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WO2008132464A2 - Procédés - Google Patents

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Publication number
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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WO
WIPO (PCT)
Prior art keywords
module
mek5
erk5
mef
neurodegenerative disease
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PCT/GB2008/001469
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English (en)
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WO2008132464A3 (fr
Inventor
John R. Mayer
Weng Choon Chan
Zahra Nooshin Rezvani
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The University Of Nottingham
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Publication of WO2008132464A2 publication Critical patent/WO2008132464A2/fr
Publication of WO2008132464A3 publication Critical patent/WO2008132464A3/fr

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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

L'invention concerne un procédé de diagnostic, de surveillance ou d'imagerie d'une maladie neurodégénérative dans un individu, le procédé comprenant la mesure des taux d'un ou plusieurs composants du module MEK5/ERK5/MEF, ou d'un activateur du module MEK5/ERK5/MEF, ou le taux d'un ou plusieurs composants du module MEK3/p38 MAPK, dans un échantillon de fluide corporel ou de tissu provenant de l'individu. L'invention concerne également un procédé de détection de composés utiles pour le diagnostic, la surveillance, l'imagerie ou le traitement d'une maladie neurodégénérative, le procédé comprenant la mise en contact d'un composé à tester avec un élément du module MEK5/ERK5/MEF et le dosage pour une augmentation de l'activité du module MEK5/ERK5/MEF. L'invention concerne en outre l'utilisation d'un composant du module MEK5/ERK5/MEF ou d'un activateur du module MEK5/ERK5/MEF ou du module MEK3/p38 MAPK dans un procédé de détection de composés utiles pour le diagnostic, la surveillance, l'imagerie ou le traitement d'une maladie neurodégénérative.
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WO2006010514A2 (fr) * 2004-07-29 2006-02-02 Bayer Healthcare Ag Diagnostic et traitement therapeutique des maladies associees a la proteine kinase kinase 5 activee par les mitogenes (map2k5) a double specificite

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US10961578B2 (en) 2010-07-23 2021-03-30 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
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