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WO2003025205A2 - Kinase regulee extracellulaire 2 (erk2) - Google Patents

Kinase regulee extracellulaire 2 (erk2) Download PDF

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Publication number
WO2003025205A2
WO2003025205A2 PCT/EP2002/010489 EP0210489W WO03025205A2 WO 2003025205 A2 WO2003025205 A2 WO 2003025205A2 EP 0210489 W EP0210489 W EP 0210489W WO 03025205 A2 WO03025205 A2 WO 03025205A2
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Prior art keywords
erk1
raf
mek1
protein
compound
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WO2003025205A3 (fr
Inventor
Barbara NAVÉ
Volker RÖNICKE
Stéphane LECLAIR
Martin Funk
Birgit Reuner
Kay Brinkmann
Thomas Henkel
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Medigene AG
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Medigene AG
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Priority to AU2002338732A priority Critical patent/AU2002338732A1/en
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Publication of WO2003025205A3 publication Critical patent/WO2003025205A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the present invention relates to a method of identifying and/or obtaining a compound or a plurality of compounds useful in the prevention or treatment of cardiac diseases which comprises the quantification of the activity of Raf-1 , MEK1/2 and/or Erkl/2 depending on said compound.
  • said compound is an inhibitor or antagonist of the signal cascade which is recruited into the sarcomere and leads to activation of Raf-1 , MEK1/2 and/or Erk1/2, since said activation results in translocation of Erk2 into sarcomeres and induction of sarcomeric remodeling.
  • the present invention relates to pharmaceutical compositions for prevention or treatment of cardiac diseases, in particular congestive heart failure (CHF) comprising the identified and/or obtained compound or a plurality of compounds as an active ingredient.
  • CHF congestive heart failure
  • the systemic alterations in heart insufficiency or congestive heart failure are reflected for instance in a dramatically reduced " exercise tolerance and pulmonary edema.
  • the macroscopic manifestation of the disease progression is the initial thickening of the left ventricular myocardium, which is followed by a severe dilatation of the thin walled left ventricular cavity. These alterations are reflected on the cellular level in distinct forms of cardiomyocyte hypertrophy.
  • the early phase (adaptive) is characterized by thick cardiomyocytes with a parallel organization of sarcomeres while the later, decompensated stage shows elongated cells with a serial sarcomere organization (Gerdes et al. 1995, J Mol Cell Cardiol Mar; 27(3): 849-56). Both stages are thought to be regulated by extracellular stimuli, which are transmitted by intracellular signaling cascades, causing sarcomeric remodeling (for an overview see: Hunter and Chien 1999, The New England Journal of Medicine: 341 (17), 1276-
  • MAP-kinase mitogen activated or microtubuli associated protein kinase
  • They generally consist of three levels of kinases: the MAP-kinases are phosphorylated by upstream MAP- kinase kinases, which are phosphorylated and activated by MAP-kinase kinase kinases. Roughly they can be divided in at least three groups, which are the extracellular regulated kinases (Erk1-5), the jun N-terminal kinases (JNK1-3) and the p38 kinases (p38 ⁇ - ⁇ ).
  • the activity and target specificity of the individual kinases is regulated by phosphorylation on specific serine/threonine and tyrosine residues and by sequestration or subcompartimentalisation via specific scaffolding proteins (for review see: Garrington and Johnson 1999, Current Opinion in Cell Biology: 11 , 211- 2).
  • MLCK myosin light chain kinase
  • Congestive heart failure is a life threatening disease.
  • the prevalence and incidence in the US are 4.7 million and 550,000 cases respectively.
  • An inevitable prerequisite for the development of new therapeutic principles in this indication is the identification and validation of new therapeutic targets. This can only be based on the observation of pathological relevant action of disease associated gene products.
  • This invention describes for the first time the identification of a signaling cascade, which is not only activated due to hypertrophic signals leading to serial sarcomere organization, but which is completely recruited into the sarcomere.
  • This selective recruitment of signaling components could be a target for therapeutic intervention.
  • Substances inhibiting the sarcomere association of signaling molecules could avoid the localized activation of Erk2 and sarcomeric remodeling.
  • the direct phosphorylation of MLCK by Erk2 could be a target for drug screening.
  • An inhibitor of Erk2 could be of high therapeutic value for cardiac diseases.
  • the technical problem underlying the present invention is to provide a method of identifying and/or obtaining a compound or a population of compounds useful in the prevention and or treatment of said heart diseases.
  • the present invention relates to a method of identifying and/or obtaining a compound useful in the prevention or treatment of cardiac diseases comprising: (i) contacting a host cell which expresses Raf-1 , MEK1/2 and/or Erk1/2 with a compound suspected to be an inhibitor or antagonist of Raf-1 , MEK1/2 and/or Erk1/2 under conditions allowing interaction of said compound with members of the signal-cascade comprising Raf-1 , MEK112 and/or Erk112;
  • Ras-1 indicates a kinase which is known by a skilled artesian to form a part of the Mitogen-Activated Protein (MAP) kinase signal pathway.
  • Said kinase is a proto-oncogene, identified as a serine/threonine-protein kinase and described to be activated by an other member of said signal-cascade which is designated Ras.
  • MEK in accordance with the present invention is used for MAP kinase kinase which represents a group of kinases which are known by a person skilled in the art to form a part of the Mitogen-Activated Protein (MAP) kinase signal pathway.
  • MEK1/2 relates to the kinases MEK1 and MEK2.
  • ⁇ rk in accordance with the present invention means extracellular regulated kinase.
  • the skilled artesian knows Erks as group of kinases consisting of at least 6 kinases which are designated in numerical order Erk1 to Erk6. Consequently, the term “Erk1/2” relates to the kinases Erk1 and Erk2.
  • the determination of the activity of the kinases as referred in the above described embodiment of the invention is described in the appended examples, e.g. for Erk1/2 activity in Example 9. Said activity may also be determined by an assay which measures the transfer of phosphate-groups from ATP to an other substrate. Also the conversion of ATP to ADP may be measured in a corresponding assay.
  • various methods known to . the skilled artesian exist. They are generally based on the fact that a known (purified) substrate of the kinase is incubated with the respective kinase under conditions suitable for the phosphorylation reaction and the phosphorylation of the substrate is measured afterwards.
  • the phosphorylation of the substrate can be determined for example by the use of radiolabeled [ ⁇ 32 P]ATP, that leads to a labeled substrate or by the use of phosphorylation-specific antibodies, that only detect the phosphorylated substrate.
  • radiolabeled [ ⁇ 32 P]ATP that leads to a labeled substrate
  • phosphorylation-specific antibodies that only detect the phosphorylated substrate.
  • Examples for such kinase assays can be found in Zimmerman, S and Moelling K (1999) Science 286, 1742-1744, Lazou, A et al. (1994) Circulation Research 75, 932-940 or Klemke, RL et al. (1997) J Cell Biol. 137, 481-492.
  • the applicant has surprisingly identified, that the whole Raf-MEK-1-Erk2 signalling chain sequestered into the sarcomere of cardiomyocytes, as described supra.
  • Erk2 as the relevant kinase for the mediation of the induction of cardiac hypertrophy has been demonstrated.
  • Said Erk2 was shown to interact with (and/or to phosphorylate) telethonin (T-Cap) (see appended example 14). Consequently, the inventive specific inhibition of the disclosed signal-cascade by an inhibitor or antagonist of the invention results in prevention and or reduction of hypertrophy.
  • the present invention relates to said method, wherein said compound useful in the prevention or treatment of human hypertrophy is an inhibitor or antagonist of the signal cascade which is recruited into the sarcomere and leads to activation of Raf-1 , MEK1/2 and/or Erk1/2, wherein said activation results in translocation of Erk2 into sarcomeres and induction of sarcomeric remodeling.
  • Said translocation of Erk2 may be determined by the method as desc ⁇ bed in the appended example 10. Similar methods can be found in Klemke, RL et al. (1997) J Cell Biol. 137, 481-492.
  • the inhibitors as mentioned in said embodiment are inhibitors or antagonists of Raf- 1 , MEK1/2 and/or Erk1/2 and/or its activity which preferably are compounds that interacts and/or interferes with Raf-1 , MEK1/2 and/or Erk1/2.
  • Said interaction may be a direct interaction, like a direct protein/protein or protein/nucleic acid molecule interaction, but it is also envisaged that said interaction may be mediated by further, additional compounds.
  • inhibitors or antagonists to be identified, screened for and/or obtained with the method of the present invention include molecules, preferably small molecules which bind to, interfere with and/or occupy relevant sites on Raf-1 , MEK1/2 and/or Erk1/2. It is furthermore envisaged that such inhibitors interfere with the synthesis/production of functional Raf-1 , MEK1/2 and/or Erk1/2, like, e.g. anti-sense constructs and the like inhibitors and /or antagonist which can be screened for and obtained in accordance with the method of the present invention include, inter alia, peptides, proteins, nucleic acids including cDNA expression libraries, antibodies, small organic compounds, small molecules ligands, PNAs and the like.
  • the inhibitor and/or antagonist of Raf-1 , MEK1/2 and/or Erk1/2 may comprises (an) antibody(ies).
  • Said antibody(ies) may comprise monoclonal antibodies as well as polyclonal antibodies.
  • chimeric antibodies, synthetic antibodies as well as antibody fragments (like Fab, F(ab)2, Fv, scFv), or a chemically modified derivative of antibodies are envisaged. It is envisaged that said antibodies bind to Raf-1 , MEK1/2 and/or Erk1/2 and/or interferes with the activity of Raf-1 , MEK1/2 and/or Erk1/2.
  • oligonucleotides and/or aptamers which specifically bind to Raf-1 , MEK1/2 and/or Erk1/2 as defined herein or which interfere with the Raf-1 , MEK1/2 and/or Erk1/2 activity are envisaged as inhibitors and/or antagonists of Raf-1 , MEK1/2 and/or Erk1/2.
  • the term "oligonucleotide” as used in accordance with the present invention comprises coding and non-coding sequences, it comprises DNA and RNA and/or comprises also any feasible derivative.
  • oligonucleotide further comprises peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkages (Nielson, Science 274 (1991), 1497-1500).
  • Oligonucleotides which may inhibit and/or antagonize Raf-1 , MEK1/2 and/or Erk1/2 and/or Raf-1 , MEK1/2 and/or Erk1/2 activity and which can be identified and/or obtained by the method of the present invention can be, inter alia, easily chemically synthesized using synthesizers which are well known in the art and are commercially available like, e.g., the ABI 394 DNA-RNA Synthesizers.
  • aptamer means nucleic acid molecules that can bind to target molecules, preferably to Raf-1 , MEK1/2 and/or Erk1/2 as defined herein.
  • Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules.
  • the preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, Ann. Rev. Biochem. 64>(1995), 763-797).
  • said inhibitor and/or antagonist of Raf-1 , MEK1/2 and/or Erk1/2 may also comprise "small molecules". This term relates, but is not limited to small peptides, inorganic and/or organic substances or peptide-like molecules, like peptide-analogs comprising D-amino acids.
  • peptidomimetics and/or computer aided design of appropriate antagonists or inhibitors may be employed in order to obtain candidate compounds to be tested in the inventive method.
  • Appropriate computer systems for the computer aided design of, e.g., proteins and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991.
  • the results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known compounds, substances or molecules.
  • Appropriate compounds can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
  • the three-dimensional and/or crystallographic structure of inhibitors or activators of Raf-1 , MEK1/2 and/or Erk1/2 or of the nucleic acid molecule encoding for Raf-1 , MEK1/2 and/or Erk1/2 can be used for the design of peptidomimetic inhibitors or antagonists Raf-1 , MEK1/2 and/or Erk1/2 to be tested in the method of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
  • the compounds to be screened with the method(s) of the present invention do not only comprise single, isolated compounds. It is also envisaged that mixtures of compounds are screened with the method of the present invention. It is also possible to employ extracts, like, inter alia, cellular extracts from prokaryotic or eukaryotic cells or organisms.
  • Host ceils to be employed in the present invention comprise, preferably cultured cells, even more preferably cardiomyocytes, most preferably primary cardiomyocytes or in vitro differentiated embryonic stem cells.
  • interaction of said compound with Raf- 1 , MEK1/2 and/or Erk1/2 does, not only comprise a direct interaction of said compound with Raf-1 , MEK1/2 and/or Erk1/2 but also comprises interaction(s) mediated by further compounds/molecules, for example in signaling pathways or in complex formations.
  • the term "quantitatively relating" as employed herein above means that the activity of Raf-1 , MEK1/2 and/or Erk1/2 which is determined in steps (ii) and (iii) of the inventive method are compared. It is, inter alia, envisaged that the activity(ies) obtained when the compound to be identified and/or obtained by the inventive method is compared to the activity(ies) of a control which comprises the same experimental setting but wherein said compound is omitted. Said “quantitative relation” may be carried out be methods known to the skilled artisan and described herein and in the appended examples, e.g. morphometric analysis, comparisons of protein patterns, phosphorylation status, etc. Furthermore, it is envisaged that said comparisons and/or quantitative relation is carried out in a computer-assisted fashion. Said comparison/quantitative relation may also comprise the analysis in high-throughput screens.
  • the inhibitors or antagonists as identified and/or obtained by the method of the present invention are particularly useful in the therapeutic management, prevention and or treatment of cardiac diseases.
  • cardiac diseases encompasses all diseases of the heart and/or heart muscle, especially those that are related to hypertrophy, especially to a maladaptive or eccentric hypertrophy.
  • Said diseases are for example cardiac hypertrophy, heart insufficiency, congestive heart failure (CHF), dilated cardiomyopathy (idiopathic), hypertrophic cardiomyopathy, restrictive cardiomyopathy, obstructive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, unclassified cardiomyopathy, ischemic cardiomyopathy, valvular cardiomyopathy, hypertensive cardiomyopathy, inflammatory cardiomyopathy, metabolic cardiomyopathy, infiitrative cardiomyopathy, muscular dystrophy-associated cardiomyopathy, neuromuscular disorder-associated cardiomyopathy, toxic cardiomyopathy or peripartum cardiomyopathy.
  • cardiovascular diseases may lead to a cardiac disease related to hypertrophy the term cardiac diseases also includes cardiovascular diseases such as coronary diseases, especially coronary artery diseases, stable angina, angina pectoris or hypertension.
  • the activity of said Raf-1 , MEK1/2 and/or Erk1/2 comprises activation of an intracellular signalling pathway.
  • the activity of said Raf-1 , MEK1/2 and/or Erk1/2 comprises increasing the activity and/or recruitment of a cellular protein into the sarcomere which is indicative for hypertrophy.
  • the host ceil expresses a marker gene indicative for hypertrophy and that, more preferred, the cellular protein is encoded by said marker gene.
  • Said marker gene may be selected from the group consisting of ⁇ -MHC, DCMAG-1 , PKB and PKC ⁇ .
  • marker/reportergenes be employed which are selected form the group consisting of GFP, YFP, BFP, Firefly luciferase, Renilla luciferase, LacZ, SEAP and hGH. These markergenes/reportergenes may be fused/linked to further proteins/peptides.
  • inventive methods may be employed, inter alia, the expression of said cellular protein and/or reporter gene by (recombinant) viral systems, as well as the measurement of said reporter gene-product in living cells, cell-supematants and/or cellular extracts.
  • reporter/reportergenes for signaling pathways are well known in the art, as inter alia documented in Shepherd (1998), Biochem J. 333, 471 -490 or in Toker (2000). Mol. Pharmacol. 57, 652-658, describing specific signalling cascades. It is envisaged that these reporter genes are, inter alia, transfected into the host cells. Furthermore, said reportergenes may be comprised in the genome of said host cells and may be incorporated in said genome by methods known in the art, like, e.g. knock-ins, as desc ⁇ bed herein below.
  • the present invention also relates to a method of identifying and/or obtaining a compound useful in the prevention or treatment of cardiac diseases which is suspected to be an inhibitor or antagonist of an intracellular signalling pathway comprising
  • corresponding activities may be measured by methods known in the art and comprise but are not limited to kinase assays, phosphorylation/dephosphorylation assays, multimerization/dimerization assays, nuclear translocation assays, reporter- gene assays with specific cis-acting elements, assays measuring the production of "second messengers", like, inter alia, AMP, cGMP or Ca 2+ .
  • the above mentioned intracellular signaling pathway comprises telethonin (T-Cap), -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2, MLCK, MNK1 , MNK2, osciilin, ribosome protein L11 , EST THC 296820, EST 262302, Erk1 dn, p38c dn, p38b dn, S6KII 2A, MEK2, antigen NY, 28S protein, EST 16.1.42, prcc, PRS6KA4, MAD2, NKX 2.5 and/or PTGR and/or that said activity, for example binding activity, of an intracellular signalling pathway is telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2, MLCK, MNK
  • novel compounds be identified and/or be obtained which are of high relevance in the treatment as well as in the prevention of heart insufficiency(ies).
  • the methods for identifying as well as validating such compounds as desc ⁇ bed herein may also be combined. It is, e.g., envisaged that in said activity assays/methods hypertrophic markermolecules are measured upon intracellular expression of Raf-1 , MEK1/2 and/or Erk1/2.
  • T-Cap telethonin
  • MHC actin
  • titin myomesin
  • nebulette tropomyosin
  • troponin troponin
  • Erk1/2 Erk1/2 and/or MLCK
  • the compound to be tested may lead to a modified activities of Raf-1 , MEK1/2 and/or Erk1/2, wherein, for example a reduced activation of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK or an reduced expression of hypertrophic markers disclosed herein are indicative for an inhibitor or antagonist of Raf-1 , MEK1/2 and/or Erk1/2 or their activity.
  • T-Cap telethonin
  • the present invention provides for a method of identifying and/or obtaining a compound useful in the prevention or treatment of cardiac diseases which is suspected to be an inhibitor or antagonist of sarcomeric remodeling and/or formation of hypertrophy comprising:
  • marker proteins for hypertrophy are telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and MLCK.
  • Said hypertrophy may be determined by microscopical and/or morphometric means. As mentioned herein above, such measuring methods are known in the art and comprise biochemical, morphometric as well as morphological (microscopical) analysis and such methods are exemplified in the appended examples.
  • said Raf-1 , MEK1/2 and/or Erk1/2 is selected from the group consisting of
  • Raf-1 , MEK1/2 and/or Erk1/2 means in context of this invention that Raf-1 , MEK1/2 and/or Erk1/2 described herein comprises at least one, preferably at least two, more preferably more than two of the functional activities of Raf-1 , MEK1/2 and/or Erk1/2.
  • These activities comprise activities as known in the art, like ATP-binding and kinase activity, but, preferably, these activities comprise the capability of Raf-1 , MEK1/2 and/or Erk1/2 to the formation of an hypertrophic phenotype and/or the activation of signaling pathway, in particular the activation of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK.
  • T-Cap telethonin
  • ⁇ -actinin ⁇ -actinin
  • MHC actin
  • actin actin
  • titin myomesin
  • nebulette tropomyosin
  • troponin troponin
  • Erk1/2 Erk1/2
  • Said hypertrophy caused by the above mentioned hormones could be induced by activating the hypertrophic function of Raf-1 , MEK1/2 and/or Erk1/2, at
  • Raf-1 , MEK1/2 and/or Erk1/2 would lead to a decreased or even completely inhibited progress of the cytokine dependent hypertrophy.
  • functional fragment of Raf-1 , MEK1/2 and/or Erk1/2 relates to the activities of Raf-1 , MEK1/2 and/or Erk1/2 as described herein. Methods for detection and/or verification of the said function of said domains are disclosed herein and in the appended examples.
  • said Raf-1 , MEK1/2 and/or Erk1/2 further comprise a tag.
  • Said tag may be selected from the group consisting of: His-tag, Flag-tag, Myc-tag, HA-tag, GST-tag, T100TM, VSV-G, V5, S-tagTM, HSV, CFP, RFP, YFP, GFP, Cellulose binding domain (CBD), Maltose binding protein (MBP), NusA-tag, thioredoxin (Trx), DsbA, DsbC and a biotinylation sequence.
  • further tags known be the person skilled in the art are envisaged in context of this invention.
  • the host cell employed in the inventive method described herein comprises a recombinant DNA molecule which comprises a DNA encoding a protein as defined herein above, e.g. Raf-1 , MEK1/2 and/or Erk1/2 as described herein with or without tag and/or any further modification.
  • a recombinant DNA molecule which comprises a DNA encoding a protein as defined herein above, e.g. Raf-1 , MEK1/2 and/or Erk1/2 as described herein with or without tag and/or any further modification.
  • said recombinant DNA molecule is, preferably, a viral vector.
  • said recombinant DNA-molecule may be a non-viral vector, a recombinant adeno-viral DNA, a (recombinant) Herpes simplex- viral DNA, a (recombinant) adeno-associated-viral (AAV) DNA, BACs, PACs, or YACs.
  • the host cell employed in the inventive method may be stimulated by biochemical, chemical or physiological means.
  • said host cell is stimulated by hormones, hormone analoga, phorbolester and/or cytokines or combinations thereof.
  • hormones are selected from a group consisting of ET-1.
  • ET-2, ET-3, angiotensin I, angiotensin II and noradrenalin comprising derivatives thereof as insulin, IGF and myotrophin, hormone analoga are selected from a group consisting of phorbolester, isoproterenol (ISO) and phenylephrin (PE) and/or cytokines are selected from a group consisting of leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), interleukin-6 (IL-6), interleukine-11 (IL-1 1 ), oncostatin M, ciliary neurotrophic factor. Said stimulation may lead to a hypertrophy.
  • ISO isoproterenol
  • PE phenylephrin
  • cytokines are selected from a group consisting of leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), interleukin-6 (IL-6), interleukine-11 (IL-1 1 ), oncostatin M, ciliary neurotrophic factor.
  • said host cells are stimulated by a combination of the hereinabove mentioned stimuli. More preferably, said combination is a combination of LIF/ISO/ET-1 or LIF/PE/ET-1.
  • the host cell employed in the present invention is preferably a mammalian host. Most preferred is a host cell which is derived from primary cardiomyocytes (pCMs) or in vitro differentiated embryonic stem cells. Methods to isolate and/or differentiate said cells are known by a person skilled in the art. However, further host cells may be employed in the inventive method. These cells comprise, but are not limited to, HEK-, Hela-, 3T3-, L-, Jurkat-, COS-, BHK- or CHO- cells. Said cells are cells derived from striated muscle or heart-muscle.
  • pCMs primary cardiomyocytes
  • pCMs primary cardiomyocytes
  • Methods to isolate and/or differentiate said cells are known by a person skilled in the art. However, further host cells may be employed in the inventive method. These cells comprise, but are not limited to, HEK-, Hela-, 3T3-, L-, Jurkat-, COS-, BHK- or CHO- cells. Said cells are
  • tissue- or cell culture systems be employed, but also in vivo test/assay systems are within the scope of the present invention.
  • In vivo and in vitro systems are well known in the art and comprise, inter alia, the production and use of non-human transgenic animals expressing Raf-1 , MEK1/2 and/or Erk1/2 as defined herein (either mutated or non- mutated), as well as corresponding tissue- and/or cell culture systems.
  • Raf-1 , MEK1/2 and/or Erk1/2 as defined be employed in in vitro systems which also comprise high-throughput screens.
  • the inventive method also comprises the use on non- human transgenic animals and cells, organs or tissues thereof. It is, inter alia, envisaged, that non-human transgenic animals be generated which overexpress Raf- 1 , MEK1/2 and/or Erk1/2, preferably human Raf-1 , MEK1/2 and/or Erk1/2.
  • This Raf- 1 , MEK1/2 and/or Erk1/2 may comprise the species-specific Raf-1 , MEK1/2 and/or Erk1/2 of said transgenic animal, but it is also envisaged that said animal expresses (overexpress) Raf-1 , MEK1/2 and/or Erk1/2 from another species, e.g. human Raf-1 , MEK1/2 and/or Erk1/2.
  • the inhibitor/antagonist of Raf-1 , MEK1/2 and/or Erk1/2 and/or its activities can than be tested, screened in said non-transgenic animals or the inventive method can be employed in isolated cells or organs from these transgenic animals.
  • Preferred non-human transgenic animals are mice, rats, sheep, cows. Yet, other transgenic animals comprising muscle cells, in particular heart muscle cells or equivalents, are envisaged.
  • the present invention also provides for a method for identifying a protein or a plurality of proteins in heart tissue whose activity is modulated by telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK comprising the steps of (i) providing telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK or a functional fragment thereof; and (ii) identifying a protein or plurality of proteins capable of interacting with telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK.
  • Proteins and or plurality of proteins capable of interacting with telethonin (T-Cap), ⁇ - actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK may be further tested for their ability to function as inhibitors and/or antagonists of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK by the inventive methods described herein above.
  • said interacting protein(s) could act as activator(s) or modulator(s) of telethonin (T-Cap)-, ⁇ -actinin-, MHC-, actin-, titin-, myomesin-, nebulette-, tropomyosin-, troponin-, Erk1/2- and/or MLCK-signaling function. All methods, uses and assays described herein are not only useful for scientific purposes but are in particular useful in drug screening methods and for pharmaceutical/pharmacological tests and assays.
  • T-Cap telethonin
  • MHC actin
  • titin myomesin
  • nebulette tropomyosin
  • troponin Erk1/2 and/or MLCK
  • vectors which encode for telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK as described herein.
  • T-Cap telethonin
  • ⁇ -actinin MHC
  • actin titin
  • myomesin nebulette
  • tropomyosin troponin
  • Erk1/2 and/or MLCK MLCK
  • vector systems are known in the art and described herein below.
  • viral system are employed, like retroviral or adenoviral systems.
  • these are recombinant viral systems, as, inter alia, for adenoviral systems described in He (1998) PNAS 95, 2509-2514.
  • Said “knock-in” assays may comprise “knock-in” in tissue culture cells, as well as in (transgenic) animals. Examples for successful “knock-ins” are known in the art (see, inter alia, Tanaka, J. Neurobiol. 41 (1999), 524-539 or Monroe, Immunity 11 (1999), 201-212).
  • biochemical assays may be employed which comprise, but are not limited to, binding of the telethonin (T-Cap), Erk1/2 and/or MEK1/2 (or (a) functional fragment(s) thereof) to other molecules/(poly)peptides and assaying said interactions by, inter alia, scintillation proximity assay (SPA) or homogenous time- resolved fluorescence assay (HTRFA).
  • FRET-assays fluorescence resonance energy transfer; as described, inter alia, in Ng, Science 283 (1999), 2085-2089 or Ubarretxena-Belandia, Biochem. 38 (1999), 7398-7405), TR-FRETs and biochemical assays as disclosed herein.
  • commercial assays like "Amplified Luminescent Proximity Homogenous AssayTM" (BioSignal Packard) may be employed. Further methods are well known in the art and, inter alia, described in Fernandez, Curr. Opin. Chem. Biol. 2 (1998), 547- 603.
  • test for interaction may also be carried out by specific immunological and/or biochemical assays which are well known in the art and which comprise, e.g., homogenous and heterogeneous assays as described herein below.
  • Said interaction assays employing read-out systems are well known in the art and comprise, inter alia, two hybrid screenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911 ; and as exemplified in the appended examples), GST- pull-down columns, co-precipitation assays from cell extracts as described, inter alia, in Kasus-Jacobi, Oncogene 19 (2000), 2052-2059, "interaction-trap" systems (as described, inter alia, in US 6,004,746) expression cloning (e.g.
  • Homogeneous (interaction) assays comprise assays wherein the binding partners remain in solution and comprise assays, like agglutination assays.
  • Heterogeneous assays comprise assays like, inter alia, immuno assays, for example, ELISAs, RiAs, IRMAs, FIAs, CLIAs or ECLs.
  • the compound identified and/or obtained by the method of the invention is further refined by peptidomimetics in an additional step according to a preferred embodiment of said method.
  • Appropriate compounds can also be identified as well as refined by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
  • the method further comprises the step of modifying the compound identified or refined by the inventive method as a lead compound to achieve, modified site of action, spectrum of activity, organ specificity, and/or improved potency, and/or decreased toxicity (improved therapeutic index), and/or decreased side effects, and/or modified onset of therapeutic action, duration of effect, and/or modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or modified physico- chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or improved general specificity, organ/tissue specificity, and/or optimized application form and route may be modified by esterification of carboxyl groups, or esterification of hydroxyl groups with carbon acids, or esterification of hydroxyl groups to, e.g.
  • phosphates, pyrophosphates or sulfates or hemi succinates or formation of pharmaceutically acceptable salts, or formation of pharmaceutically acceptable complexes, or synthesis of pharmacologically active polymers, or introduction of hydrophilic moieties, or introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or modification by introduction of isosteric or bioisosteric moieties, or synthesis of homologous compounds, or introduction of branched side chains, or conversion of alkyl substituents to cyclic analogues, or derivatisation of hydroxyl group to ketales, acetales, or N-acetylation to amides, phenylcarbamates, or synthesis of Mannich bases, imines, or transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxa ⁇ olidines, thiozolidines or combinations thereof.
  • the present invention also provides a method for the production of a pharmaceutical composition comprising the steps of the method the invention as described herein above and the further step of formulating the compound identified, obtained, refined and/or modified in a pharmaceutical acceptable carrier or diluent. Therefore, the present invention also relates to a pharmaceutical composition comprising a compound which is obtainable by the method described herein.
  • a pharmaceutical composition of the present invention comprises an inhibitor and/or antagonist of Raf-1 , MEK1/2 and/or Erk1/2 and/or an inhibitor and/or antagonist of the Raf-1 , MEK1/2 and/or Erk1/2 activity as described herein said inhibitor/antagonist may inhibit the formation of hypertrophy and/or inhibit/influence the signaling pathways as described herein. Yet, it is furthermore envisaged that, optionally, said inhibitor/antagonists modifies/interferes with/inhibits the binding capacity of Raf-1 , MEK1/2 and/or Erk1/2 to their corresponding substrates.
  • the present invention provides for the use of a compound which is obtainable by the method as described herein for the preparation of a pharmaceutical composition for prevention or treatment of cardiac diseases.
  • the invention relates to the use of a compound which is obtainable by the inventive method described herein for the preparation of a pharmaceutical composition for treating a disease related to impaired or increased activation of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK.
  • T-Cap telethonin
  • MHC actin
  • titin myomesin
  • nebulette tropomyosin
  • troponin Erk1/2
  • Erk1/2 Erk1/2
  • MLCK MLCK
  • the inhibitor/antagonist Raf-1 , MEK1/2 and/or Erk1/2 as identified and/or obtained by the inventive method also inhibits activation of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK in cardiomyoctes.
  • T-Cap telethonin
  • ⁇ -actinin ⁇ -actinin
  • MHC actin
  • actin actin
  • titin myomesin
  • nebulette tropomyosin
  • troponin troponin
  • Erk1/2 Erk1/2
  • MLCK cardiomyoctes
  • a protein as defined herein above a functional fragment thereof be used for the preparation of a diagnostic composition for diagnosing a disease or a predisposition for a disease related to hypertrophy.
  • a nucleic acid molecule encoding said protein be employed in diagnostic methods. It is particularly preferred that said proteins and/or nucleic acid molecules be employed for the preparation of a diagnostic composition for diagnosing a disease or a predisposition for a disease related to impaired or increased activation of telethonin (T-Cap), ⁇ -actinin, MHC, actin, titin, myomesin, nebulette, tropomyosin, troponin, Erk1/2 and/or MLCK.
  • Said diagnostic composition may be employed, inter alia, for measuring serum markers which are directly or indirectly related to the level of activation of Raf-1 , MEK1/2 and/or Erk1/2 as defined herein.
  • the present invention relates to the use of (an) antibody(ies) which specifically recognize(s) the activated/phosphorylated form of a polypeptide for the preparation of a diagnostic composition.
  • Said diagnostic composition is envisaged for diagnosing a disease or a predisposition for a disease related to hypertrophy.
  • Said polypeptide which is specifically recognized by said antibody(ies) is encoded by a polynucleotide comprising a polynucleotide selected from the group consisting of:
  • polynucleotide which comprises a fragment encoding at least a specific functional domain of the polypeptide encoded by the polynucleotide of (a) or (b);
  • a polynucleotide which is more than 64.99% identical to a nucleic acid sequence as shown in SEQ ID NO: 1 , 3, 5, 7or 9 or encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8 or 10;
  • a polynucleotide which hybridizes with the polynucleotide of any one of (a) to (e), wherein said polynucleotide encodes a polypeptide having the biological function of a polypeptide encoded by the polynucleotide of any one of (a) to (e).
  • the general methodology for producing antibodies is well-known and has, for monoclonal antibodies, been described in, for example, Kohler and Milstein, loc.cit and reviewed in J.G.R. Hurrel, loc.cit.).
  • antibody relates to monoclonal or polyclonal antibodies. Polyclonal antibodies (antiserum) can be obtained according to conventional protocols.
  • Antibody fragments or derivatives have been described herein above and comprise F(ab') 2 , Fab, Fv or scFv fragments; see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, NY.
  • the antibody of the invention is a monoclonal antibody.
  • the derivatives of the invention can be produced by peptidomimetics.
  • Aptamers are well known in the art and, inter alia, described in Famulok, Curr. Op. Chem. Biol. 2 (1998), 320-327.
  • the preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, loc.cit)
  • Said other receptors may, for example, be derived from said antibody etc. by peptidomimetics.
  • the specificity of the recognition implies that other known proteins, molecules are not bound.
  • a suitable host for assessing the specificity would imply contacting the above recited compound comprising an epitope of the nucleic acid molecule or the (poly)peptide of the invention as well as corresponding compounds e.g. from protein or nucleic acid molecules known in the art, for example in an ELISA format and identifying those antibodies etc. that only bind to the compound of the invention but do not or to no significant extent cross-react with said corresponding compounds.
  • An other preferred embodiment of the present invention relates to the use of an aforementioned protein for the preparation of a diagnostic composition for diagnosing a disease or a predisposition for a disease related to impaired or increased activation of Raf-1 , MEK1/2 and/or Erk1/2.
  • the aforementioned disease is congestive heart failure (CHF).
  • CHF congestive heart failure
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned polynucleotide(s), polypeptide(s) and/or an antibody(ies).
  • a diagnostic composition of the invention comprises the aforementioned polynucleotide(s), polypeptide(s) and/or an antibody(ies).
  • Figure 1 shows the hybridization pattern of Erk-2 on HG-U95A oligo arrays (Affymetrix) in different tissues. Individual relative signal intensities of Erk-2 as well as ratios between tissues are given.
  • FIG. 2 shows the analysis of stimulated and CFP expressing cardiomyocytes. Cardiomyocytes were stimulated, infected with a recombinant adenovirus (AV 151 , Flag-CFP) and analysed 48 hrs later.
  • PE Phenylephrine (100 ⁇ M); LIF: Leukemia inhibitory factor (1 ng/ml); ET-1 : Endothelin-1 (10 nM); ISO: Isoprenaline (10 ⁇ M).
  • Figure 3a depicts a representative western blot of the phosphorylated or activated form of Erkl and Erk2 detected with a phospho-specific antibody from NEB after different time periods of stimulation.
  • Figure 3b shows the same blot as shown in figure 2 a, but bands for Erk1 and Erk2 are detected with an antibody recognizing the whole protein, irrespective of phosphorylation and thus quantitates the total amount of Erk protein.
  • FIG. 4 depicts the subcellular localization of c-raf in hypertrophic cardiomyocytes. Cardiomyocytes were transiently transfected with a HA1 -c-raf-YFP expression construct, PE stimulated and analysed 48 hrs later. PE: Phenylephrine (100 ⁇ M).
  • FIG. 5 shows ⁇ -actinin staining of cardiomyocytes. Cardiomyocytes were stimulated with ET-1/ISO/LIF and analysed 48 hrs later by immunostaining with an anti ⁇ -actinin antibody and a Cy3-conjugated secondary antibody.
  • LIF Leukemia inhibitory factor (1 ng/ml); ET-1 : Endothelin-1 (10 nM); ISO: Isoprenaline (10 ⁇ M).
  • FIG. 6 shows the subcellular localization of ca-c-raf in stimulated cardiomyocytes.
  • Cardiomyocytes were transiently transfected with an expression construct for a constitutive active c-raf mutant (HA1-ca-c-raf-YFP), stimulated and analysed 48 hrs later.
  • PE Phenylephrine (100 ⁇ M);
  • LIF Leukemia inhibitory factor (1 ng/ml); ET-1 : Endotheiin-1 (10 nM); ISO: Isoprenaline (10 ⁇ M).
  • Figure 7a shows a representative image of a cardiomyocyte stimulated and stained as desc ⁇ bed in methods for 48h.
  • Figure 7b shows a picture of cells treated with 50 ⁇ M of the MEK inhibitor PD98059 in addition to the stimuli shown in Figure 6a.
  • FIG 8 depicts the over-expression of a dominant negative MEK-1 in cardiomyocytes.
  • Cardiomyocytes were transiently transfected with an expression construct for a dominant negative MEK-1 mutant (HA1-dn-MEK1-YFP), stimulated with ET-1/ISO/LIF and analysed 48 hrs later.
  • LIF Leukemia inhibitory factor (1 ng/ml); ET-1 : Endothelin-1 (10 nM); ISO: Isoprenaline (10 ⁇ M).
  • Figure 9a shows an image of cells stained with anti-MLCK antibody as described in methods.
  • Figure 9b shows an image of a cell stained with phospho-Erk antibody as described in methods.
  • Figure 10a depicts a representative gei of an MLCK kinase assay. In the left two lanes only Erk was present, in the middle two lanes only MLCK and in the right two lanes both were incubated together in the presence of ⁇ -P33-ATP.
  • Figure 10b shows a western blot of the same samples as depicted in figure 9a.
  • Example 1 Isolation of total RNA from heart tissue
  • Trizol Trizol
  • a rotor-stator homogenizer Ultra-Turrax T8, IKA Labortechnik
  • the sample was incubated for 5 min at room temperature (RT) and mixed with 200 ⁇ l chloroform per 1 ml Trizol.
  • RT room temperature
  • the sample was incubated for 2-3 min at RT and centrifuged at 12000g for 15 min at 4 °C.
  • the aqueous phase was transferred to a fresh 50 ml polypropylene tube and centrifuged again for 15 min.
  • RNA pellet was washed with 1 ml 75% EtOH per 1 ml Trizol and dried at RT for 5-10 min.
  • DEPC-treated water per 1 ml Trizol was added and the sample was incubated at 60 °C for 10 min, final storage was at -80 °C.
  • An aliquot was used for quantification by A 26 o measurement and separation on a formaldehyde agarose gel (Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) to check integrity and size distribution.
  • RNA of adult human patients from the following suppliers was used: skeletal muscle (Invitrogen); liver (Stratagene), kidney (Stratagene), brain (Clontech).
  • skeletal muscle Invitrogen
  • liver Stratagene
  • kidney Stratagene
  • brain Clontech
  • RNA of three human NF 520 ⁇ g
  • five DCM patients 900 ⁇ g was prepared as desc ⁇ bed and mixed.
  • Example 3 Affymetrix array technology 50 ⁇ g total RNA was purified using RNeasy Mini columns (Qiagen) as desc ⁇ bed by the manufacturer. 5 ⁇ g of purified total RNA was used for synthesis of first and second strand cDNA and double stranded cDNA then used to synthesize the biotinylated cRNA probe as recommended by Affymetrix. 15 ⁇ g of fragmented and labeled cRNA was hybridized to the human genome U95A array and stained by streptavidin-phycoerythrin (SAPE). Signal intensities were amplified using a biotinylated anti-streptavidin antibody and a second SAPE staining step. Data were analyzed by means of Microarray Suite and Data Mining Tool software provided by Affymetrix. Fold changes in gene expression were analyzed by comparing average intensity values.
  • SAPE streptavidin-phycoerythrin
  • Figure. 1 shows the hybridization pattern of Erk2 on HG-U95A oligo arrays (Affymetrix) in different tissues.
  • Example 4 Isolation of primary cardiomyocytes from neonatal rats
  • Neonatal rats (P2-P7) were sacrificed by cervical dislocation.
  • the ventricles of the beating hearts were removed and cardiomyocytes were isolated with the "Neonatal Cardiomyocyte Isolation System" (Worthington Biochemicals Corporation, Lakewood, New Jersey) according to the protocol. Briefly, the ventricles were washed twice with ice cold Hank's Balanced Salt Solution without Potassium and Magnesium (CMF- HBBS) and minced with a scalpel to an average volume of one cubic millimeter .
  • the heart tissue was further digested over night with trypsin at 10°C. On the following " morning trypsin inhibitor and collagenase were added.
  • the cells were dispersed by pipetting.
  • the solution was further purified by filtering through a 70 ⁇ m mesh (Cell Strainer) and centrifuged twice for 5 minutes at 60 x g.
  • the cell pellet was resuspended in plating medium and counted.
  • Cells were seeded with a density of 2 x 10 4 /cm 2 on gelatine (Sigma, Deisenhofen) coated dishes. The next morning cells were washed twice with DMEM and maintenance medium was added.
  • Plating medium DMEM/M-199 (4/1 ); 10% Horse serum, 5% Fetal calf serum; 1 mM Sodiumpyruvate; Antibiotics and antimycotics
  • Example 5 Stimulation of isolated cardiomyocytes from neonatal rats
  • pCMs primary cardiomyocytes from neonatal rats
  • Example 6 Generation of recombinant adenoviruses
  • Recombinant adenoviruses were produced according to the simplified system developed by He et al. (1998, Proc. Natl. Acad. Sci. USA. 95: 2509-2514).
  • GFP green- fluorescent-protein
  • the pAdTrack vector was combined with the pAdEasy-1 plasmid by homologous recombination. Briefly, 5 ⁇ g of the pAdTrack plasmid were linearized with the restriction enzyme Pme I and gel-purified. Approximately 100 ng of the linearized vector were combined with 100 ng of the pAdEasy-1 plasmid and aqua bidest.
  • Plasmid DNA was prepared by the Tip-500 column (Qiagen, Hilden, Germany) according to the manufacturers instructions. Then 10 ⁇ g DNA were digested with Pac I, ethanol precipitated and resuspended in 40 ⁇ l H 2 0 (cell culture grade).
  • the packaging was performed in HEK 293 cells (ATCC: CRL-1573 ) by lipofectjon.
  • the day before transfection cells were seeded into two T-25 flask (2 x 10 6 cells each) in DMEM (10% fetal calf serum).
  • DMEM fetal calf serum
  • For each flask 20 ⁇ l of the Pac I digested adenovirus genome was mixed with 20 ⁇ l of Lipofectamine (GIBCO BRL) in 500 ⁇ l OptiMem I medium and incubated for 15 minutes at room temperature. Meanwhile cells were washed twice with 4 ml serum-free DMEM. Then 2.5 ml OptiMem I was added to each flask followed by the DNA Lipofectamine solution.
  • DMEM fetal calf serum
  • the first amplification was performed in one T-25 flask with HEK 293 cells at a density of 70 to 80%. Cells were harvested and lysed as described above. The virus titer was determined after two to four rounds of further amplification in T-75 flasks.
  • the titer of infectious particles was determined by end-point-dilution with HEK 293 cells based on the TCID 50 method (Mahy and Kangro, Virology Methods Manual, Academic Press, p37).
  • adenoviruses for the monocistronic expression of recombinant proteins were generated based on the pShuttle vector.
  • the pShuttle plasmid was cut with Sal I and Kpn I, gel purified, blunted with T4-polymerase and reiigated to eliminate the multiple cloning site.
  • the vector was digested with EcoR I, blunted with T4-polymerase and reiigated to get rid of the single EcoR I site in the vector backbone.
  • the vector was linearized with Bgl II and desphosphorylated.
  • a whole expression cassette for a Flag-CFP (cyano variant of GFP) fusion protein derived from a modified pCI vector was inserted into the Bgl II site.
  • the new construct was named plasmid #151.
  • the pCI vector (Promega) was modified in the following way. It was cut with BsrG I, blunted with Klenow-fragment in the presence of dNTPs and reiigated to eliminate the BsrG I site. The new vector was cut with Nhe I and Not I and gel purified. A PCR fragment containing the coding region for the CFP and the following restriction sites was inserted into the Nhe I and Not I sites.
  • the pECFP-C1 plasmid (Clontech) served as a template for the PCR amplification.
  • the new vector was cut with Xba I and EcoR I and gel purified.
  • the coding region for the Flag epitope was constructed by oligo annealing and inserted into the Xba I and EcoR I sites.
  • Flag oligo 5' CT ⁇ GAT CCA CCA TGG ATT ACA AGG ATG ACG ACG ATA AGG
  • Flag oligo 3' AAT TCC TTA TCG TCG TCA TCC TTG TAA TCC ATG GTG GAT (the first ATG of the coding region is indicated in bold)
  • pCI expression vector Promega was used for transient transfections of pCMs.
  • the vector was constructed as described in chapter example 3 with minor modifications. Briefly, the yellow variant instead of the cyano variant of GFP was introduced.
  • the template for the PCR amplification using the same oligonucleotide primers and conditions in this case was the pEYFP vector (Clontech).
  • the resultant plasmid was cut with Xbal and EcoRI and gel purified.
  • the coding region for the influenza Hemagglutinine 1 epitope (HAi-tag) was inserted in order to express cDNAs as fusion proteins, which bear N-terminally the HAi-tag and C- terminally the CFP sequence.
  • the cDNAs without stop codon were inserted into the EcoRI and Xhol sites.
  • HAi-epitope TCT AGA ATG TAC CCA TAC GAT GTA CCT GAC TAT GCG GGC TAT ccc TAT GAC GTC CCG GAC TAT GCA GGA TAT CCG TAT
  • Example 4 Cells were seeded in a density of 6 x 10 5 cell per well in 6-well plates as described (example 4). Transfections were performed 24 hours after plating using Lipofectamine (Life Technologies) according to the protocol. Briefly, samples for six wells were prepared in a pool in the following way. First 6 ⁇ g of plasmid DNA (0.5 ⁇ g/ ⁇ l) were combined with 120 ⁇ l 2 x BBS and 600 ⁇ l maintenance medium without antibiotics and incubated for 15 minutes at room temperature. Meanwhile 24 ⁇ l of Lipofectamine was combined with 4 ml maintenance medium in a polystyrol tube. After 15 minutes the DNA sample was added to the Lipofectamine solution, mixed by inverting and incubated for another 15 minutes.
  • Lipofectamine Life Technologies
  • the MAP-kinase Erk2 was found to be recruited to the sarcomere after stimulation of pCMs. In addition, a diffuse or punctuated staining could be observed.
  • the MAP-kinase kinase MEK-1 was also found in a striated pattern. This was also true for a dominant negative mutant of MEK-1. Also the MAP-kinase kinase kinases A-raf and c-raf could be identified as sarcomere associated in all stimulation conditions.
  • Example 9 Time-dependent activation of Erk1/2 in neonatal cardiomyocytes pCMs were prepared as described (example 4) and plated in 6 well plates at a density of 1x10 6 cells/well. Cells were stimulated for the times indicated with Leucocyte Inhibitor Factor (Lif; 10ng/ml), Isoproterenol (100 ⁇ M) and Endothelin-.1 (ET-1 ; 200nM). Cells were harvested by washing once in ice-cold PBS and scraping into 100 ⁇ l/well 1x Western Loading Dye (10% glycerol, 2% SDS, 150mM DTT in 300mM Tris, pH 6.8) and transferring to Eppendorf tubes.
  • Leucocyte Inhibitor Factor Lif; 10ng/ml
  • Isoproterenol 100 ⁇ M
  • E-1 Endothelin-.1
  • the tubes were vortexed to shear DNA and boiled for 5 min. 40 ⁇ l were loaded per lane (corresponds to 4x10 5 cells per lane) onto a 10% gel and proteins separated by SDS-polyacrylamid gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane and Western blotted for phosphorylated Erk1/2.
  • the Western blot procedure was as follows: The nitrocellulose membrane was blocked in 1 % BSA (Sigma, Fraction V) for 1 h.
  • Membranes were then incubated with the phospho-specific antibody for Erk1/2 (NEB; dilution 1 :1000) in 1%BSA-0.1 %Tween in Tris-buffered saline solution (TBS-T) overnight at 4°C. Membranes were washed 4-5 times in TBS-T and then incubated with anti-rabbit-HRP conjugated secondary antibody (Amersham; Dilution 1 :10,000) for 2 hr at RT. Membranes were washed again 4-5- times in TBS-T and then incubated with LumiGlo (NEB), the ECL developing agent as per instructions. Bands were visualized using the digital Fuji LAS1000 system and quantified using the AIDA software.
  • the analysis of the subcellular localization of the HA1-c-raf-YFP fusion protein was performed on a fluorescence microscope (Axiovert 100S, Zeiss, Jena) in combination with a YFP-filter set (AF-Analysetechnik, Tubingen), a digital camera (Fuji LAS-1000) and the AIDA image analysis software (Raytest).
  • the fluorescence signal was arranged in a striated pattern (figure 4), which closely resembled the organization of ⁇ -actinin (figure 5).
  • the ⁇ -actinin is the main component of the sarcomeric Z-band, which represents the most important organization center for remodeling.
  • a similar distribution could be observed for a constitutive active mutant of c-raf- 1 as depicted in figure 6.
  • Cells were prepared and treated as described in example 4 and 5, but plated on glass coverslips in a density of 6.5 x 10 4 cells/m 2 .
  • Two days after stimulation and infection cells were washed twice with cold (4°C) phosphate buffered saline (PBS) incubated with cold (-20°C) Methanol/Aceton (7/3 vol/vol) for 20 minutes at -20°C, and washed three times with PBS (room temperature (RT)) for 5 minutes each.
  • PBS room temperature
  • Fixed cells were incubated with 10 % fetal calf serum in PBS for one hour in a humidified chamber at RT and incubated with first antibody for 1 hour at 37°C followed by three washing steps with PBS, 5 minutes each.
  • 1 st antibody monoclonal anti- ⁇ -actinin (A7811 , Sigma) 1 :800 or monoclonal anti myosin heavy chain (M8421 , Sigma)
  • Example 11 Inhibition of MEK1/2 leads to reduction of elongated phenotype
  • Cells were prepared as previously described and plated on gelatine coated coverslips in 12 well plates at a density of 1x10 5 cells/well. Cells were stimulated with LiF/ISO/ET-1 or LIF/PE/Et-1 and then incubated with either DMSO or 50 ⁇ M PD98059 for 48h. Cells were then washed in PBS and incubated for 20min with 3% paraformaldehyde, washed three times, then incubated for 2x10 min with 100mM ammonium chloride, washed again 3 times and incubated with 0.2% Triton in PBS for 5 minutes. Cells were washed again in PBS three times and incubated over night with gelatine (0.1 % in PBS).
  • Cells were then stained with a-actinin or anti-myosin heavy chain to facilitate the visualization of heart cells, (Sigma; dilution 1 :500-1 :800 in PBS, 1 % tween, 0.1%BSA) for 1 h at RT. Coverslips were washed three times in PBS and then incubated with 2° antibody (mouse-FITC from Dianova, dilution 1 :250 in PBS, 1 % tween, 0.1 %BSA) for 1 h at RT. Cells were again washed in PBS and coverslips mounted with Mowiol and analyzed using a Zeiss Axiovert 100 S Fluorescent microscope.
  • a-actinin or anti-myosin heavy chain to facilitate the visualization of heart cells, (Sigma; dilution 1 :500-1 :800 in PBS, 1 % tween, 0.1%BSA) for 1 h at RT. Coverslips were washed
  • Figure 7a shows stimulated cells incubated with carrier and figure 7b stimulated cells incubated with PD980159.
  • PD980159 the inhibitor of the kinase activating Erk1/2, and thus leading to Erk1/2 inhibition, resulted in an inhibition of sarcomeric reorganization and the elongated phenotype.
  • Example 12 W1LCK and P-Erk1/2 are in sarcomere Location of MLCK and phosphorylated, i.e. activated, Erk1/2 were tested by immunofiuorescence according to the protocol described above, except that the cells were fixed and permeabilised in methanol/acetone 7:3 for 10 min at -2Q°C. MLCK antibody was used at 1 :500 and phospho-Erk1/2 antibody was used at 1 :250 dilution.
  • MLCK was located in the sarcomere as has been described previously (Cavadore JC (1982) Proc Natl Acad Sci USA Jun;79(11):3475-3479). Similarly, but surprisingly, phosphorylated Erk1/2 could also be found in a striated pattern, especially in Triple stimulated heart cells after 48h stimulation (figure 9a and 9b).
  • Cells were plated at a density of 1x10 6 per well of a 6-well plate. Cell were lysed in 50mM Tris, pH 7,4, 1 % TritonX100, 100mM NaF, 10mM glycerophosphate and protease inhibitors (Boehringer Mini), then centrifuged for 5 min at 4°C, 13,000 rpm to pellet cell debris and nuclei.
  • n-Erk antibody NEB, 1 :100
  • MLCK antibody Sigma, 1 :100
  • Immunoprecipitations were washed twice in lysis buffer and once in kinase assay buffer (30mM Tris, pH 8.0, 200mM MgCI 2 , 2mM MnCI 2 ) and resuspended in 20 ⁇ l kinase assay buffer.
  • Example 14 Yeast two hybrid interactors of Erk2 14.1 Material and Methods Bait vectors
  • the vector 413MetLexN0 was constructed by cloning a PCR generated full length LexA repressor cDNA (with Xbal/BamHI overhangs) into the vector 413Met25 (Mumberg et al. 1994, Nucleic Acids Res. Dec 25;22(25):5767-8) cut Xbal/BamHI.
  • the vector 424GBN0 was constructed by cloning a PCR generated full length B42 transactivation domain cDNA (with Xbal/BamHI overhangs) derived from the vector pJG4-5 into the vector 424GAL1 (Mumberg et al. 1994, supra) cut Spel/BamHl.
  • the genes were cloned by RT-PCR using human heart tissue RNA or by PCR using the library pJG#9 as template.
  • the name of the genes, the accession number (Swiss prot entry) and the sequences of primers used for the amplification of the genes are listed in table 1.
  • Table 1 Primer list used for cloning of genes involved in the Erk pathway. The endonuclease restriction sites are marked in bold.
  • PCR products were cloned between the EcoRI and Xhol restriction site in the mammalian shuttle vector pCIH, and in the yeast shuttle vectors pEG202 or/and 413MetLexN0 or/and pJG4-5 and/or 424GBN0.
  • Erk2 and Mek1 dominant negative mutants were generated by PCR mutagenesis.
  • the sequences the mutations introduced are listed table 2.
  • Table 2 List of mutations introduced for the dominant negative Erk2 and Mek1 mutants. The mutated amino acids are marked in bold.
  • the c-raf constitutive active mutant is comprised of the C-terminus of c-raf protein spanning amino acids 331 to 648.
  • yeast two-hybrid vectors are described in section above.
  • Yeast strains used were EGY48LacZ-GFP (ura3::6*LexOp-lacZ, /ys2::6*LexOpCYC1GFP, ' his3, trpl , 6*LexAOp-LEU2, mat ⁇ ) and EGY199UL (t/ra3::6 * LexOp-lacZ, his3, trpl , 6*LexAOp- LEU2, mat a).
  • Yeast were grown in YPD or selective minimal medium as described by Sherman (1986), Basic Life Sci. ;40:533-44. Transformations were done using the high-efficiency method described by Gietz et al.
  • Bait plasmids were introduced into the yeast strain EGY48LacZ-GFP resulting in the strain EGY48LacZ-GFP-bait. Self activation of the bait was checked by plating the yeast on minimal glucose medium X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside) with or without leucine. In parallel protein expression was verified by western blot analysis using a polyclonal rabbit anti LexA antiserum. A human heart cDNA library (pJG#9) constructed in the vector pJG4-5 was introduced in the EGY48LacZ-GFP-bait strain.
  • yeast After transformation (4.10 4 colonies per plate) yeast were plated on selective medium (-Histidine, -tryptophane, +methionine, glucose). Colonies were harvested and an aliquot was plated on selective medium (- Histidine, -tryptophane, -uracil, raffinose, galactose, X-gal). Interactions were assayed by colony growth on selective medium as well as by ⁇ -galactosidase activity on the plate. Positive clones were plated over night on medium (-Histidine, - tryptophane, -uracil, glucose, X-gal) in order to repress the expression of the prey.
  • Verification of the interaction was performed by plating the colonies on medium A: (- Histidine, -tryptophane, -uracil, glucose, X-gal) and medium B: (-Histidine, - tryptophane, -uracil, raffinose, galactose, X-gal). Only blue colonies growing on medium B but not on medium A were further analysed by yeast-colony-PCR. Plasmid DNA was isolated from the yeast clones and introduced in E.coli (Robzyk and Kassir, 1992 Nucleic Acids Res 20, 3790). DNA was isolated from the bacteria and the 5 prime end of the cloned cDNA was sequenced.
  • a collection of yeast two-hybrid plasmids carrying a collection of kinases or activity altered mutants of these kinases from different MAP-kinase pathways (Cloned in the pEG202 and/or 413MetLexN0; pJG4-5 and/or 424GBN0) constructed at MediGene was introduced into EGY48LacZ-GFP and EGY199UL respectively. Each EGY48LacZ-GFP-bait were tested against each EGY199UL-prey for interaction via mating (Golemis and Khazak 1997, supra). This procedure was performed with an in house collection of full length ORFs of different kinases or of a large collection of other selected heart proteins. Positive interactions were detected by growth on selective medium and by ⁇ -galactosidase activity.
  • Table 3 Interactors found by screening of the pJG9 library.
  • accession numbers correspond to either Swiss prot or NCBI entries. Interactions are classified into strong interactions (++) and weak interactions (+) based on the ⁇ -galactosidase activity observed on plates.
  • Table 4 Direct Interactors of Erk2 found using the yeast two-hybrid kinase matrix. Interactions are classified into strong interactions (++) and weak interactions (+) based on the ⁇ -galactosidase activity observed on plates.
  • Table 5 Direct interactors of Erk2 found using the yeast two-hybrid 40K matrix. Accession number correspond to either Swiss prot or NCBI entries. Interactions are classified into strong interactions (++) and weak interactions (+) based on the ⁇ - galactosidase activity observed on plates.
  • the RSK (ribosomal S6 kinase) family MAP-kinases comprises growth factor- regulated serine/threonine kinases, also known as p90(rsk). Trivier et al. (1996, Nature 384: 567-570) noted that homologs of RSK exist in several species. The highly conserved feature of all members of the RSK family is the presence of 2 non- identical kinase catalytic domains. RSKs are implicated in the activation of the mitogen-activated kinase (MAPK) cascade and the stimulation of cell proliferation (at the transition between phases GO and G1 of the cell cycle) and differentiation.
  • MAPK mitogen-activated kinase
  • Telethonin is known as a capping protein of the sarcomeric protein titin (Mues et al. 1998, M FEBS Lett May 22;428 (1-2):11 1 -114). More over, in skeletal muscle telethonin (T-Cap) was found to interact with a FATZ protein (Faulkner et al. 2000, J Biol Chem. Dec 29;275(52):41234-42). The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin (Faulkner et al. 2000, supra). These data correlate with the fact that titin also appeared very early in the sarcomer formation.
  • Nkx2-5 is a transcription factor also known as CSX. This protein contains the NK family homeodomain. This protein is found in normal adult myocyte (Thompson et al. ' 1998, Am J Physiol.; 274(5 Pt 2):H1569-73). The same authors used pressure overload as primary stimulus in the feline pulmonary artery banding model to produce right ventricular hypertrophy (RVH). In this model, Nkx2-5 RNA expression level increased 5.1 fold in the right ventricle and 3.9 fold in the corresponding left ventricle after 2 to 7 days. By two weeks when the RVH had been completed Nkx2-5 level return to normal.
  • RVH right ventricular hypertrophy
  • PTRG is also know as prostacyclin 1.2 receptor.
  • Prostacyclin elicits potent vasodilation and inhibition of platelet aggregation through binding to its membrane receptor.
  • the impairment of prostacyclin receptor activity is implicated in various human cardiovascular diseases (Nakagawa et al. 1995, Clin Exp Pharmacol Physiol 22:S270-S272).
  • These authors studied the expression of the prostacyclin receptor in rat heart. They showed that prostacyclin receptor is express in various rat tissues but not in brain, and they further demonstrated the receptor mRNA expression in hypertensive cardiac hypertrophy. However no differential gene expression was identified in this study.
  • Activity of the prostacyclin receptor could be modulated via Erk2 activity in DCM or CHF.
  • the 28S protein also known as paraneoplastic cerebrellar degeneration (PCD) associated antigen.
  • PCD paraneoplastic cerebrellar degeneration
  • MA Methyl-accepting chemotaxis-like domains
  • This neural antigen is recognized by antibodies known as anti-Purkinje cell antibody type.
  • the exact role of 28S protein in heart cells is not clearly defined and there is no further evidence that this protein might also play a role in DCM or CHF.
  • CHIP Interaction with antigen NY Antigen NY is also known as CHIP.
  • CHIP interact with HSP70 and negatively regulates it chaperone functions.
  • HSP70 is known to be induced by stress.
  • HSP70 protein level was found to be up regulated during ischemia (Osaki et al. 1998, J Hypertens.16(8): 1193-200) as well as in the dog pacing model (Heinke et al.1998 Electrophoresis Aug;19(1 1 ):2021-30). Therefore by inhibiting the phosphorylation of CHIP, one could interfere with the activation of the heat shock machinery.
  • PRCC is ubiquitously expressed in normal adult and fetal tissues.
  • the PRCC protein of mouse and chicken show homology with formin (accession Q05858) over its whole length. It seems highly improbable that the PRCC gene encodes the human formin counterpart.
  • the predicted contains a relatively high percentage of prolineswith several P-P-P-P motifs present. In the SH3 domain of Crkll, binding was observed to proteins containing the P-X-X-P motif. A consensus site for tyrosine kinases is also present, indicating that this predicted protein may function in a signaling cascade and, as such, may contribute to tumor formation as part of a fusion product.
  • Osciilin glucosamine-6-phosphate deaminase
  • MAD2 is a protein involved in the mitotic cell cycle control (ref). So far there is no further evidence for an involvement of MAD2 in DCM or CHF.

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Abstract

La présente invention se rapporte à un procédé d'identification et/ou d'obtention d'un composé, ou d'une pluralité de composés, permettant de prévenir ou de traiter des maladies cardiaques, qui consiste à effectuer une évaluation quantitative de l'activité de Raf-1, MEK1/2, et/ou Erk1/2, dépendante dudit composé. Ledit composé est de préférence un inhibiteur ou un antagoniste de la cascade de signaux, qui est recruté dans le sarcomère et entraîne l'activation de Raf-1, de MEK1/2 et/ou d'Erk1/2, étant donné que ladite activation aboutit à la translocation d'Erk2 dans les sarcomères et à l'induction du remodelage de ces derniers. De plus, la présente invention a trait à des compositions pharmaceutiques destinées à prévenir ou à traiter l'hypertrophie, en particulier l'insuffisance cardiaque congestive, lesdites compositions renfermant le composé identifié et/ou obtenu, ou une pluralité de composés, en tant qu'ingrédient actif.
PCT/EP2002/010489 2001-09-19 2002-09-18 Kinase regulee extracellulaire 2 (erk2) Ceased WO2003025205A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007014033A3 (fr) * 2005-07-22 2007-07-12 Univ Colorado Regents Inhibition de la kinase erk 1/2 ('extracellular signal-regulated kinase') servant a traiter l'hypertrophie cardiaque et l'insuffisance cardiaque
EP2270196B1 (fr) 2004-05-11 2016-04-20 Axiogenesis Ag Methodes pour la découverte de médicaments utilisant des cellules différentiées in vitro
US10507228B2 (en) * 2015-04-20 2019-12-17 H. Lee Moffitt Cancer Center And Research Institute, Inc. Methods and compositions related to KRAS inhibitors

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WO1997039132A1 (fr) * 1996-04-16 1997-10-23 University Of Miami Preparations stabilisees de troponines humaines et modifications de ces troponines, procedes diagnostiques de dosage et trousses de dosage
AU764094B2 (en) * 1998-07-21 2003-08-07 Aventis Pharma S.A. MEKK1(serine threonine kinases)-interacting FHA (forkhead associated domain) protein 1 (MIF1)
AUPP785098A0 (en) * 1998-12-21 1999-01-21 Victor Chang Cardiac Research Institute, The Treatment of heart disease
CA2382045A1 (fr) * 1999-08-20 2001-03-01 Board Of Regents, The University Of Texas System Methodes et compositions relatives a la regulation par hdac 4 ou 5 de l'expression genique au niveau cardiaque
DE19962154A1 (de) * 1999-12-22 2001-07-12 Medigene Ag Krankhaft veränderte Herzmuskelzelle, ihre Herstellung und Verwendung

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2270196B1 (fr) 2004-05-11 2016-04-20 Axiogenesis Ag Methodes pour la découverte de médicaments utilisant des cellules différentiées in vitro
WO2007014033A3 (fr) * 2005-07-22 2007-07-12 Univ Colorado Regents Inhibition de la kinase erk 1/2 ('extracellular signal-regulated kinase') servant a traiter l'hypertrophie cardiaque et l'insuffisance cardiaque
US10507228B2 (en) * 2015-04-20 2019-12-17 H. Lee Moffitt Cancer Center And Research Institute, Inc. Methods and compositions related to KRAS inhibitors

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