASLLADW LEGHGMNPAD IESLQREIQM
DSPMLLADLP DLQDP 0
The human MEF polypeptide sequence is given in Genbank Accession Q06413 and presented below. The term "MEF" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEF and are included within the scope of5 this term. Preferably, by MEF we mean the MEF polypeptide sequence shown below.
MEF polypeptide sequence (Genbank Accession Q06413)
MGRKKIQITR IMDERNRQVT FTKRKFGLMK KAYELSVLCD CEIALIIFNS TNKLFQYAST DMDKVLLKYT EYNEPHESRT NSDIVETLRK KGLNGCDSPD PDADDSVGHS PESEDKYRKI NEDIDLMISR QRLCAVPPPN FEMPVSIPVS SHNSLVYSNP VSSLGNPNLL PLAHPSLQRN SMSPGVTHRP PSAGNTGGLM GGDLTSGAGT SAGNGYGNPR NSPGLLVSPG NLNKNMQAKS PPPMNLGMNN
RKPDLRVLIP PGSKNTMPSV SEDVDLLLNQ RINNSQSAQS LATPVVSVAT
PTLPGQGMGG YPSAISTTYG TEYSLSSADL SSLSGFNTAS ALHLGSVTGW
QQQHLHNMPP SALSQLGACT STHLSQSSNL SLPSTQSLNI KSEPVSPPRD
RTTTPSRYPQ HTRHEAGRS P VDSLS SCSS S YDGS DREDHR NE FHS PIGLT RPSPDERESP SVKRMRLSEG WAT
By "an activator of the MEK5/ERK5/MEF module" we include molecules which can interact with and activate the function of the MEK5/ERK5/MEF module, i.e. lead to the activation of the MEK5/ERK5/MEF module. Methods of measuring the activation of components of the MEK5/ERK5/MEF module are discussed below in relation to the screening methods of the invention.
Examples of such activators include p62, the MEKK2,3/Tpl2 protein kinase and
"atypical protein kinase C" (aPKC). ρ62, MEKK2,3/Tρl2 and aPKC are well known in the art (Segfried et at (2005) MoI Cell Biol 25, 9820-9828; Hirano et al
(2004) J Biol Chem 279, 31883-31890). Therefore information about the p62,
MEKK2,3/Tpl2 and aPKC genes and polypeptides is readily available to the skilled person. MEKK2,3/Tpl2 is a protein kinase which interacts with and phosphorylates MEK5. On phosphorylation MEK5 becomes "active".
The human p62 polypeptide sequence is given in" Genbank Accession Ql 3501 and presented below. The term "p62" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to p62 and are included within the scope of this term. Preferably, by p62 we mean the p62 polypeptide sequence shown below.
p62 polypeptide sequence fGeribank Accession 013501)
MASLTVKAΫL LGKEDAAREI. RRFSFCCSPE PEAEAEAAAG PGPCERLLSR VAALFPALRP GGFQAHYRDE DGDLVAFSSD EELTMAMSYV KDDIFRIYIK EKKECRRDHR PPCAQEAPRN MVHPNVICDG CNGPWGTRY KCSVCPDYDL CSVCEGKGLH RGHTKLAFPS PFGHLSEGFS HSRWLRKVKH GHFGWPGWEM
GPPGNWSPRP PRAGEARPGP TAESASGPSE DPSVNFLKNV GESVAAALSP
■ LGIEVDIDVE HGGKRSRLTP VSPESSSTEE KSSSQPSSCC SDPSKPGGNV
EGATQSLΆEQ MRKIALESEG RPEEQMESDN CSGGDDDWTH LSSKEVDPST GELQSLQMPE SEGPSSLDPS QEGPTGLKEA ALYPHLPPEA DPRLIESLSQ MLSMGFSDEG GWLTRLLQTK NYDIGAALDT IQYSKHPPPL
The human MEKK2,3/Tpl2 polypeptide sequence is given in Genbank Accession Q9Y2U5 and presented below. The term "MEKK2,3/Tpl2" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEKK2,3/Tpl2 and are included within the scope of this term. Preferably, by MEKK2,3/Tpl2 we mean the MEKIC2,3/Tpl2 polypeptide sequence shown below.
MEKK2.3/Tpl2 polypeptide sequence (Genbank Accession O9Y2U5)
MDDOQALNSI MQDLAVLHKA SRPALSLQET RKAKSSSPKK QNDVRVKFEH RGEKRILQFP RPVKLEDLRS KAKIAFGQSM DLHYTNNELV IPLTTQDDLD KALELLDRSI HMKSLKILLV INGSTQATNL EPLPSLEDLD NTVFGAERKK RLSIIGPTSR DRSSPPPGYI PDELHQVARN GSFTSINSEG EFIPESMEQM LDPLSLSSPE NSGSGSCPSL DSPLGGESYP KSRMPRAQSY PDNHQEFSDY DNPIFEKFGK GGTYPRRYHV SYHHKDNDGR KTFPRARRTQ GNQLTSPVSF SPTDHSLSTS SGSSIFTPEY DDSRIRRRGS DIDNPTLTVM DISPPSRSPR APTNWRLGKL LGQGAFGRVY LCYDVDTGRE LAVKQVQFDP DSPETSKEVN ALECEIQLLK NFLHERIVQY YGCLRDPQEK TLSIFMEYMP GGSIKDQLKA YGALTENGTR KYTRQILEGV HYLHSNMILH RDIKGANILR DSTGNVKLGD FGASKRLQTI CLSGTGMKSV TGTPYWMSPE VISGQGYGRK ADIWSVACTV VEMLTEKPPW AEFEAMAAIF KIATQPTNPK LPPHVSDYTR DFLKRIFVEA KLRPSADELL RHMFVHYH
Where used herein in all aspects of the invention, by "components of the MEK3/p38 MAPK module" we include MEK3 and ρ38 MAPK, both of which are well known in the art. Therefore information about the MEK3 and p38 MAPK genes and polypeptides is readily available to the skilled person.
For example, the human MEK3 polypeptide sequence is given in Genbank Accession P46734 and presented below. The term "MEK3" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEK3 and are included within the scope of this term. Preferably, by MEK3 we mean the MEK3 polypeptide sequence shown below.
MEK3 polypeptide sequence fP46734)
MESPASSQPASMPQSKGKSKRKKDLRISCMSKPPAPNPTPPRNLDSRTFITIGDR NFEVEADDLVTISELGRGAYGVVEKVRHAQSGTIMAVKRIRATVNSQEQKRLLMD LDINMRTVDCFYTVTFYGALFREGDVWICMELMDTSLDKFYRKVLDKNMTIPEDI LGEIAVSIVRALEHLHSKLSVIHRDVKPSNVLINKEGHVKMCDFGISGYLVDSVA KTMDAGCKPYMAPERINPELNQKGYNVKSDVWSLGITMIEMAILRFPYESWGTPF QQLKQWEEPSPQLPADRFSPEFVDFTAQCLRKNPAERMSYLELMEHPFFTLHKT KKTDIAAFVKEILGEDS -
The human p38 MAPK polypeptide sequence is given in Genbank Accession Q15759 and P53778 and presented below. The term "p38 MAPK" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to p38 MAPK and are included within the scope of this term. Preferably, by to p38 MAPK we mean the to p38 MAPK polypeptide sequence shown below.
p38 MAPK polypeptide sequence (2) (O15759)
MSGPRAGFYR QELNKTVWEV PQRLQGLRPV GSGAYGSVCS AYDARLRQKV
AVKKLSRPFQ SLIHARRTYR ELRLLKHLKH ENVIGLLDVF TPATSIEDFS
EVYLVTTLMG ADLNNIVKCQ AGAHQGARLA LDEHVQFLVY QLLRGLKYIH
SAGIIHRDLK PSNVAVNEDC ELRILDFGLA RQADEEMTGY VATRWYRAPE IMLNWMHYNQ TVDIWSVGCI MAELLQGKAL FPGSDYIDQL KRIMEVVGTP SPEVLAKISS EHARTYIQSL PPMPQKDLSS IFRGANPLAI DLLGRMLVLD
SDQRVSAAEA LAHAYFSQYH DPEDEPEAEP YDESVEAKER TLEEWKELTY
■ QEVLSFKPPE PPKPPGSLEI EQ
p38 MAPK polypeptide sequence (3) (P53778*)
MSSPPPARSG FYRQEVTKTA WEVRAVYRDL QPVGSGAYGA VCSAVDGRTG AKVAIKKLYR PFQSELFAKR AYRELRLLKH MRHENVIGLL DVFTPDETLD DFTDFYLVMP FMGTDLGKLM KHEKLGEDRI QFLVYQMLKG LRYIHAΆGII
HRDLKPGNLA VNEDCELKIL DFGLARQADS EMTGYVVTRW YRAPEVILNW
MRYTQTVDIW SVGCIMAEMI TGKTLFKGSD HLDQLKEIMK VTGTPPAEFV
QRLQSDEAKN YMKGLPELEK KDFASILTNA SPLAVNLLEK MLVLDAEQRV
TAGEALAHPY FESLHDTEDE PQVQKYDDSF DDVDRTLDEW KRVTYKEVLS FKPPRQLGAR VSKETPL
The human MEKl polypeptide sequence is given in Genbank Accession Q02750 and presented below. The term "MEKl" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to MEKl and are included within the scope of this term. Preferably, by MEKl we mean the MEKl polypeptide sequence shown below.
MEKl polypeptide sequence (Genbank Accession 002750) MPKKKPTPIQ LNPAPDGSAV NGTSSAETNL EALQKKLEEL ELDEQQRKRL EAFLTQKQKV GELKDDDFEK ISELGAGNGG VVFKVSHKPS GLVMARKLIH LEIKPAIRNQ IIRELQVLHE CNSPYIVGFY GAFYSDGEIS ICMEHMDGGS
LDQVLKKAGR IPEQILGKVS IAVIKGLTYL REKHKIMHRD VKPSNILVNS
RGEIKLCDFG VSGQLIDSMA NSFVGTRSYM SPERLQGTHY SVQSDIWSMG LSLVEMAVGR YPIPPPDAKE LELMFGCQVE GDAAETPPRP RTPGRPLSSY
GMDSRPPMAI FELLDYIVNE PPPKLPSGVF SLEFQDFVNK CLIKNPAERA
DLKQLMVHAF IKRSDAEEVD FAGWLCSTIG LNQPSTPTHA AGV Activators of MEKl include growth factors/Ras/Raf. The human Ras polypeptide sequence is given in Genbank Accession AAMl 2633 for N-Ras and presented below. The term "Ras" as used herein includes this polypeptide sequence as well as other members of the Ras family such as K-Ras and H-Ras and naturally occurring variants thereof. Further animal species also have equivalent polypeptides to Ras and are included within the scope of this term. Preferably, by Ras we mean the Ras polypeptide sequence shown below.
N-Ras polypeptide sequence (Genbank Accession AAM12633)
MTEYKLWVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQWIDGET
CLLDiLDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ΆDINLYREQI
KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETΞAKTRQ GVEDAFYTLV REIRQYRMKK LNSSDDGTQG CMGLPCWM
The human Raf polypeptide sequence is given in Genbank Accession P04049 and presented below. The term "Ras" as used herein includes this polypeptide sequence as well as naturally occurring variants thereof. Further animal species also have equivalent polypeptides to Raf and are included within the scope of this ■ term. Preferably, by Rafwe mean the Raf polypeptide sequence shown below. - - •--
Raf polypeptide sequence (Genbank Accession P04049)
MEHIQGAWKT ISNGFGFKDA VFDGSSCISP TIVQQFGYQR RASDDGKLTD PSKTSNTIRV FLPNKQRTW NVRNGMSLHD CLMKALKVRG LQPECCAVFR LLHEHKGKKA RLDWNTDAAS LIGEELQVDF LDHVPLTTHN FARKTFLKLA FCDICQKFLL NGFRCQTCGY KFHEHCSTKV PTMCVDWSNI RQLLLFPNST IGDSGVPALP SLTMRRMRES VSRMPVSSQH RYSTPHAFTF NTΞSPSSEGS LSQRQRSTST PNVHMVSTTL PVDSRMIEDA IRSHSESASP SALSSSPNNL
SPTGWSQPKT PVPAQRERAP VSGTQEKNKI RPRGQRDSSY YWEIEASEVM LSTRIGSGSF GTVYKGKWHG DVAVKILKW DPTPEQFQAF RNEVAVLRKT RHVNILLFMG YMTKDNLAIV TQWCEGSSLY KHLHVQETKF QMFQLIDIAR QTAQGMDYLH AKNIIHRDMK SNNIFLHEGL TVKIGDFGLA TVKSRWSGSQ
QVEQPTGSVL WMAPEVIRMQ DNNPFSFQSD VYSYGIVLYE LMTGELPYSH INNRDQIIFM VGRGYASPDL SKLYKNCPKA MKRLVADCVK KVKEERPLFP QILSSIELLQ HSLPKINRSA SEPSLHRAAH TEDINACTLT TSPRLPVF
In an embodiment of these aspects of the invention, the component of the MEK5/ERK5/MEF module is MEK5. In a further embodiment of these aspects of the invention the activator of the MEK5/ERK5/MEF module is p62 or MEKK2,3/Tpl2.
The methods of the invention include the step of measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module in a sample of body fluid. Preferably the component is a polypeptide.
Assaying protein levels in a biological sample can occur using any art-known method. Preferred for assaying protein levels in a biological sample are antibody- based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. In these, the specific recognition is- provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination. is obtained. Tissues can also be extracted, e.g. with urea and neutral detergent, for the liberation of protein for Western-blot or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). In this technique, which is based on the use of cationic solid phases, quantitation of protein can be accomplished using isolated protein as a standard. This technique can also be applied to body fluids. With these samples, a molar concentration of protein will aid to set standard values of protein content for different body fluids, like serum, plasma, urine, spinal fluid, etc. The normal appearance of protein amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject. Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify the protein. The level of protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm. Such an ELISA for detecting a rumor antigen is described in Iacobelli et al., Breast Cancer Research and Treatment 11: 19-30 (1988). In another ELISA assay, two distinct specific monoclonal antibodies can be used to detect protein in a body fluid. In this assay, one of the antibodies is used as the immunoadsorbent and the other as the enzyme- labelled probe.
The above techniques may be conducted essentially as a "one-step" or "two-step" assay. The "one-step" assay involves contacting protein with immobilized antibody and, without washing, contacting the mixture with the labelled antibody.
The "two-step" assay involves washing before contacting the mixture with the labelled antibody. Other conventional methods may also be employed as suitable.
It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technecium (99mTc), and fluorescent labels, such as • fluorescein and rhodamine, and biotin. Hence examples of the methods that can be used to measure the relative amount of polypeptides in a sample of body fluid include any suitable protein quantitation method, for example ELISA, electrophoresis, chromatography or mass spectography. Further protein quantitation methods include radioimmunoassay (RIA)5 immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Antibody staining of cells on slides may be used, using antibodies to the MEK5/ERK5/MEF module in methods well known in cytology laboratory diagnostic tests, as well known to those skilled in the art.
As will be appreciated, such techniques are routine laboratory methods and are well known to the skilled person.
The level of the polypeptide may be determined using a molecule which selectively binds to the polypeptide.
It is particularly preferred that said molecule is an antibody. Antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A " manual of techniques", H ZoIa (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference. Suitable antibodies include the anti- MEK5 antibody (KAP-MA003) made available by. Bioquote Ltd., (York, U.K.); the ERK5 antibody from Santa Cruz Biotechology Inc; an anti-p62 antibody (p62 lck ligand) from BD Biosciences (Oxford, U.K.).
It will be appreciated that other antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al; J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
The body fluid may be cerebrospinal fluid (CSF) or blood or the tissue is nasal neuro-epithelial tissue. Means of collecting such body samples from an individual are well known to those skilled in the art.
A further aspect of the invention provides a method of diagnosing or monitoring a neurodegenerative disease in an individual comprising detecting the level of one or more components of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in the brain of the individual, for example in neurons in the brain of the individual. Components of the MEK5/ERK5 pathway are considered to be elevated in neurons in chronic neurodegenerative diseases.
A further aspect of the invention provides a method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising detecting the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in the brain of the individual, for example in neurons in the brain of the individual. The components of the MEK5/ERK5 pathway are considered to be elevated in neurons. However, protein components of this pathway may be released into cerebral spinal fluid where they may be detected with suitable assays.
By "comρonent(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or one or more components of the MEK3/ρ38 MAPK module" we include those components and activators listed above in relation to the earlier aspects of the invention. The level of said component(s) may be measured using neuroimaging, preferably magnetic resonance imaging (MRI). Other types of imaging that may be useful include positron emission scanning (PET), for example using a 124I- or 18F-labelled compound; single photon emission computed tomography (SPECT or SPET), for example using a 99mTc- or 123I-labelled compound; or nuclear magnetic resonance (NMR), for example using a 19F-labelled compound.
Methods of neuroimaging, including magnetic resonance imaging, of proteins in the neurons in the brain of an individual are well known. Such methods include where an agent (s) which can bind to the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/p38 MAPK module (or to component(s) of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is supplied to a patient. The agent then passes the blood-brain barrier and binds to the said component. The level of said agent in the brain of a patient can then be detected, thus providing an indication of the level of said component(s) in the patient's brain. Examples of agents which can selectively bind to said component(s) are provided below in Example 3.
A further aspect of the invention provides a method of measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in neurons in the brain of the individual comprising measuring the level of an agent(s) which can bind to 'the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/ρ38 MAPK module (or the one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in the neurons in the brain of a patient. Such a method can include where an agent(s) which can bind to the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the component(s) of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is supplied to a patient. The agent then passes the blood-brain barrier and binds to the said
■ component. The level of said agent(s) may be measured using neuroimaging, preferably magnetic resonance imaging or other imaging technique as indicated above.
As set out above, the levels of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, are elevated in neuronal cells in the brain which have high levels of protein aggregation. Similarly, down regulation of MEK3 complements the activities of the MEK5/ERK5/MEF module since the MEK3/ρ38 MAPK module facilitates cell death by apoptosis. Hence the level of such a component in the brain neurons of an individual can correlate with the state of the neurodegenerative disorder. The level of MEKl (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) is similarly considered to correlate with the state of the neurodegenerative disorder. Thus these methods of the invention may also be considered as aiding in the diagnosis of neurodegenerative diseases.
The level of the component(s) of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module (or of component(s) of the MEKl module (for" example MEKl) or the activator(s) of the MEKl module) which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least IH fold higher, or it may be at least 2-fold or 3-fold higher, in brain neurons than the level of the same component in brain neurons of an individual who is not suffering from a neurodegenerative disease.
The level of the component(s) of the MEK3/p38 MAPK module which can be an indicator of neurodegenerative disease or susceptibility to developing a neurodegenerative disease may be, for example, at least IVi fold lower, or it may be at least 2-fold or 3-fold lower, in brain neurons than the level of the same component in brain neurons of an individual who is not suffering from a neurodegenerative disease. Hence an embodiment of these aspects of the invention is wherein the method further -comprises the step of comparing the level of one or more component(s) of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or one or more component(s) of the MEK3/p38 MAPK module (or one or more components of the MEKl module (for example MEKl) or the activator(s) of the MEKl module) in neuronal cells in the patient's brain to the level of the same component(s) in a normal brain, i.e. a brain of an individual who does not have a neurodegenerative disease. Alternatively or in addition, the method may comprise comparing the level of the component with the level observed previously in the patient's brain. This may be useful in, for example, assessing progression of the patient.
Preferably the component of the MEK5/ERK5/MEF module is MEK5. Preferably the activator of the MEK5/ERK5/MEF module is p62 or MEKK2,3/Tpl2. Preferably the component of the MEKl module is MEKl.
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module and assaying for activation of the MEK5/ERK5/MEF module component(s).
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERK5/MEF module and assaying for inhibition of the MEK5/ERK5/MEF inhibitor.
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for activation of the activator. A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK3/p38 MAPK module and assaying for inhibition of the MEK3/p38 MAPK module component(s).
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of one or more components MEK5/ERK5/MEF module.
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of a MEK5/ERK5/MEF module inhibitor.
A further aspect of the invention is a method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of an activator MEK5/ERK5/MEF module.
A further aspect of the invention is a method for detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of one or more components of the MEK3/p38 MAPK module.
A further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module (or MEKl module) and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF module (or MEKl module) comρonent(s).
A further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERIC5/MEF module ■ ' and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF inhibitor.
5 A further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for binding to, activation or inhibition of the activator.
10 A further aspect of the invention is a method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK3/p38 MAPK module and assaying for binding to, activation or inhibition of the MEK3/p38 MAPK module component(s).
15
In relation to identifying compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease, it is considered that useful compounds are able to bind to the specified component. Activation or inhibition of that component is not considered to be essential, but ability to activate or inhibit the
20 " component may serve as a useful indicator of ability to bind' to the component. The techniques that are useful in measuring binding directory are well known in the art and are, for example, based on radiolabeled ligands. The level of binding affinity should be at least in the μM range.
25 The methods of the invention described above are "screening assays". Methods of detecting the activity of the protein kinases MEK5 and ERK5 (or MEKl and ERKl, for example) are well known in the art. Such methods routinely use phosphoepitope antibodies, which recognise when a substrate of the protein ldnase has been phosphorylated and hence the activity of the kinase itself. In such a
30 method an experiment is performed in which a ldnase is incubated with a substrate with and without the presence of the test compound. The activity of the kinase to its substrate is then measured using the particular phosphoepitope antibody. In this way the effect of the test compound on the activity of the kinase can be measured.
An example of an assay of ERK5 activity is provided in the examples below. Hence the effect of a test compound on protein kinase activity can be readily achieved by assessing the effect of the test compound on the function of the protein kinase.
Alternatively, the screening methods may measure any changes in the level of one or more polypeptide component(s) of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module, one or more polypeptide component (s) of the MEK3/p38 MAPK module. Methods by which polypeptide levels can be measured are provided above in relation to earlier aspects of the invention.
Furthermore, the screening methods may measure any changes in the level of one or more polynucleotides encoding component(s) of the MEK5/ERK5/MEF module (or MEKl module), or an activator of the MEK5/ERK5/MEF module (or MEKl module), one or more polypeptide component (s) of the MEK3/p38 MAPK module. Methods of measuring the change in the level of polynucleotides in a cell are routine in the art and include, for example, northern blotting and RT-PCT.
It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and 32P-ATP or 33P-ATP and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for 32P or 33P SPA assays. Only 32P or 33P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used.
It is preferred that where the neurodegenerative disease is Alzheimer's disease, the therapeutic screening methods of the invention do not include detecting compounds which upregulate ERK5 activity.
As mentioned above, MEF is a transcription factor. An assay of MEF activity may be conducted by measuring the effect of a test compound on the amount of a
MEF-regulated reporter gene, as would be appreciated by a person skilled in the art. For example, a gene encoding the Luciferase polypeptide can be fused next to a MEF-responsive nucleic acid sequence and the resulting reporter gene construct integrated into a suitable cell. Then an experiment is set up in which a cell with this reporter gene is incubated with a test compound and the effect of the compound on Luciferase activity in the cell is assessed and compared to an equivalent cell which has not been exposed to the test compound. In this way the effect of the test compound on the activity of MEF can be measured. An example of a reporter gene assay which could be used to measure MEF activity is provided in Martin et άl (1993) Proc. Natl. Acad. Sci USA 90, 5282-5286.
As described above, down regulation of MEK3/p38 MAPK complements the activities of the MEK5/ERK5/MEF module since the MEK3/ρ38 MAPK module facilitates cell death by apoptosis. Hence compounds which inhibit the activity of the MEK3/p38 MAPK module may be useful in preventing apoptosis in neuronal cells and therefore useful for the treatment of neurodegenerative disease. Representative genes and polypeptide sequences for the MEK3/p38 MAPK module polypeptide are provided herein. MEK3 is also a protein kinase and hence similar protein kinase assays to those set out above can be used to assay for MEK3 activity.
By "activation of the MEK5/ERK5/MEF kinase module (or MEKl module)", "an increase in the activity of the MEK5/ERK5/MEF module (or MEKl module)" and "upregulation of the MEK5/ERK5/MEF module (or MEKl module" we include where the test compound increases the activity or expression of that component by 1.5x, 2x, 5x, 1Ox5 2Ox, 5Ox, 10Ox, 20Ox, 300X, 40Ox, 50Ox, 60Ox5 70Ox5 80Ox, 900 or 100Ox or more compared to a component of the MEK5/ERK5/MEF module (or MEKl module, as appropriate) not exposed to the test compound.
By "an inhibitor of the MEK5/ERK5/MEF module" we include 2'-amino-3'- methoxyfiavone or an O-alkyl iV-arylanthranilyl hydroxamic acid or analogues thereof which are capable of binding to MEK5 (and MEKl). Example 3 presented below provides further information on these compounds.
By "inhibition of the MEK5/ERK5/MEF module (or MEKl module) inhibitor" and "inhibition of the MEK3/p38 MAPK module" we include where the test compound decreases the functional activity or expression of the inhibitor by 1.5x, 2x, 5x, 10x, 2Ox, 50x, 10Ox or more compared to an inhibitor of the MEK5/ERK5/MEF module (or MEKl module) or MEK3/ρ38 MAPK module not exposed to the test compound.
By "activation of the activator of the MEK5/ERK5/MEF module (or MEKl module)" we include where the test compound increases the activity or expression of that component by 1.5x, 2x, 5x, 1Ox5 20x, 5Ox, 10Ox5 20Ox, 300X, 40Ox5 50Ox5 60Ox5 70Ox5 800x, 900x or 100Ox or more compared to an activator of the MEK5/ERK5/MEF module (or MEKl module) not exposed to the test compound. ■
By "cell" we include a cell which contains a MEK5/ERK5/MEF module (or MEKl module), and/or an activator of the MEK5/ERK5/MEF module (or MEKl module), and/or an MEK5/ERK5/MEF module (or MEKl module) inhibitor, and/or a MEK3/p38 MAPK module. Examples of such cells include primary neurones and neuronal cell lines. Such cells are well known in the art and include, for example, mouse N2a and PC12 cell lines. The above aspects of the invention are screening methods to identify drugs or lead compounds of use in treating, diagnosing, -monitoring or imaging neurodegenerative diseases.
It will be appreciated that in the methods described herein, which may be drug screening methods, a term well known to those skilled in the art, the compound may be a drug-like compound or lead compound for the development of a drug- like compound.
The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential.
The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non- selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
Suitable characteristics for an imaging compound will also be known to those skilled in the art, and may include the ability to be labelled with a suitable isotope, for example a fluorine isotope. Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of the polypeptides mentioned herein. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result.
It will be appreciated that several cycles of identifying pools of polynucleotides comprising a polynucleotide having the required property and then rescreening those polynucleotides may be required in order to identify a single species of polynucleotide with the required property.
It will be appreciated that further tests and/or sequence analysis may be required in order to distinguish polynucleotides encoding a functional equivalent of polypeptides mentioned herein from polynucleotides encoding an activator of a said functional equivalent.
Methods of preparing a suitable expression library for screening are well known to those skilled in the art. The library may preferably be from the same source as the said functional equivalent that is expressed in the said host cell, ie. a human expression library may be screened for effects on host cells expressing human equivalent proteins.
A further aspect of the invention provides a compound detected according to any one of the screening assay aspects of the invention for use in medicine.
A further aspect of the invention provides a pharmaceutical composition comprising a compound as detected according to any one of the screening assay aspects of the invention and a pharmaceutically acceptable excipient.
The aforementioned compounds or a formulation thereof may be administered by any conventional method hicluding oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses' over a period of time.
Whilst it is possible for a compound to be adrninistered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
A further aspect of the invention provides the use of a material which binds to a component of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module or binds to a component of the MEF3/p38 MAPK module in the diagnosis or monitoring of a neurodegenerative disease in an individual or to determine the susceptibility of an individual to developing a neurodegenerative disease or to detect compounds useful for the treatment of neurodegenerative disease. For example, the diagnostic or imaging techniques described herein may be useful for assessing the effect of a treatment regime on a patient and may therefore be useful in evaluating that treatment regime and/or in formulating and testing alternative treatment regimes. The diagnostic or imaging techniques may therefore be useful in clinical trials and may provide a more rapid and quantifiable outcome measurement than, for example, measurement of early behavioural changes.
A further aspect of the invention provides a kit of parts useful for diagnosing (or monitoring, as discussed above) neurodegenerative disease comprising a material which is capable for use in determining the level of one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEF3/p38 MAPK module.
Preferably the kit of parts comprises a control sample containing one or more components of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEF3/p38 MAPK module where in the control sample may be a negative control (which contains a level of said component which is not associated with a neurodegenerative disease) or it may be a positive control (which contains a level of said component which is associated with a neurodegenerative disease). The kit may contain both negative and positive controls. The ldt may be usefully contain controls of said components polypeptide which correspond to different levels in a sample such that a calibration curve may be made.
Suitable, the kit further comprises means for obtaining sample of body fluid or tissue from a patient.
Examples of materials which bind to components of MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module, (or MEKl module) or binds to the MEF3/p38 MAPK module are provided above.
Examples of methods of diagnosing a neurodegenerative disease in an individual and methods for determining the susceptibility of an individual to developing a neurodegenerative disease and methods of detecting compounds useful for the treatment of neurodegenerative disease are presented above. ■ ■ •
The material may be an antibody. Preferably the material binds to MEK5 (or MEKl), more preferably the material is an anti-MEK5 (or anti-MEKl) antibody, as described above and in the accompanying examples.
Alternatively the material binds to MEK3; preferably the material is an anti- MEK3 antibody.
An embodiment of these aspects of the invention is wherein the material is a compound which binds to components of the MEK5/ERK5/MEF module and which is modified with a phase contrast agent.
An embodiment of these aspects of the invention is. wherein the material is 2'- amino-3'-methoxyflavone or an 0-alkyl JV-aryl anthranilyl hydroxamic acid or analogues thereof which are capable of binding to MEK5. Example 3 presented below provides further information on these compounds. Such compounds can be further modified to include a phase-contrast agent such as F19 (and heteronuclei C 3-F19) for use in MRI visualisation of neurodegeneration (Higuchi supra).
A further aspect of the invention provides the use of an oligonucleotide encoding one or more components of the MEK5/ERK5/MEF .module (or MEKl module) or an activator the MEK5/ERK5/MEF module (or MEKl module) or one or more components of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease.
As set out above, the members of the MEK5/ERK5/MEF module, MEKl module, activators of the MEK5/ERIC5/MEF module or MEKl module and the MEK3/p38 MAPK module are well known to those skilled in the art. The skilled person could identify an appropriate oligonucleotide sequence from Genbank with no inventive contribution required.
For example, a polynucleotide sequence encoding MEK5 is provided in Genbanlc accession number BC008838; ERK5 polynucleotide sequence is provided in Genbank accession number BC030134; MEF polynucleotide sequence is provided in Genbank accession number BC026341; p62 polynucleotide sequence is provided in Genbank accession number BC003139; MEKK2,3/Tρl2 polynucleotide sequence is provided in Geribank accession number NM_006609; p38 MAPK polynucleotide sequence is provided in Genbank accession number BC027933 and BCOl 5742; MEK3 polynucleotide sequence is provided in Genbank accession number BC032478.
Methods of attaching an oligonucleotide to a solid support are well known to those skilled in the art.
A further aspect of the invention provides the use of a component of the MEK5/ERK5/MEF module (or MEKl module) or an activator of the MEK5/ERK5/MEF module (or MEKl module) or a component of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease. Preferably the component is MEK5.
A further aspect of the invention provides a method of treating an individual suffering from a neurodegenerative disease comprising supplying to a patient an ' appropriate quantity of a compound detected according to any one of the previous screening methods of the invention.
A further aspect of the invention provides the use of a compound detected according to any one of the previous therapeutic screening methods of the invention in the manufacture of a medicament for preventing or treating a neurodegenerative disease.
A further aspect of the invention provides the use of a compound detected according to any one of the previous imaging screening methods of the invention in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
"A further aspect of the invention provides a method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of upregulating MEK5/ERK5/MEF module (or MEKl module) activity either directly or by downregulating the activity of one or more inhibitors of MEK5/ERK5/MEF module (or MEKl module) activity.
A further aspect of the invention provides the use of an agent or agents capable of upregulating MEK5/ERK5/MEF module activity (or MEKl module activity) either directly or by downregulating the activity of one or more inhibitors of MEK5/ERK5/MEF module activity (or MEKl module activity) in the manufacture of a medicament for treating a neurodegenerative illness in a human
' or animal. A further aspect of the invention provides a method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of inhibiting MEK3/p38 MAPK module activity.
A further aspect of the invention provides the. use of a pharmaceutically effective amount of an agent or agents capable of inhibiting MEK3/p38 MAPK module activity in the manufacture of a medicament for treating a neurodegenerative illness in a human or animal.
Examples of agents which can be used in the above aspects of the invention are provided herein.
It is preferred that where the neurodegenerative illness is Alzheimer's disease, the method or medicament of the previous aspects of the invention does not comprise an agent or a compound which upregulates ERK5 activity.
A further aspect of the invention provides a method of preventing neuronal cell death comprising upregulating the activity of the MEK5/ERK5/MEF module (or MEKl module) and/or downregulating the activity of the MEK3/p38 MAPK module in the neuronal cell.
In relation to any one of the previous aspects of the invention, a preferred embodiment is wherein the neurodegenerative disease or illness is progressive. Preferably the progressive neurodegenerative disease is Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy, multisystem atrophy, motor neurone disease (amyotrophic lateral sclerosis) or frontotemporal dementia.
In relation to any one of the previous aspects of the invention, the individual may be a human or animal; preferably the individual is a human. The compounds described in Example 3 or compounds identified by the screening • methods of the invention in relation to the MEK5 module may also be useful in treating, diagnosis, monitoring or imaging (as appropriate) of cancer, particularly prostate cancer. MEK5 has been implicated in cancer, particularly prostate cancer.
Accordingly, a further aspect of the invention provides a compound which binds to a component of the MEK5/ERK5/MEF modulate or to an activator of the MEK5/ERK5/MEF module or binds to a component of the MEF3/p38PAK module, for example, for use in treating, imaging, diagnosing or monitoring cancer, for example prostate cancer. The compounds useful in treating and imaging prostrate cancer are the same as for neurodegeneration.
A relevant reference is Dudderidge et al. Br. J. Cancer, 2007 May 7; 96(9): 1384- 93. Mitogenic growth signalling, DNA replication licensing and survival are linked to prostrate cancer. Other relevant references include Mehta et al (2003) MEK5 overexpression is associated with metastatic prostate cancer, and stimulates proliferation, MMP-9 expression and invasion. Oncogene 22(9) 1381-1389; Robinson et al (1996) A tyrosine kinase profile of prostate carcinoma PNAS USA 93(12), 5958-5962; McCracken et al (2007) Aberrant expression of extracellular signal-regulated kinase 5 in" human prostate cancer. Oncogene Epub ahead -of print.
A further aspect of the invention provides the anthranilyl hydroxamic acid analogue JV-(Cyclopropylmethoxy)-3 ,4-difluoro-2-(2-fluoro-4- iodophenylamino)benzamide; 2-(4-Bromo-2-fluorophenylamino)-7vr-
(cyclopropylmethoxy)-3 ,4-difluorobenzamide; 2-(4-Chloro-2- fluorophenylarnino)-N-(cyclopropylmethoxy)-3,4-difluorobenzarnide; 3,4-
Difluoro-2-(2-fluoro-4-iodophenylarnino)-7V'-(2-hydroxyethoxy)benzamide; 2-(4- Bromo-2-fluorophenylamino)-3,4-difluoro-N-(2-hydroxyethoxy)benzamide; 2-(4- Chloro-2-fluorophenylammo)-3 ,4-difluoro-N-(2-hydroxyethoxy)benzamide; TV- (Emoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; 2-(4-Chloro-2- fluorophenylammo)-N-emoxy-3,4-difluorobenzamide; (3,4-Difluoro-2-(2-fLuoiO- 4-iodophenylamino)phenyl)methanol; N-(2-(Dimethylamino)ethyI)-3,4-difluoro- 2-(2-fluoro-4-iodophenylamino)benzamide; 2-(4-Bromo-3-methoxyphenylamino)- iV-ethoxy-3,4-difluorobenzamide; 2-(4-Chloro-3-methoxyphenylamino)-N-ethoxy- 3,4-difiuorobenzamide; (2-(4-Bromo-3-methoxyρhenylamino)-3,4- difluorophenyl)methanol; (2-(4-Chloro-3-methoxyphenylamino)-3 ,A- difluorophenyl)methanol; or 7Vα-(Fluorescein-5-caxbonyl)-iVε-(3,4-difluoro-2-(2- fluoro-4-iodophenylamino)benzoyl)-L-lysyl amide; or radiolabelled or fluorescently labelled derivative any thereof. These analogues are discussed in Examples 3 to 5.
hi an embodiment, the anthranilyl hydroxamic acid analogue is 2-(4-Chloro-3- memoxyphenylarnino)-7V-ethoxy-3 ,4-difluorobenzamide; (2-(4-Bromo-3 - methoxyphenylamino)-3,4-dirluorophenyl)methanol; or (2-(4-Chloro-3- methoxyphenylammo)-3,4-difluorophenyl)methanol; or radiolabelled or fluorescently labelled derivative any thereof.
A further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention for use in medicine. A further aspect of the invention provides a pharmaceutical composition comprising an anthranilyl hydroxamic acid analogue of the invention and a pharmaceutically acceptable excipient.
A further aspect of the invention provides the anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition for use in aiding diagnosing, imaging or monitoring of a neurodegenerative disease. A further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention or related pharmaceutical composition in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease. The neurodegenerative disease may be a progressive neurodegenerative disease. The progressive neurodegenerative disease may be Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy, multisystem atrophy, motor neurone disease (amyotrophic lateral sclerosis) or frontotemporal dementia. The individual may be a human or animal patient. A further aspect of the invention provides an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition for use in aiding diagnosing, imaging or monitoring of prostate cancer. A further aspect of the invention provides, the use of an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of prostate cancer. A furfltier aspect of the invention provides a method of aiding diagnosing, imaging or monitoring of prostate cancer, wherein the patient is administered an anthranilyl hydroxamic acid analogue of the invention or relevant pharmaceutical composition.
The invention will now be described by reference to the following, non-limiting Examples and Figures.
Figure 1: Immunohistochemical detection of MEK5, p62 and ERK5 in neurones containing Lewy bodies, from the temporal cortex from a case of Dementia with Lewy Bodies.
Figure 2: Immunohistochemical detection of MEK5, p62 and ERK5 in neurones containing neurofibrillary tangles, from the temporal cortex from a case of Dementia with Lewy Bodies.
Figure 3: Imrnunohistochemical detection of MEK5, p62 and ERK5 in neurones showing granulovacuolar degeneration (GVD), from the hippocampus from a case of Dementia with Lewy Bodies.
Figure 4: A. Expression profile of the MEK5 gene. At the beginning of the analysis of gene expression protein aggregation has already started (Supplementary Methods). The expression of MEK5 exhibits a clear up-regulation that proceeds gradually and peaks at 24 h, before the peak of maximal aggregate load (at 48 h) and subsequently subsides. Data are from pooled RNA samples derived from 6 culture plates (control or co-transfected) transfected simultaneously and harvested at the designated times.
B. Western analyses of MEK5 levels in cells expressing S8-GFP and His-FLAG-mGluRlα . The increase in MEK5 levels at 48 h (E) is followed by subsequent attenuation at 72 h (F). MEK5 was expressed at increasing levels in cells expressing GFP and FLAG epitope alone which is expected since the
MEK5/ERK5 module responds to serum growth factors in the medium (11).
Figure 5: Aggregate formation exhibits high specificity even during times of considerable aggregate load (here at 16 h post transfection). A 3D projection of the interior of a co-transfected cell shows that S 8 ATP ase and mGluRlα receptor aggregate individually and do not intermix. Aggregates emitting one fluorescent signal were encircled on the panels on the left side, at different depths, and then projected on to the panels of the other fluorescent signal on the right side. Signals were not observed to coincide in any case. This observation was also verified by 3D rotational examination (not shown).
Figure 6: The schematic structure of MEKl with Mg, ATP {thin stick) and PD318088 (thick stick) bound, showing a 'closed' conformation involving the 'activation loop' (yellow) and helix C (green) (Ohren, 2004).
Figure 7: The ligand PD318088 bound to MEKl, in which the ligand B-ring via van der Waals interaction makes contact with the hydrophobic side-chain of Met143 (ball-and-stick) (Ohren, 2004). The bound ATP was removed in order to facilitate visualisation of the ligand interactions.
Figure 8: Synthetic chemical agents discussed in Example 3.
Figure 9: Methods for production of radioimaging ligands for SPECT and PET Figure 10: A. Expression analysis of MEKl. BL21(DE3)pLys cells were lysed by sonication and 50 μl of soluble fraction was analysed on 5-20% SDS-PAGE and stained with Coomassie blue.
The arrow, MEKl protein (MW: 43 kD). Lane 1: BL21(DE3)pLys E-coli cells before induction; Lane 2: BL21(DE3)ρLys cells induced by 250 μM IPTG (3 hours); Lane 3: BL21(DE3)pLys cells induced by 500 μM IPTG (3 hours); Lane 4: BL21(DE3)pLys cells induced by 500 μM IPTG (6 hours). All cells were grown at 37 0C.
B. The corresponding lanes were immunobloted with the MEKl antibody. As the figure shows induction with 500 μM IPTG for 6 hours gives the highest protein expression.
Figure 11: Analysis of the imidazole eluted His-tagged MEKl recombinant protein from the nickle-charged resin. 50 μl eluted protein was loaded on each. Lane. 1: elution using 500 mM imidazole; Lane 2: elution using 50 mM EDTA. The figure shows 500 mM imidazole is slightly more efficient than EDTA.
Figure 12: Fluorescence polarization assay was employed to assess the binding of chemical reagents to the MEKl recombinant protein in competition with fluorescent probe. • ■
A. Association of fluorescence-labelled compound to the MEKl protein. Figure shows that the fluorescent compound binding is increased with increasing MEKl concentration as the ΔmP signal enhances correspondingly.
Binding reaction was carried out for 30 minutes at room temperature in a 384- well plate (384 ShallowWell, Nunc™) in final volume of 150 μl with 2 nM fluorescent probe and increasing concentration of MEKl protein as indicated. The binding affinity was also measured with 500 nM, 200 nM and 100 nM fluorescent probe (data not shown) which gave similar affinity constant (Zd) 485 nM.
B to L. Competitive binding to MEKl for examples of chemical reagents, in the presence of 2 nM fluorescent probe.
M & N. Negative controls were performed with EPvKl recombinant protein, albumin and 5-carboxyfluorescein (5-FAM, Merck Chemicals Ltd # 01-63-0112). The figure shows there is no specific binding of the fluorescent probe to the ERKl and albumin. Binding of the 5-FAM to the MEKl recombinant protein is not specific.
Example 1: Localisation of MEK5, p62, ERK5 in neurones
Immunohistochemistry materials and methods
Routine preparation of paraffin-embedded sections, and antigen retrieval by microwaving, was used for the irnmunocytochemical analyses16. Immunohistochemcial detection of MEK5 in paraffin sections was performed with rabbit anti-MEK5 polyclonal antibody (KAP-MA003 : directed to the N-terminus of the protein; residues 59-74) purchased from Bioquote Ltd., (York, U.K.). Immunohistochemical detection of p62 in paraffin sections was performed with mouse monoclonal anti-p62 (p62 lck ligand) directed to the C-terminus of the protein purchased from BD Biosciences (Oxford, U.K.). Immunohistochemical detection of ERK5 was performed with ERK5 antibody from Santa Cruz Biotechology Inc. and is rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 516-815 of the carboxy terminus of human ERK5. .
Results and discussion.
Immunohistochemical detection of MEK5, p62 and ERK5 was carried out in neurones from the temporal cortex from a case of Dementia with Lewy Bodies. p62 is a scaffolding protein that can bind multiubiquitin chains attached to misfolded and aggregated proteins and which is found in all intracellular inclusions containing ubiquitylated proteins in chronic neurodegenerative diseases. The deposits of MEK5 and p62 accumulate in overlapping deposits in neurones containing Lewy bodies (Figure 1) and neurofibrillary tangles (Figure 2), which is expected since MEK5 binds to p62. The MEK5 effector kinase ERK5 is also detected in the nuclei of afflicted cells (Figure 1 and 2). MEK5 and ERK5 are also present in areas of granulovacuolar degeneration (GVD) in the hippocampus which do not contain the p62 protein (Figure 3). These areas are thought to represent areas of intense autophagic activity to eliminate aggregate-prone proteins by the autophagosome-lysosome system. These findings support the notion that the MEK5/ERK5 pathway has a central role in neurones in the temporal cortex in the pathogenesis of chronic neurodegenerative disease.
Figures 1-3: Immunohisto chemical detection of MEIC5 and ERK5 in neurones from the temporal cortex from a case of dementia with Lewy bodies. (Fig 1) immunostaining of MEK5 in a neurone containing a Lewy body (Fig 2) MEK5 in a neurone containing a neurofibrillary tangle (Fig 3) MEK5 in a neurone showing granulovacuolar degeneration (GVD) (Fig 1) ERK5 in neurone containing a Lewy body (Fig 2) ERK5 in a neurone containing a neurofibrillary tangle (Fig 3) ERK5 in a neurone containing GVD. An anti-MEK5 antibody directed to the N- terminus of the protein (see Materials and Methods) was used for immunocytochemical detection of the protein
Example 2: Gene expression changes in response to aggregate-prone proteins identifies mitogen activated protein kinase kinase 5 (MEK5) which, with ERK5, is abundant in neurones in Alzheimer-related disease.
Sumrnaiy Intra-neuronal protein aggregation is central to chronic neurodegenerative disease. The genetic response to aggregate-prone proteins has not been previously investigated. A pan-genomic screen of expression changes to protein aggregates reveals that the expression of MEK5 matches the accumulation and dissolution of protein aggregates. Immunochemical extrapolation to Alzheimer-related disorders showed that MEK5α and ERK5 are abundant in afflicted neurons in the brain. The findings have novel implications for neuronal survival.
Experimental data and discussion
Intraneuronal protein aggregates are the single most consistent molecular feature of all the chronic neurodegenerative disorders. This diverse range of neurological conditions including Alzheimer's disease (AD), Parkinson's disease (PD) and
Huntington's disease (HD), is characterised by intraneuronal aggregates of different proteins associated with clinically distinct disease presentation and progression. A significant development in understanding neurodegeneration came with the experimental demonstration of a novel type of intracellular protein deposit termed the "aggresome". The aggresome, originally identified by protein microinjection studies1'2 in cultured cells, is an intracellular structure formed in response to aberrantly folded3 or mutated protein4. Aggresome formation may be cytotoxic and contribute to neuronal cell death. However, reports advocating a cytoprotective rather than cytotoxic function for aggresomes have been published with increasing frequency in recent years5' . Aggresomes may be an end-point in the cellular response to aggregate-prone proteins. Changes in gene expression in response to protein aggregation have not been studied. Specific aggregate-prone proteins cause neurodegeneration. However, a generic response to protein aggregation may occur i.e. not confined to proteins aggregating in neurodegenerative disease. Therefore, the effects of the aggregation of proteins unrelated to neurodegeneration on gene expression in non-neuronal HEK cells was investigated. Cells were co-transfected with plasmids to express the aggregate prone proteasomal S8 (rpt6) ATPase together with the metabotropic glutamate receptor mGluRlα which had previously been shown to specifically interact via long Homer 3 proteins with the ATPase7. In spite of evidence that S8 and mGluRlα can bind via Homer proteins specific independent protein aggregates
• formed (Figure 5) in cells as previously described for other proteins8. The accumulation and dissolution of the aggregates occurs over as period of 72 hours presumably by a combination of the activities of the ύbiquitin proteasome system and autophagy9. Gene profiling was carried out with the Affymetrix micro array system. As expected there were modest changes (+/- 2-fold) in the expression of many genes (see Supplementary Methods). However, careful inspection of the gene expression profiles showed unexpectedly much larger changes (Fig. 4A) in the expression of a mitogen activated protein ldnase kinase 5 (MEK5).The transcriptional changes in MEK5 were also seen at the level of MEK5α protein (Fig. 4B) which mirrored the time course of accumulation and removal of protein aggregates. MEK5 has one known down-stream phosphorylation target, ERK5, and this kinase' module is specifically activated by axonally-delivered neurotrophins to cause the expression of neuronal survival genes10. The MEK5/ERK5 module activates the myocyte enhancer factor to trigger neuronal expression of the survival genes. MEF2C has a major role in survival of neurons during development11 and the MEK5/ERK5 module appears to be central in the response to cellular insults including oxidative stress12. The findings presented here may suggest the existence of a previously unsuspected cellular mechanism for detecting aggregate-prone proteins that includes the MEK5/ERK5 signalling pathway. If true, evidence for a role of this MAP kinase module should be found by molecular neuropathological investigation. This notion is confirmed by immunohistochemical demonstration of abundant MEK5α and ERK5, as well as p62, in neurones harbouring the hallmarks of Alzheimer-related disease: neurofibrillary tangles, Lewy bodies and granulovacuolar degeneration (Fig. 5).
No immunohistochemical evidence for the presence of the MEK5/ERK5 module was found in inclusion-free neurones. Immunostaining for the p62 protein was carried out because p62 is found in all inclusions in chronic neurodegenerative disease13. Intraneuronal inclusions also contain ubiquitylated proteins14.Previously, it has been shown that the p62 protein binds, through a UBA domain, to polyubiquitin chains5 and also binds to MEK5 homotypically through their shared PBl domains15. MEK5 may be the limiting factor in such complexes whose expression would consequently have to become up-regulated in response to protein aggregation in the cell as well as in neuronal inclusions (Fig. 5). This study is the first to examine a generic pan-genomic response to aggregate- prone proteins. Future work needs to test the notion that proteins may be ubiquitylated in intraneuronal inclusions, not for degradative purposes, but to act as a scaffold for a p62/MEK5/ERK5 signalling system to ensure cell survival.
Supplementary Methods Expression vectors and antibodies
The GFP-S 8 ATPase fusion protein cDNA was expressed ligated in the pEGFP- Cl vector (Promega, Southampton, UK). The FLAG-tagged mGluRlα receptor cDNA was expressed in the pcDNA 3.1 Zeo (+/-) plasmid vector (Invitrogen Corp., Carlsbad, CA, USA). Mouse monoclonal anti-FLAG (M2) was purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK) and used to detect FLAG- tagged mGluRlα. Immunostaining was performed using anti-mouse Alexafluor 594-conjugated antibody supplied by Molecular Probes (Eugene, Oregon, USA) as secondary antibody. Western blots were performed using rabbit anti-MEK5 polyclonal anti-peptide antibody (H-94: directed to the C-terminus of the protein; residues 351-444) purchased from Santa Cruz Biosciences Inc. (Santa Cruz, CA). Immunohistochemical detection of MEK5 in paraffin sections was performed with rabbit anti-MEK5 polyclonal antibody (KAP-MA003: directed to the N-terminus of the protein; residues 59-74) that detects MEK5α only purchased from Bioquote Ltd., (York, U.K.). Immunohistochemical detection of p62 in paraffin sections was performed with mouse monoclonal anti-p62 (p62 lck ligand) directed to the C-terminus of the protein purchased from BD Biosciences (Oxford, U.K.). Immunohistochemical detection of ERK5 in paraffin sections was performed with rabbit polyclonal antibody (H300: directed to the carboxy-terminus of ERK5) purchased from Auto gen Bioclear, (Came, Wiltshire, U.K).
Cell culture co-transfections
HEK293 cells were co-transfected with vectors expressing S 8 ATPase/mGluRlα by the calcium phosphate method. Culture plates (100 mm) at 50-70% confluence were selected and media changed 3 h prior to transfection. To prepare the initial CaCl2-DNA solution for each 100 mm plate, sterile deionised H2O, 10' μg of purified DNA (each vector) and 83 μl of 1.5 M CaCl2 were added sequentially. The CaCl2-DNA solutions were made up to 500 μl with sterile water. An aliquot of 500 μl of 2x HBS (HEPES-Buffered-Saline; 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.1) was also prepared for each 100mm transfection plate. The prepared solutions of CaCl2-DNA and 2x HBS were then slowly mixed in a dropwise fashion with concomitant, vigorous shaking (or slow vortexing). The resulting Ca-PO4-DNA solution (1 ml), was left to incubate for 15-20 min at room temperature. Cell pellets derived from each trypsinised confluent plate were re- suspended in 3 ml of medium (DMEM/10%FBS) and mixed thoroughly with 1 ml of the Ca-PO4-DNA solution. The final mixture was made up to 10 ml with further addition of medium and was split evenly into 2 plates. All plates containing transfected cells were finally placed in a 5% CO2 (37 °C) incubator and harvested at the tiπies indicated in the experimental design (below).
Experimental design The experimental design included a minimum post-transfection incubation period (4 h) for DNA absorption and cell adherence to the plates. Aggresome formation was assessed in three experiments by confocal microscopy, gene expression profiling and apoptosis evaluation. Cells were harvested at O h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h and 72 h after the post-transfection incubation period.
Immunofluorescence and confocal microscopy
FLAG-mGluRlα and GFP-S 8 ATPase were detected via immunofluorescence on fixed cell preparations. Cells were fixed with paraformaldehyde (4%) followed by serial application of staining antibodies and mounting in DAPI-Vectashield immunofluorescence medium (Vector Laboratories, Burlingame, CA, USA), shortly before visualization by confocal microscopy. Cells were cultured on plates containing cover slips. At each time point coverslips were removed from the plate and carefully washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 1.47 mM KH2PO4, pH 7.3). The cells on each coverslip were fixed in ImI of fresh paraformaldehyde (4%) in PBS. Each coverslip was left for 40 min at room temperature. Subsequently, cells were washed with 10 mM Glycine in PBS and then twice with PBS. An aliquot of freshly made 0.1% (v/v) Nonidet-P40 (IGEPAL-63; ICN Biomedical Inc., Aurora, Ohio, USA) in PBS was subsequently applied to each coverslip to permeabilise the cells. Permeabilisation was allowed to proceed for 5 min at room temperature followed by washing 3 times with PBS. Cells were then incubated with 500 μl of Blocking Buffer (1% donkey serum in PBS) for 1 h before application of primary antibody. The anti-FLAG primary antibody was applied in 1:100/ 1 :200 dilutions in blocking buffer and the coverslips were incubated overnight in a humid chamber at 4 0C, in the dark. Following incubation with the primary antibody, coverslips were washed 3 times with PBS and incubated with the secondary anti-mouse Alexafluor 594 antibody , prepared as a 1 :50 dilution in blocking buffer. The secondary antibody incubation was carried out for 1 h at room temperature, followed by PBS washing and mounting in DAPI-Vectashield immunofluorescence medium. All immunostaining reactions were carried out with minimum exposure of cells to light to avoid fading of the contained GFP-S8 ATPase fluorescent signal. Confocal microscopy image analyses were performed using the LSM510 Browser software supplied by Carl Zeiss company (Oberkochen, Germany).
HG-U133A Affymetrix gene expression analysis
Total RNA was extracted from six 100 mm plates of cells expressing GFP and His-FLAG (control cells) and six plates of cells expressing GFP-S8 ATPase and FLAG-mGluRl α (test cells) at each time point. Six millilitres of TRIzol™ reagent (Life Technologies) was added to each plate and the RNA was extracted using the manufacturer's protocol. RNA samples from control cells or test cells were pooled and resuspended in RNAse-free water and quantified spectrophotometrically. The integrity of all samples was confirmed by gel electrophoresis, demonstrating a 2- fold excess of 28 S to 18S RNA, with no evidence of degradation. RNA Samples were then subjected to further purification with Qiagen RNeasy rninikit and measured spectrophotometrically to ensure that only those samples with A26O/2so= 1.9.2.0 were used for subsequent cRNA generation and microarray hybridization,
Affymetrix HG-U133 microarray chip
Samples of RNA (13 μg) were used for cRNA preparation. The first and second strand cDNA synthesis was performed using the Superscript Choice System (Life Technologies) according to the manufacturer's instructions except using an oligo(dT) primer containing a T7 RNA polymerase promoter site. Labelled cRNA was prepared using the BioArray™ RNA Transcript labelling kit (Enzo Life Sciences, Farmingdale, NY, USA) in total reaction volumes of 40μl. Biotin- labelled CTP and UTP (Enzo) were used in the reaction together with unlabelled NTPs. Following in vitro transcription, the unincorporated nucleotides were removed using RNeasy columns (Qiagen). Fifteen micrograms of cRNA were fragmented at 94 0C for 35 min in a fragmentation buffer containing 40 mM Tris- acetate pH 8.1, 100 mM KOAc, 3O mM MgOAc. Prior to hybridization, the fragmented cRNA in a 6x SSPE-T hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton X-100) was heated to 95 °C for 5 min and subsequently to 40°C for 5 min, before loading onto the Affymetrix probe array cartridge. The probe array was then incubated for 16 h at 40 0C with constant rotation (60 r.p.m.). Washing and staining procedures were performed in the Affymetrix Fluidics
• 5 Station. The probe array was exposed to 10 washes in 6x SSPE-T at 25 °C followed by four washes in 0.5x SSPE-T at 50 0C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, 10 mg/ml (Molecular Probes, Eugene, OR), in 6x SSPE-T for 30 min at 25 0C followed by 10 washes in 6x SSPE-T at 25 0C. The probe arrays were scanned at 560 nm using a confocal laser- 10 scanning microscope with an argon ion laser as the excitation source (Hewlett Packard GeneArray Scanner G2500A). The readings from quantitative scanning were analysed by the Expressionist Analyst software (version 5.0.3) from Genedata AG (Basel, Switzerland), which was also used for normalization of the different arrays prior to the analysis.
15
The complete changes in gene expression in this experiment are logged at the European Bioinformatcs Institute Array Expression site (h.ttp://www.ebi.ac.uk/arrayexpress/querv/entrv) with accession number E-MEXP- 389.
20
Western analyses
Cells were harvested at 4, 48 and 72 hours after co-transfection, washed twice with phosphate buffered saline (PBS) and rocked with 500 μl of RIPA buffer [PBS containing NP40 (l%v/v) and sodium deoxycholate (0.5% w/v)] for 5
25 minutes at room temperature. Cells were collected from the plates into microtubes (1.5 ml) and freeze/thawed 4 times in (liquid nitrogen/37 0C). Microtubes were then centrifuged for 10 min at 13,000 rpm. Supernatants were collected, transferred to a fresh microrαbe and centrifuged again. The protein concentration of supernatants were determined by the Bradford assay. Supernatant protein (125
30 μg) from each time point was subjected to SDS-PAGE (10%w/v). After electrophoresis proteins were transferred overnight to nitrocellulose membrane and blocked in Marvel (5% w/v) in tris buffered saline (TBS). The membrane was probed with anti-MEK5 antibody [1:200 in Marvel (5%) in TBS] at room temperature for 1 hour, and washed (3 x 5 minutes) in Tween (0.1% v/v) in TBS (TTBS). Horse radish peroxidase (HRP) conjugated goat anti-rabbit [1:5000 in Marvel (1% w/v) in TTBS] was then applied to the membrane for 1 hour at room • temperature followed by washing (2 x 10 minutes) in TTBS and finally in TBS for 10 minutes. Antigen was visualized by ECL with film exposure for 5 minutes.
Immunohistochemistry
Routine paraffin-embedded sections and antigen retrieval techniques were used for the immunocytochemical analyses of a laboratory control tissue block from a case of Dementia with Lewy bodies.
References for Example 2
1. Doherty, FJ., Wassell, J.A., Mayer, RJ. Biochem J., 241, 793-800 (1987).
2. Earl, R.T., Mangiapane, E.H., Billett, E.E., Mayer, RJ. Biochem J., 241, 809- 815 (1987).
3. Johnston, J.A., Ward, C.L., Kopito, R.R. J. Cell Biol., 143, 1883-1898 (199).
4. Gu, WJ. et al. Neurobiol. Dis., 14, 357-364 (2003). 5. Tanaka, M et al. J Biol Chem., 279, 4625-4631 (2004).
6. Taylor, J. et al. Hum MoI Genet., 12, 749-757 (2003).
7. Rezvani, K et al. Biochem Soc Trans., 31, 470-473 (2003).
8. Rajan, R.S et al. PNAS, 98, 13060-13065 (2001).
9. Bjorkoy, G. et al. J. Cell Biol. 171, 603-614 (2005). 10. Watson, F.L et al. Nat Neurosci., 4, 981-988 (2001).
11. Liu, L et al. (2003) Proc. Natl. Acad. Sci. 100, 8532-8537 (2001).
12. Cavanaugh, J.E. Eur. J. Biochem. 271, 2056-2059 (2004).
13. Zatloukal, K. et al. Am. J. Pathol.160, 255-263 (2002).
14. Lowe, J. et al, J. Pathol. 155, 9-15 (1988). 15. Noda,Y. et.al. J. Biol. Chem. 278, 43516-43524 (2003). Example 3: Novel chemical tools for MEK5: diagnostic and therapeutic implications
The bolded figures "used herein refer to the numbered compounds shown in Figures 8 and 9.
The synthetic molecules 2'-amino-3'-methoxyflavone (also known as PD98059) and 2'-amino-3'-methoxy-thiofiavone are highly selective and potent inhibitors of MEKl (Dudley, 1995; Kataoka, 2004). These flavones selectively block the activation of MEKl, without inhibitory effects on the phosphorylation activities of both upstream and downstream kinases. The high selectivity for MEKl is not surprising since in contrast to many of the well established kinase inhibitors, e.g. flavopiridol and fisentin (De Azevedo, 1996; Mapelli, 2005), the flavones do not competitively interact with the ATP -binding cavity (Dudley, 1995; Alessi, 1995). In fact, these inhibitors appear to bind to a novel allosteric pocket in the inactive form of MEKl. A series of O-alkyl iV-arylanthranilyl hydroxamic acids, e.g. PD184352 and PD318088, were also recently discovered to be selective inhibitors of MEK-I (Sebolt-Leopold, 1999; Chen, 2002). Similar to the flavones, these compounds bind to inactive MEKl via a non-competitive mechanism and selectively block the activation of MEKl. In fact, PD318088 binds to a unique allosteric pocket within the hinge region (i.e. between the N-terminal β-sheets and the C-terminal α-helical lobes), which is separate from but adjacent to the Mg- ATP binding cavity (Ohren, 2004). The bound ligand induced significant conformational changes (see Figure 6; Ohren, 2004) of the MEKl helical 'activation loop' and helix C, as well as deformation of the catalytic site.
The flavone and JV-arylanthranilyl rrydroxamic acids have previously been shown to be weak inhibitors of MEK5 (Mody, 2001). MEK-5 is approximately 40% ' identical to MEKl (Zhou, 1995), increasing to ca. 81% similarity in the PD318088-binding site, with a high degree of fold similarity. Carefully re- examination of the PD318088 binding pocket led to the identification of several subtle differences between MEKl and MEK5. For example, instead of Met143 in MEKl (see Figure 7), a Thr241 residue is located in the same position in the folded MEK5. Thus, chemical analogues of anthranilyl hydroxamic acids (1 - 14) shown in Figure 8 and Table 1 were constructed and used to determine binding affinity to MEKl and MEK5.
Preparation ofiV-(cyclopropylmethoxy)~3,4-difluoro-2-(2-fluoro~4- iodophenylamino)benzamide
Figure imgf000053_0001
A solution of pentafluorophenyl 3,4-difluoro-2-(2-fiuoro-4- iodophenylamino)benzoate (792 mg, 1.41 mmol) in CH2Cl2 (10 ml) was added i\yV-diisopropylethylamine (0.50 ml, 2.83 mmol) followed by O- cyclopropylmethylhydroxylamine hydrochloride (350 mg, 2.83 mmol). The mixture was stirred at ambient temperature for 72 h, which was then washed with water (3 x 15 ml), brine (15 ml), dried over MgSO4, and the organic extract was evaporated to dryness to give an orange gum. The gum was purified by silica column chromatography, using EtOAc as the eluent to give the title compound as an orange .fluffy solid (107 mg, 16%); m.p. 121-123 0C. 1H NMR (300 MHz, D6-
' DMSO) δ 0.18 (m, 2H), 0.46 (m, 2H), 1.03 (m, IH)5 3.57 (d, J = 6.8 Hz, 2H), 6.65
(m, IH), 7.19 (m, IH), 7.33 (m, 2H), 7.55 (d, J = 10.9 Hz, IH), 8.67 (s, IH)5 11.74 (s, IH); MS (ES+) calcd for Ci7H15F3IN2O2 (MH+) 463.01, found 462.84.
Preparation of 2-(4-bromo-2-fluorophenylamino)-iV-(cyclopropylmethoxy)- 3,4-difluorobenzamide
Figure imgf000053_0002
A solution of 2-(4-bromo-2-fluorophenylamino)-3,4-difluorobenzoic acid (370 mg, 1.07 mmol) in THF (3 ml) and DMF (3 ml) was added iV-[(li7-benzotriazol- l-yl)(dimemylairdno)memylene]-N-methyhnemanarniiiium hexafluorophosphate iV-oxide (HBTU) (446 mg, 1.18 mmol) and i\yV-diisopropylethylamine (0.56 ml, 3.21 mmol). The mixture was stirred at ambient temperature for 1 h, after which O-cyclopropylmethylhydroxylamine hydrochloride (200 mg, 1.61 mmol) was added and the mixture was stirred for a further 48 h. The mixture was then concentrated in vacuo, diluted with EtOAc (20 ml), washed with water (4 x 15 ml), dried over Na2SO4, and the organic extract was evaporated to dryness to give a coloured residue material. The residue material was suspended in CH2Cl2 (5 ml), filtered and the filtrate was loaded directly onto a silica chromatography column, which was then eluted with CH2Cl2 to give the title compound as a white solid (72 mg, 16%); m.p. 116-118 0C. 1H NMR (300 MHz, D6-DMSO) δ 0.18 (m, 2H), 0.46 (m, 2H), 1.04 (m, IH), 3.58 (d, J = 7.2 Hz, 2H), 6.79 (m, IH), 7.19 (m, 2H), 7.34 (m, IH), 7.49 (d, J = 8.9 Hz, IH), 8.73 (s, IH), 11.74 (s, IH); MS (ES+) calcd for C17Hi5BrF3N2O2 (MH+) 415.03 and 417.02, found 414.85 and 416.88.
Preparation of 2-(4-chloro-2-fluorophenylamino)-iV-(cy clopropylmethoxy)- 3 ,4-difluorobenzamide
Figure imgf000054_0001
A solution of 2-(4-chloro-2~fluorophenylammo)-3,4-difluoroberizoic acid (325 mg, 1.07 mmol) in THF (3 ml) and DMF (3 ml) was added HBTU (446 mg, 1.18 mmol) and ΛζiV-diisopropylethylamine (0.56 ml, 3.21 mmol). The mixture was stirred at ambient temperature for 1 h, after which O- cyclopropylmethylhydroxylamine hydrochloride (200 mg, 1.61 mmol) was added and the mixture was stirred for a further 48 h. The mixture was then concentrated in vacuo, diluted with EtOAc (20 ml), washed with water (4 x 15 ml), dried over Na2SO4, and the organic extract was evaporated to dryness to give a coloured residue material. The residue material was suspended in CH2Cl2 (5 ml), filtered and the filtrate was loaded directly onto a silica chromatography column, which was then eluted with CH2Cl2 to give the title compound as a pale yellow solid (90 mg, 23%); m.p. 101-103 0C. 1HNMR (300 MHz, D6-DMSO) δ 0.18 (m, 2H), 0.47 (m, 2H), 1.04 (m, IH)5 3.58 (d, J = 6.9 Hz3 2H), 6.85 (m, IH), 7.10 (m, IH), 7.16 (m, IH), 7.37 (m, 2H), 8.75 (s, IH), 11.76 (s, IH); MS (ES+) calcd for C17H15ClF3N2O2 (MH+) 371.08, found 370.94.
Preparation of 3,4~difluoro-2-(2-fluoro-4-iodophenylamino)-iV-(2- hydroxyethoxy)benzamide
Figure imgf000055_0001
A solution of 3,4-difluoro-2~(2-fluoro-4-iodophenylamino)benzoic acid (500 mg, 1.27 mmol) in THF (3 ml) and DMF (3 ml) was added HBTU (529 mg, 1.40 mmol) and 7\yV-diisopropylethylamine (0.22 ml, 1.27 mmol). The mixture was stirred at ambient temperature for 1 h, after which O-hydroxyethylhydroxylamine (195 mg, 2.54 mmol) and i\yV-diisopropylethylamine (0.45 ml, 2.54 mmol) were added and the mixture was stirred for a further 48 h. The mixture was then concentrated in vacuo and loaded directly onto a silica chromatography column, which was then sequentially eluted with CH2Cl2 and 10% v/v MeOH in CH2Cl2 to give a buff solid (180 mg). The sample was recrystallised from CH2Cl2-hexane to
Figure imgf000055_0002
1H NMR (300 MHz, CD3OD) δ 3.69 (t, J = 4.8 Hz, 2H), 3.92 (t, J = 4.8 Hz, 2H), 6.74 (m, IH), 7.00 (m, IH), 7.16 (m, IH), 7.31 (dd, J - 2.2 and 10.8 Hz, IH), 7.38 (m, IH); MS (ES+) calcd for C15Hi3ClF3N2O3 (MH+) 452.99, found 452.77.
Preparation of 2-(4-bromo-2-fluorophenylamino)-3,4-difluoro-7V-(2- hydroxyethoxy)benzamide
Figure imgf000055_0003
A solution of 2-(4-bromo-2-fluorophenylamino)-3,4-difiuorobenzoic acid (500 mg, 1.23 mmol) in THF (3 ml) and DMF (3 ml) was added HBTU (514 mg, 1.23 mmol) and ΛζΛLdiisoproρylethylamine (0.21 ml, 1.23 mmol). The mixture was stirred at ambient temperature for 1 h, after which O-hydroxyethylhydroxylamine (189 mg, 2.46 mmol) and AyV-diisopropylethylamine (0.43 ml, 2.46 mmol) were added and the mixture was stirred for a further 48 h. The mixture was then concentrated in vacuo and loaded directly onto a silica chromatography column, which was then sequentially eluted with CH2Cl2 and 10% v/v MeOH in CH2Cl2 to give a solid. The solid material was recrystallised from CH2Cl2- hexane to afford the title compound as a pale pink solid (140 mg, 28%); m.p. 138-140 0C. 1H NMR (300 MHz, D6-DMSO) δ 3.53 (br s, 2H)5 3.80 (br s, 2H), 6.78 (m, IH)5 7.18 (m, 2H)5 7.36 (m, IH)5 7.48 (dd, J = 2.1 and 10.9 Hz5 IH)5 8.68 (s, IH)5 11.83 (s, IH); MS (ES+) calcd for C15Hi3BrF3N2O3 (MH+) 405.01 and 407.00, found 404.83 and 406.82.
Preparation of 2-(4-chIoro-2-fluorophenylamino)-3,4-difluoro-iV-(2- hydroxyethoxy)benzamide
Figure imgf000056_0001
A solution of 2-(4-chloro-2-fluorophenylamino)-354-difluorobenzoic acid (500 mg, 1.66 mmol) in THF (5 ml) was added l5l'-carbonyl diimidazole (538 mg, 3.32 mmol). The mixture was stirred at ambient temperature for 1 h5 after which C-hydroxyethylhydroxylamine (511 mg, 6.64 mmol) was added and the mixture was stirred for a further 18 h. The mixture was evaporated to dryness. The residual material was dissolved in EtOAc (20 ml), washed with 2 M aqueous HCl (10 ml), water (4 x 15 ml), dried over Na2SO4, and the organic extract was evaporated to dryness to give a coloured oil, which was purified by silica chromatography column, using EtOAc as eluent, to afford the title compound as an off-white solid (306 mg, 51%); m.p. 144-146 0C. 1H NMR (300 MHz5 D6-DMSO) δ 3.53 (br s, 2H)5 3.81 (br s, 2H)5 4.68 (br S5 IH), 6.85 (m, IH)5 7.06-7.19 (m5 2H), 7.40 (m, 2H), 8.70 (S5 IH)5 11.83 (s, IH); MS (ES+) calcd for C15H13ClF3N2O3 (MH+) 361.06, found 360.95. Preparation of 2-(4-chloro-2-fluorophenylamino)-iV-ethoxy-3,4- difluorobenzamide
Figure imgf000057_0001
A solution of 2-(4-chloro-2-fluorophenylamino)-3,4-difluorobenzoic acid (500 mg, 1.66 mmol) in THF (2.5 ml) and DMF (2.5 ml) was added HBTU (691 mg, 1.82 mmol) and N,7V-diisopropyletliylamine (0.28 ml, 1.66 mmol). The mixture was stirred at ambient temperature for 1 h, after which 0-ethylhydroxylamine hydrochloride (322 mg, 3.32 mmol) and N^-diisopropylethylamine (0.57 ml, 3.32 mmol) were added and the mixture was stirred for a further 48 h. The mixture was concentrated in vacuo to give a thick oil, which was dissolved in CH2Cl2 (5 ml) and then purified by silica chromatography, using CH2Cl2 as eluent, to give a pale yellow solid. The solid material was recrystallised from CH2Cl2-hexane to afford the title compound as a pale yellow solid (152 mg, 27%); m.p. 104-106 0C. 1H NMR (300 MHz5 D6-DMSO) δ 1.12 (t, J = 7.0 Hz, 3H), 3.80 (q, J = 7.2 Hz, 2H), 6.85 (m, IH), 7.06-7.20 (m, 2H)5 7.39 (m, 2H)5 8.72 (s, IH), 11.74 (s, IH); MS (ES+) calcd for C15H13ClF3N2O2 (MH+) 345.06, found 345.01.
Preparation of (3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyI)methanol
Figure imgf000057_0002
A solution of 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzoic acid (1.00 g, 2.54 mmol) in THF (10 ml) and DMF (10 ml) was added HBTU (1.06 g, 2.79 mmol) and i\yV-diisopropylethylamine (0.44 ml, 2.54 mmol). The mixture was stirred at ambient temperature for 1 h, after which N-hydroxysuccinimide (586 mg, 5.09 mmol) and N,N-diisopropylethylamine (0.88 ml, 5.08 mmol) were added and the mixture was stirred for a further 48 h. The mixture was concentrated in vacuo to give a thick oil, which was dissolved in CH2Cl2 (5 ml) and then purified by silica chromatography, using CH2Cl2 as eluent, to give succinimidyl 3,4- difluoro-2-(2-fiuoro-4-iodophenylamino)benzoate as an off-white solid (440 mg, 35%). A sample of the isolated solid (424 mg, 0.86 mmol) was dissolved in a mixture of THF (10 ml) and water (2.5 ml). To the solution was added portionwise sodium borohydride (163 mg, 4.3 mmol) and the resultant mixture was refluxed for 6 h, cooled to ambient temperature, and allowed to stand overnight. Water (20 ml) and EtOAc (20 ml) were added followed by 2 M aqueous HCl (20 ml). The organic phase was extracted, washed with brine (30 ml) and concentrated in vacuo to give a pink oily material, which was purified by silica column chromatography, using EtOAc-hexane as eluent, to afford the title compound as a pale pink solid (181 mg, 56%); m.p. 78-79 °C. 1H NMR (300 MHz, CDCl3) δ 1.95 (br s, IH)5 4.65 (s, 2H), 6.39 (m, IH), 6.50 (s, IH), 6.95 (m, IH), 7.07 (m, IH), 7.26 (m, IH), 7.39 (dd, J = 2.1 and 10.6 Hz, IH); HR-ToF MS (ES+) calcd for Ci3H10F3INO (MH+) 379.9754, found 379.9833.
Preparation of 2-(4-chloro-3-methoxyphenylamino)-iV-ethoxy-3,4- difliiorobenzamide
Figure imgf000058_0001
To a solution of 2-chloro-5-nitroanisole (8.0 g, 42.7 mmol) in CH2Cl2 (300 ml) was added 5% platinum on carbon (25 g) and ammonium formate (27 g, 427 mmol), and the mixture was refluxed for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite, and concentrated in vacuo to give 5- amino-2-chloroanisole (6.47 g, 96%).
A solution of LiHIsO)S was prepared by the addition of 2.5 M butyllithium in hexanes (26.8 ml, 67 mmol) to a solution of hexamethyldisilazane (14.1 ml, 67 mmol)' in THF (50 ml) at -78 °C. This solution was warmed to ambient temperature over 1 h, which was then gradually added to a solution of 5-amino-2- chloroanisole (7.1 g, 45.0 mmol) in THF (100 ml) at -78 °C. The resulting dark suspension was then stirred for a further 1 h. To the mixture was then added a solution of 2,3,4-trifluorobenzoic acid (3.96 g, 22.5 mmol) in THF (50 ml). The mixture was warmed to room temperature over 2 h, and stirred a further 6 days. The mixture was then evaporated to dryness in vacuo to give a dark tar, which was partitioned between 2 M aqueous HCl (200 ml) and EtOAc (150 ml). The organic . extract was separated, the aqueous phase was extracted with EtOAc (2 x 100 ml), and the combined organic extracts were dried over Na2SO4 and finally evaporated to dryness in vacuo to give a dark solid. Trituration of the solid with CH2Cl2 gave the first crop of the desired 2-(4-chloro-3-methoxyphenylamino)-3,4- difluorobenzoic acid (2.13 g, 30%) and the second crop (0.34 g, 5%) was obtained by silica column chromatography of the CH2Cl2-supernatant, using CH2Cl2- MeOH (9:1) as eluent. A solution of 2-(4-chloro-3-methoxyphenylamino)-3,4-difiuorobenzoic acid (250 mg, 0.80 mmol) in THF (5 ml) was added l,l'-carbonyl diimidazole (227 mg, 1.40 mmol). The mixture was stirred at ambient temperature for 1 h, after which (9-ethylhydroxylamine hydrochloride (169 mg, 1.75 mmol) was added and the mixture was stirred for a 1 h. A further sample of O-ethylhydroxylamine - hydrochloride (77 mg, 0.8 mmol) was added along with N,N- diisopropylethylamine (0.39 ml, 2.80 mmol), and the mixture was stirred for a further 12 h. The mixture was evaporated to dryness. The residual material was dissolved in EtOAc (10 ml), washed with water (2 x 10 ml), dried over Na2SO4, and the organic extract was evaporated to dryness. The residual material was purified by silica chromatography column, using EtOAc-MeOH (9:1) as eluent, to give the title compound as a pale brown solid (120 mg, 42%); m.p. 122-124 °C. 1H NMR (300 MHz, CDCl3) δ 1.28 (t, J = 7.2 Hz, 3H), 3.84 (s, 3H), 3.99 (q, J = 7.0 Hz, 2H), 6.35 (m, IH), 6.48 (d, J = 2 Hz, IH), 6.85 (m, IH), 7.19 (d, J = 8.6 Hz, IH), 7.34 (m, IH), 8.10 (s, IH)5 8.75 (s, IH); MS (ES+) calcd for C16H16C^2N2O3 (MH+) 357.08, found 357.00.
Preparation of (2-(4-chloro-3-methoxyphenylamino)-3,4- difluorophenyl)methanol
Figure imgf000059_0001
A solution of 2-(4-chloro-3-metlioxyρhenylamino)-3;4-difluorobenzoic acid (400 mg, 1.28 mmol) in THF (10 ml) was added l,l '-carbonyl diimidazole (415 mg, 2.56 mmol). The mixture was stirred at ambient temperature for 1 h, after which N-hydroxysuccinimide (369 mg, 3.20 mmol) was added and the mixture was 5 stirred for a further 16 h. The mixture was evaporated to dryness to give a thick coloured oil, which was purified by silica column chromatography, using hexane- diethyl ether as eluent, to give succinimidyl 2-(4-chloro-3-methoxyphenylamino)- 3,4-difluorobenzoate as a brown solid (330 mg, 63%). A sample of the isolated solid (300 mg, 0.73 mmol) was dissolved in a mixture of THF (7 ml) and water (2
10 ml). To the solution was added sodium borohydride (139 mg, 3.65 mmol) and the resultant mixture was stirred for 6 h. To the mixture was then added water (20 ml) and EtOAc (20 ml), and the organic phase was extracted, washed with brine (30 ml) and evaporated to dryness to give a coloured solid, which was purified by silica column chromatography to yield the title compound as a purple solid (98
15 mg, 45%); m.p. 96-97 °C. 1H NMR (300 MHz, CDCl3) δ 1.82 (br s, IH), 3.83 (s', 3H), 4.64 (s, 2H), 6.27 (d, J = 8.5 Hz, IH), 6.38 (s, IH), 6.92 (m, IH), 7.05 (m, IH), 7.17 (d, J = 8.2 Hz, IH); HR-ToF MS (ES+) calcd for C14H13ClF2NO2 (MH+) 300.0597, found 300.0583.
'20 Preparation of (2-(4-bromo-3-methoxyphenylamino)-3,4- difluorophenyl)methanol
Figure imgf000060_0001
A solution of 2-(4-bromo-3-methoxyphenylamino)-3,4-difiuoroberiZoic acid (1.4 g, 3.91 mmol) in THF (10 ml) was added l,l '-carbonyl diimidazole (1.27 g, 7.81
25 mmol). The mixture was stirred at ambient temperature for 1 h, after which N- hydroxysuccinimide (1.13 g, 9.80 mmol) was added and the mixture was stirred for a further 16 h. The mixture was evaporated to dryness, to give a thick orange oil, which was purified by silica column chromatography, using hexane-EtOAc as eluent, to give succinimidyl 2-(4-bromo-3-methoxyphenylamino)-354-
30 difluorobenzoate as a golden yellow solid (807 mg, 45%). A sample of the isolated solid (666 mg, 1.46 rαmol) was dissolved in a mixture of THF (5 ml) and water (1 ml). To the solution was added sodium borohydride (275 mg, 7.32 mmol) and the resultant mixture was stirred for 6 h. To the mixture was then added water (20 ml) and EtOAc (20 ml), and the organic phase was extracted, washed with brine (30 ml) and evaporated to dryness to give a coloured material, which was purified by silica column chromatography to yield the title compound as a pink solid (180 mg5 36%); m.p. 99-101 0C. 1H NMR (300 MHz, D6-DMSO) δ 3.71 (s, 3H), 4.39 (d, J = 5.1 Hz, 2H), 5.27 (t, J = 5.4 Hz, IH), 6.00 (d, J = 8.6 Hz, IH), 6.41 (s, IH), 7.21-7.31 (m, 3H), 7.78 (s, IH); HR-ToF MS (ES+) calcd for C14H13BrF2NO2 (MH+) 344.0098 and 346.0077, found 344.0059 and 345.9974.
Table 1
Example Compound Code M.p. MS ES+ No. (°C) (MH+) iV-(Cyclopropylmethoxy)-3,4-difluoro-2-(2- WCCOOl 121-123 462.84 fluoro-4-iodophenylamino)benzamide
2-(4-Bromo-2-fluorophenylamino)-Λr- WCC003 116-118 414.85,
(cyclopropylmethoxy)-3,4-difluorobenzamide 416.88
2-(4-Cmoro-2-fluorophenylamino)-N- WCC004 101-103 370.94
(cyclopropylmethoxy)-3,4-difluorobenzamide
3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-iV- WCC005 126-128 452.77
(2-hydroxyethoxy)benzamide
2-(4-Bromo-2-fluorophenylamino)-3,4-difluoro- WCC006 138-140 404.83, iV-(2-hydroxyethoxy)benzamide 406.82
2-(4-Chloro-2-j-luoroph.enylamino)-354-difluoro- WCC007 144-146 360.95
N-(2-hy droxyethoxyjb enzamide
JV-(Ethoxy)-3,4-difluoro-2-(2-fluoro-4- WCC009 108-110 436.86 iodophenylamino)benzamide I
2-(4-CMoro-2-fluorophenylamino)-iV"-ethoxy- WCC009 104-106 345.01
3 ,4-difluorobenzainide Cl
(3,4-Difluoro-2-(2-fluoro-4- WCC019 78-79 379.9833 iodophenylamino)phenyl)methanol
10 N-(2-(Dimethylamino)ethyl)-3,4-difluoro-2-(2- WCC020 gum 463.85 fluoro-4-iodophenylamino)benzamide
11 2-(4-Bromo-3-methoxyphenylamino)-7V-ethoxy- WCCOI l 128-130 400.91,
3 ,4-difluorobenzamide 402.88
12 2-(4-CMoro-3-methoxyphenylamino)-7V'-ethoxy- WCC012 122-124 357.00
3 ,4-difluorobenzamide
13 (2-(4-Bromo-3 -methoxyphenylamino)-3 ,A- WCCOl 5 99-101 344.00595 difluorophenyl)methanol 345.9974
14 (2-(4-Chloro-3 -methoxyphenylamino)-3 ,4- WCCO 16 96-97 300.0583 difluoiOpb.enyl)methanol The examples of compounds shown in Figure 8 and listed in Table 1 can be readily converted to unique radiolabeled ligands or radiopharmaceuticals, which can be used for in vivo investigation and diagnosis by applying any established radioimaging modalities such as SPECT and PET. The specific selection and subsequent installation of the radio-isotope will depend on a number of factors including the desired radioimaging modality, resolution and the practicality of the production of the radiopharmaceutical.
For example, outlined in Figure 9 are two simple methods for the production of the radiopharmaceuticals, JV-(ethoxy)-3 ,4-difluoro-2-(2-fluoro-4-[123I]iodophenyl- amino)benzamide and 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-iV-(2- [18F]fluoroethoxy)benzamide, from the compounds 7 and 4, for use in SPECT and PET respectively. Thus, treatment of compound 7 with bis(tributyltin) in the presence of tetrakis(triphenylphosphine)palladium (0) gave a stable tributylstannyl analogue as the radiopharmaceutical precursor. Radioiododestannylation of the precursor chemical in the presence of an oxidant such as hydrogen peroxide or fert-butylhydroperoxide afforded the required radioligand jV-(ethoxy)-3,4- difluoro-2-(2-fluoro-4-[123I]iodophenylamino)benzamide. Alternatively, treatment of compound 4 with^-tosyl chloride under mild basic condition gave the activated- _p-tosyl derivative, which is subsequently converted to the radioligand 3,4- difluoro-2-(2-fluoro-4riodophenylamino)-iV"-(2-[lsF]fluoroethoxy)benzamide by nucleophilic displacement
References
Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995). PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270, 27489-27494.
Chen, M., Gu, H., Davis, E. M., Magano, J., Nanninga, T. N., and Winklw, D. D. (2002). International patent WO 02/18319 Al. De Azevedo, W. F., Jr., Mueller-Dieckmann, H. J., Schulze-Gahmen, U., Worland, P. J., Sausville, E., and Kim, S. H. (1996). Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proc Natl Acad Sci U S A 93, 2735-2740.
Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. I, and SaltieL A. R. (1995). A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 92, 7686-7689.
Kataoka, T., Watanabe, S., Mori, E., Kadomoto, R, Tanimura, S., and Kohno, M. (2004). Synthesis and structure- activity relationships of thioflavone derivatives as specific inhibitors of the ERK-MAP kinase signaling pathway. Bioorg Med Chem 12, 2397-2407.
Mapelli, M., Massimiliano, L., Crovace, C, Seeliger, M. A., Tsai, L. H., Meijer, L., and Musacchio, A. (2005). Mechanism of CDK5/p25 binding by CDK inhibitors. J Med Chem 48, 671-679.
Mody, N., Leitch, J., Armstrong, C, Dixon, J. E., and Cohen, P. (2001). Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett 502, 21- 24.
Ohren, J. F., Chen, H., Pavlovsky, A., Whitehead, C, Zhang, E., Kuffa, P., Yan, C, McConnell, P., Spessard, C, Banotai, C, et al. (2004). Structures of human MAP kinase kinase 1 (MEKl) and MEK2 describe novel noncompetitive kinase inhibition. Nat Struct MoI Biol IJ, 1192-1197.
Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., Gowan, R. C, Tecle, H., Barrett, S. D., Bridges, A., Przybranowski, S., et al. (1999). Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med 5, 810-816. Zhou, G., Bao, Z. Q., and Dixon, J. E. (1995). Components of a new human protein kinase signal transduction pathway. Journal of Biological Chemistry 270, 12665-12669.
Example 4: Assay of ERK5 activity
Cells were lysed in lysis buffer. Endogenous ERK5 or tagged-ERK5 was immunoprecipitated with anti-ERK5 antibody (Ab 1) or anti-tag antibody and incubated for 10 min at 3O0C with myelin basic protein (final concentration, 2 mg/ml) in reaction buffer (20 mM Tris-Cl (jpH 7.5], 10 mM MgCl2, and 100 μM ATP, or 2 μCi of [γ-32P]ATP).
Antibodies were purchased as follows: rabbit anti-ERK5 antibody from Sigma (Ab 1) and Calbiochem (Ab 2); rabbit anti-phospho-ERK5 antibody from
Calbiochem; mouse anti-Myc (9E10), rabbit anti-Myc (A- 14), and rabbit anti- glutathione S-transferase (anti-GST) (Z-5) antibodies from Santa Cruz; mouse anti-HA (16B12) antibody from Covance; Alexa Fluor 488-goat anti-mouse or rabbit IgG, Alexa Fluor-594 goat anti-mouse, or rabbit IgG from Molecular Probes.
Example 5: Imaging compound assessment
RNA isolation and first strand DNA synthesis
Total RNA was purified from HeLa cells using Qiagen RNeasy extraction kit (Cat # 74104). The quality of the RNA was checked by native agarose gel electrophoresis. First strand cDNA was synthesized using the Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.
Cloning Human MEKl
The human MEKl (Gene Bank Accession number: L11284.1) spanning 2,167 bps and contains 11 exons, was cloned by PCR utilizing cDNA derived form HeLa cells as the template. The primers utilized for full length MEKl amplification were: MEKl forward :5'CGC GGA TCC ATG CCC AAG AAG AAG CCG3';
MEKl reversed 'GCAA GCT TCG TTA GAC GCC AGC AGC ATG3'. Direct ligation of the Ml length MEKl cDNA into the ρRSET-A plasmid (Invitrogen) was performed using BamHI and HindIII restriction sites introduced into the forward and reverse primers respectively. By employing pRSET-A plasmid, the
MEKl recombinant protein is N-terminally tagged with 6 histidine residues for further purification. The final construct was verified by DNA sequence analysis.
MEKl cDNA was inserted into different plasmids, pET-41a, pET-28a (Novagen) and pGEX-4T-l (Amersham Biosciences) to produce the MEKl recombinant protein with different tags.
Expression of human MEKl
BL21(DE3)pLyse E. coli competent cells (Novagen) were utilized as a bacterial host for expression of the recombinant MEKl protein. Optimization of the expression was completed. The BL21(DE3)pLyse cells were grown in the LB medium at 37 0C and induced with 500 μM isopropyl-β-D-thiogalactopyranoside
(IPTG) for 6 h to express the MEKl protein in highest yield (Figure 10A).
Immunoblotting with the MEKl antibody was performed using the polyclonal MEKl -antibody (Abeam # 32091) which confirms the expression of the MEKl recombinant protein (Figure 10B).
The pRSET-A plasmid gave the best expression of MEKl recombinant protein. The other plasmids did not yield a sufficient amount of protein in comparison to pRSET-A. Amongst different growth temperatures which were also tried; cell growth at 370C produced the best result.
Purification of recombinant His-tagged MEKl
To purify sufficient amount of purified recombinant MEKl protein for binding assay, 1 litre LB culture was utilized. After 6 h of IPTG induction, the cell pellet was harvested (3000g, 10 rnin at room temperature) and bacterial cells lysed in ' lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40 and 0.1% SDS) and then sonicated on ice for 5 x at high output setting. The lysate was centrifuged at high speed (1000Og, 10 min at room temp), insoluble fraction was spun down and supernatant was collected.
To purify the His-tagged protein under native conditions, nickel-charged resin was employed. Imidazole was added to the supernatant to a final concentration of 10 mM and applied to the prepared 700 μl nickel-charged resin. The column was incubated with gentle shaking overnight at 4 0C. The resin was then washed 3x with TBS, TBS-20 mM Tris and TBS-40 mM Tris (pH 7.5) respectively. .The recombinant MEKl protein was eluted using 1000 μl of 500 mM imidazole. The purified protein was analysed on 5-20% SDS-PAGE electrophoresis (Figure 11).
Preparation of iVα-(fluorescein-5-carbonyl)-iVε-(3,4-difluoro-2-(2-fluoro-4- iodophenylamino)benzoyl)-L-lysyl amide
Figure imgf000067_0001
..
A suspension of the NovaPEG Rink Amide resin (100 mg, 0.064 mmol; 0.64 mmol Merck Chemicals Ltd. # 01-64-0473) in DMF (2 ml) in a reaction column was washed with DMF (2.5 ml min"1, 10 min), 20% v/v piperidine in DMF (2.5 ml min'1, 5 min) and finally with DMF (2.5 ml min"1, 15 min). The excess DMF was removed, and the resin in the reaction column was treated with a pre-prepared mixture of Dde-L-Lys(Fmoc)-OH (133 mg, 0.25 mmol, Merck Chemicals Ltd. # 04-12-5201; Bycroft et al (1993) J Chem Soc, Chem Commun, 778-779), N-[(dimethylamino)-li?-l,2,3-triazolo[4,5-&]pyridiri-l-ylmethylene]-N- methyhnethanaminium hexafluorophosphate TV-oxide (HATU) (95 mg, 0.25 mmol) and ΛyV-diisopropylethylamine (0.087 ml, 0.5 mmol) in DMF (1 ml). The resultant mixture was gently stirred for 14 h, followed by washing with DMF (2.5 ml min"1, 15 min). The resin product in the reaction column was treated with 20% piperidine (2.5 ml mm"1, 10 min), washed with DMF (2.5 ml min"1, 15 min), excess DMF removed and treated with a pre-prepared mixture of 3,4-difluoro-2- (2-fluoro-4-iodophenylamino)benzoic acid (98 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol) and ΛyV-diisopropylethylamine (0.087 ml, 0.5 mmol) in DMF (1 ml) for 1O h. The resin product was washed with DMF (2.5 ml min'1, 10 min), treated with 4% hydrazine monohydrate in DMF (2.5 ml min"1, 30 min), washed with DMF (2.5 ml min'1, 15 min), excess DMF removed and then treated with a pre- prepared mixture of 5 -carboxy fluorescein (100 mg, 0.26 mmol), 7-aza-l- hydroxybentriazole (34 mg, 0.25 mmol) and ΛyV'-diisopropylcarbodiimide (0.110 ml, 0.25 mmol) in DMF (1 ml) for 14 h. The resin product was then successively washed with DMF (2.5 ml min"1, 15 min), 20% piperidine in DMF (2.5 ml min"1, 15 min), and DMF (2.5 ml min"1, 15 min).
The coloured resin product was collected, rinsed with CH2Cl2, dried in vacuo, and then treated with the acidolytic mixture trifluoroacetic acid (6 ml) - H2O (0.6 ml) - iPr3SiH (0.2 ml) for 2 h at ambient temperature. The suspension was filtered, the filtrate was evaporated to dryness in vacuo and the residual material was triturated with diethyl ether (3 x 5 ml) to afford the title compound as a bright yellow solid (48 mg).
Reversed-phase HPLC was carried out using Waters™ 510 pumps, Waters™ 484 detector and Hypersil™ Pep 100-C1S analytical column (150 x 4.6 mm, 5 μm) at a flow rate of 1.20 ml min"1 and the effluent was monitored at 220 nm. Gradient elution was from 50 % to 100 % B in 20 min, and the eluents used were: solvent A (0.06% v/v TFA in MiUi-Q water) and solvent B (0.06% v/v TFA in MeCN - Milli-Q water, 9:1 v/v). The solid product was established by HPLC to be >90% pure; Λt .8.48 min. RP-HPLC purified sample: 1H MMR (400 MHz, CD3OD) δ 1.50 (m, 2H), 1.60 (m, 2H), 1.88 (m, IH), 1.96 (m, IH), 4.58 (m, IH), 6.51-6.60 (m, 4 H), 6.71 (d, J = 2.0 Hz, 2H), 6.99 (m, IH), 7.29 (m, 2H), 7.41-7.46 (m, 2H), 8.23 (dd, J = 1.6 and 8.0 Hz, IH), 8.50 (s, IH), 8.71 (d, J = 7.8 Hz, IH); HR-ToF ' MS (ES+) calcd for C40H31F3IN4O8 (MH+) 879.1133, found 879.1170.
Fluorescence polarization binding assay for MEKl
To determine the binding of chemical agents listed in Table 1 to the human MEKl recombinant protein, a fluorescence polarization assay (FP) was employed. The assay is based on competition between the fluorescent compound [TV0- (fluorescein-5-carbonyl)-iVE-(3,4-difluoro-2-(2-fluoro-4- iodophenylamino)benzoyl)-L-lysyl amide; Compound Code: WCC030F] and increasing amounts of competing compound, determined as fluorescence polarization intensity (milli-Polarization, mP). Binding of different concentrations of the fluorescein-labelled compound (WCC030F) to increasing amounts of MEKl recombinant protein was first analysed. The fluorescent compound (WCC030F) binding to MEKl showed a K& 485 nM (Figure 12A; 2 nM concentration of fluorescent compound). Negative controls were also performed using ERKl recombinant protein, albumin and 5-carboxyfluorescein (5-FAM; Merck Chemicals Ltd. # 01-63-0112) (Figure 12M & N). The competition assays for a panel of chemical reagents were carried out with the 2 nM fluorescence probe, 1000 nM MEKl with increasing concentration of the competing ligands. The inhibitory concentration 50% (ICs0) was calculated using Prism™ statistical software (Figures 12B to L).
Table 2. Summary of competitive inhibitory concentrations (IC5o) determined using fluorescence polarization method.
Figure imgf000070_0001
The following are examples of assessments that can be performed on a target . imaging compound.
1. competitive binding assays with non radiolabeled ligands.
2. Selected compounds from 1 that are cell permeable. 3. Autoradiography with selected radiolabeled compounds from 2.
4. Transgenic mice over-expressing Tau and Alzheimer precursor proteins (APP) with selected label compounds and either PET or SPECT or MMR as indicated earlier.
The objective of the work is to identify compounds that bind to MEK5 better than MEKl for the neuroimaging analyses. The compounds are designed to fit into the MEK1/MEK5 allosteric regulation site.
Compounds were tested against MEKl in the first instance but it is anticipated that these compounds have similar, but not identical, activity against MEK5.
References for the main description.
1. Mayer, RJ. et al. Ubiquitin and dementia. Nature 340, 193 (1989).
2. Lennox, G. et al. Diffuse Lewy body disease: correlative neuropathology using anti-ubiquitin irnmunocytochemistry. J Neurol Neurosurg Psychiatry ■ 52,
1236-47 (1989).
3. Lennox, G., Lowe, J.S., Godwin-Austen, R.B., Landon, M. & Mayer, RJ. Diffuse Lewy body disease: an important differential diagnosis in dementia with extrapyramidal features. Prog Clin Biol Res 317, 121-30 (1989). 4. Steiner, H. & Haass, C. Intramembrane proteolysis by presenilins. Nature Reviews in Molecular Cell Biology 1, 217-224 (2000).
5. Liou, Y.C. et al. Role of the prolyl isomerase Pinl in protecting against age-dependent neurodegeneration. Nature 424, 556-61 (2003). • •
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Claims

1. A method of diagnosing a neurodegenerative disease in an individual comprising measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the patient.
2. A method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising measuring the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module, in a sample of body fluid or tissue from the patient.
3. The method of claim 1 or 2 further comprising the step of comparing the level of one or more component(s) of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module, or the level of one or more components of the MEK3/p38 MAPK module in the sample to the level of the same component(s) hi a normal sample.
4. The method of any one of the previous claims wherein the component of the MEK5/ERK5/MEF module is MEK5.
5. The method of any one of the previous claims wherein the activator of the MEK5/ERK5/MEF module is p62 or MEKK2,3/Tpl2.
6. The method of any one of the previous claims wherein the method measures the level of the polypeptide of one or more component(s) of the MEK5/ERK5/MEF module or the activator of the MEK5/ERK5/MEF module or the level of the polypeptide of one or more components of the MEK3/p38 MAPK module.
7. The method of any one of the previous claims wherein the level of the component of the MEK5/ERK5/MEF module or the activator of the MEK5/ERK5/MEF module or the level of one or more components of the MEK3/p38 MAPK module is measured using ELISA, electrophoresis, chromatography or mass spectrography.
8. The method of claim 6 or 7 wherein the level of the polypeptide is determined using a molecule which selectively binds to the polypeptide.
9. The method of claim 8 wherein the molecule is an antibody.
10. The method of any one of the previous claims wherein the body fluid is cerebrospinal fluid or blood or the tissue is nasal neuro-epithelial tissue.
11. A method of diagnosing or monitoring a neurodegenerative disease in an individual comprising detecting the level of one or more components of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in the brain of the individual.
12. A method of determining the susceptibility of an individual to developing a neurodegenerative disease comprising detecting the level of one or more components of the MEK5/ERK5/MEF module, or an activator of the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in neurons in the brain of the individual.
13. The method of claim 11 or 12 wherein the level of the component(s) of the MEK5/ERK5/MEF module or the activator(s) of the MEK5/ERK5/MEF module or the level of the component(s) of the MEK3/p38 MAPK module is measured using neuroimaging.
14. The method of claim 13 wherein the neuroimaging is performed using magnetic resonance imaging.
15. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module and assaying for activation of the MEK5/ERK5/MEF module component(s).
16. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERK5/MEF module and assaying for inhibition of the MEK5/ERK5/MEF inhibitor.
17. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for activation of said activator.
18. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a test compound with a one or more components of the MEK3/p38 MAPK module and assaying for inhibition of the MEK3/p38 MAPK module component(s).
19. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of one or more components of the MEK5/ERK5/MEF module.
20. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of a MEK5/ERK5/MEF module inhibitor.
21. A method of detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for upregulation of an activator MEK5/ERK5/MEF module.
22. A method for detecting compounds useful for the treatment of neurodegenerative disease comprising contacting a cell with a test compound and assaying for inhibition of one or more components of the MEK3/p38 MAPK module.
23. A method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK5/ERK5/MEF module (or MEKl module) and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF module (or MEKl module) component(s).
24. A method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an inhibitor of the MEK5/ERK5/MEF module and assaying for binding to, activation or inhibition of the MEK5/ERK5/MEF inhibitor.
25. A method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with an activator of the MEK5/ERK5/MEF module and assaying for binding to, activation or inhibition of the activator.
26. A method of detecting compounds useful for the diagnosis, monitoring or imaging of neurodegenerative disease comprising contacting a test compound with one or more component(s) of the MEK3/p38 MAPK module and assaying for binding to, activation or inhibition of the MEK3/p38 MAPK module component(s).
27. A compound detected according to any one of claims 15 to 26 for use in medicine.
28. A pharmaceutical composition comprising a compound as defined in claim 27 and a pharmaceutically acceptable excipient.
29. Use of a material which binds to a component of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or binds to a component of the MEF3/p38 MAPK module in the diagnosis or monitoring of a neurodegenerative disease in an individual or to determine the susceptibility of an individual to developing a neurodegenerative disease or to detect compounds useful for the treatment of neurodegenerative disease.
30. A kit of parts useful for diagnosing or monitoring neurodegenerative disease comprising a material which is capable for use in determining the level of one or more components of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or one or more components of the MEF3/ρ38 MAPK module
31. The use of claim 29 or kit of parts of claim 30 wherein the material is an antibody.
32. The use of claim 29 or kit of parts of claim 30 wherein the material binds to MEK5.
33. The use or kit of parts of claim 32 wherein the material is an anti-MEK5 antibody.
34. The use of claim 29 or kit of parts of claim 30 wherein the material binds to MEK3.
35. The use or kit of parts of claim 34 wherein the material is an anti-MEK3 antibody.
36. The use of claim 29 or kit of parts of claim 30 wherein the material is a compound which binds to components of the MEK5/ERK5/MEF module.
37. The use or kit of parts of claim 36 wherein the material is 2'-amino-3'- methoxyflavone or an O-alkyl iV-aryl anthranilyl hydroxamic acid or analogues thereof which are capable of binding to MEK5, for example as indicated in Figure 8 and table 1 for the list of compounds.
38. Use of an oligonucleotide encoding one or more components of the MEK5/ERK5/MEF module or an activator the MEK5/ERK5/MEF module or one or more components of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease, wherein the oligonucleotide is immobilised on a solid support.
39. Use of a component of the MEK5/ERK5/MEF module or an activator of the MEK5/ERK5/MEF module or a component of the MEK3/p38 MAPK module in a method of detecting compounds useful for the treatment of neurodegenerative disease.
40. The use of claim 39 wherein the component is MEK5.
41. A method of treating an individual suffering from a neurodegenerative disease comprising supplying to the individual an appropriate quantity of a compound detected according to any one of the methods of claims 15 to 22.
42. Use of a compound detected according to any one of the methods of claims 15 to 22 in the manufacture of a medicament for preventing or treating a neurodegenerative disease.
43. A method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of upregulating MEK5/ERK5/MEF module activity either directly or by downregulating the activity of one or more inhibitors of MEK5/ERK5/MEF module activity.
44. A method of treating a neurodegenerative illness in a human or animal comprising administering to the human or animal a pharmaceutically effective amount of an agent or agents capable of inhibiting MEK3/p38 MAPK module activity.
45. A method of preventing neuronal cell death comprising upregulating the activity of the MEK5/ERK5/MEF module and/or downregulating the activity of the MEK3/p38 MAPK module in the neuronal cell.
46. The use of a compound detected according to the screening methods of any one of claims 23 to 26 in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
47. The anthranilyl hydroxamic acid analogue N-(Cyclopropylmethoxy)-3,4- difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; 2-(4-Bromo-2- fluorophenylammo)-JV-(cyclopropylmethoxy)-3 ,4-difluorobenzamide; 2-(4-
CMoro-2-fluorophenylamino)-iV-(cyclopropyhnethoxy)-3,4-difluorobenzamide; 3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-Λ'-(2-hydroxyethoxy)benzamide; 2- (4-Bromo-2-fluorophenylamino)-3,4-difluoro-Λ'-(2-hydroxyethoxy)benzamide; 2- (4-Chloro-2-fluorophenylamino)-3 ,4-difluoro-N-(2-hydroxyethoxy)benzamide; N- (Ethoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide; 2-(4-Chloro-2- fluorophenylamino)-iV-ethoxy-3,4-difluorobenzamide; (3,4-Difluoro-2-(2-fluoro- 4-iodophenylamino)phenyl)methanol; N-(2-(Dimethylamino)ethyl)-3,4-difluoro- 2-(2-fluoro-4-iodophenylamino)benzamide; 2-(4-Bromo-3-methoxyphenylamino)- Λf-ethoxy-3,4-difiuorobenzarnide; 2-(4-Chloro-3-methoxyphenylamino)-iV-ethoxy- 3 ,4-difluorobenzamide; (2-(4-Bromo-3 -methoxyphenylamino)-3 ,4- difluorophenyl)methanol ; (2-(4-Chloro-3 -methoxypheny lamino)-3 ,A- difluorophenyl)methanol; or iVα-(Fluorescein-5-carbonyl)-iVε-(3,4-difluoro-2-(2- fluoro-4-iodophenylamino)benzoyl)-L-lysyl amide; or radiolabelled or fluorescently labelled derivative any thereof.
48. The anthranilyl hydroxamic acid analogue of claim 46 wherein the analogue is 2-(4-Chloro-3-methoxyphenylamino)-N-ethoxy-3,4- difluorobenzamide; (2-(4-Bromo-3-methoxyphenylamino)-3,4- difluorophenyl)methanol; or (2-(4-Chloro-3-methoxyphenylamino)-3,4- difluorophenyl)methanol; or radiolabeled or fluorescently labelled derivative any thereof.
49. The anthranilyl hydroxamic acid analogue of claim 47 or 48 for use in medicine.
50. A pharmaceutical composition comprising an anthranilyl hydroxamic acid analogue of claim 47 or 48 and a pharmaceutically acceptable excipient.
51. The anthranilyl hydroxamic acid analogue of claim 47 or 48 or pharmaceutical composition of claim 50 for use in aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
52. Use of an anthranilyl hydroxamic acid analogue of claim 47 or 48 or pharmaceutical composition of claim 50 in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of a neurodegenerative disease.
53. The method or use of any one of claims 1 to 46, 51 or 52 wherein the neurodegenerative disease is a progressive neurodegenerative disease.
54. The method or use of claim 53 wherein the progressive neurodegenerative disease is Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy, multisystem atrophy, motor neurone disease (amyotrophic lateral sclerosis) or frontotemporal dementia.
55. A method of any of the previous claims wherein the individual is a human or animal patient.
56. The anthranilyl hydroxamic acid analogue of claim 47 or 48 or pharmaceutical composition of claim 50 for use in aiding diagnosing, imaging or monitoring of prostate cancer.
57. Use of an anthranilyl hydroxamic acid analogue of claim 47 or 48 or pharmaceutical composition of claim 50 in the manufacture of a medicament for aiding diagnosing, imaging or monitoring of prostate cancer.
58. A method of aiding diagnosing, imaging or monitoring of prostate cancer, wherein the patient is administered an anthranilyl hydroxamic acid analogue of claim 47 or 48 or pharmaceutical composition of claim 50.
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