WO2007137864A1 - Use of inhibitors of pp2c for treating or preventing arteriosclerosis - Google Patents

Use of inhibitors of pp2c for treating or preventing arteriosclerosis Download PDF

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WO2007137864A1
WO2007137864A1 PCT/EP2007/004870 EP2007004870W WO2007137864A1 WO 2007137864 A1 WO2007137864 A1 WO 2007137864A1 EP 2007004870 W EP2007004870 W EP 2007004870W WO 2007137864 A1 WO2007137864 A1 WO 2007137864A1
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pp2c
acid
fatty acid
inhibitor
inhibitor according
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French (fr)
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WO2007137864A9 (en
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Wolfgang Greb
Susanne Klumpp
Josef Krieglstein
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Eucro European Contract Research Gmbh & Co. Kg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/231—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having one or two double bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to serine-/threonine protein phosphatase type 2C (PP2C) inhibitors comprising at least a derivative of a fatty acid. Furthermore, the invention relates to a pharmaceutical composition comprising respective inhibitors and their use in therapy.
  • P2C serine-/threonine protein phosphatase type 2C
  • Reversible protein phosphorylation is an ubiquitous strategy used to control many of the intracellular events in eukaryotic cells. It is estimated that more than ten percent of proteins active in a typical mammalian cell are phosphorylated.
  • Kinases catalyze the transfer of high-energy phosphate groups from adenosine triphosphate (ATP) to target proteins on the hydroxyamino acid residues serine, threonine, or tyrosine. Phosphatases, in contrast, remove these phosphate groups.
  • Extracellular signals including hormones, neurotransmitters, and growth and differentiation factors can activate kinases, which can occur as cell surface receptors or as the activator of the final effector protein, but can also occur along the signal transduction pathway.
  • Cascades of kinases are, as well as kinases sensitive to second messenger molecules. This system allows for the amplification of weak signals (low abundance growth factor molecules, for example), as well as the synthesis of many weak signals into an all-or-nothing response.
  • Phosphatases then, are essential in determining the extent of phosphorylation in the cell and, together with kinases, regulate key cellular processes such as metabolic enzyme activity, proliferation, cell growth and differentiation, cell adhesion, cell cycle progression and apoptosis.
  • Apoptosis or programmed cell death, is involved in fundamental biological processes. It also plays a fundamental role in pathological processes like atherogenesis (Geng et al., 1997; Kockx et al., 2000; Kockx et al., 1998). More than 25 years ago it was proposed that arteriosclerosis arises as a response of the vascular wall to endothelial injury. Evidence accumulated in the last decade showed that this injury could be due to endothelial apoptosis (R ⁇ ssig et al., 2001; Choy et al., 2001; Stefanec et al., 2000).
  • Endothel cell (EC) apoptosis may result in increased permeability of the endothelial monolayer through the loss of EC number. This loss of integrity could facilitate the migration and deposition of lipids, like oxidized LDL, or monocytes in the subintimal space, further damaging vascular development and propagating plaque development. Since apoptotic cells are procoagulant, atherogenesis may also depend on increased coagulation of platelets resulting from EC apoptosis (McDonald et al., 1999). Therefore, enhanced apoptosis of endothelial cells (ECs) and macrophages is an important pathophysiologic mechanism involved in atherogenesis.
  • Apoptosis is an energy dependent process that may be regulable at several levels. However, identifying the role of the regulators of the apoptotic pathway may lead to a greater understanding as to how cellular interactions contribute to atherogenesis, as well as providing new insights into potential therapeutic targets for the treatment of arteriosclerosis.
  • Reversible phosphorylation is the most common post-translational modification of proteins. By phosphorylation almost all aspects of cellular life from classical metabolic pathways and even cell death are regulated. As mentioned before, many cellular constituents are switched "on” or “off” by phosphorylation and dephosphorylation. These processes are catalyzed by various specific protein kinases and phosphatases.
  • Protein phosphatases are generally characterized as either serine/threonine or tyrosine specific, based on their preferred phospho-amino acid substrate. However, some phosphatases (DSPs, for dual specificity phosphatases) can act on phosphorylated tyrosine, serine, or threonine residues.
  • DSPs dual specificity phosphatases
  • Serine/threonine protein phosphatases are subdivided phylogenetic into the PPP family (comprising PP1 , PP2A, PP2B) and the PPM family (Mg ⁇ 2+>- dependent) (Cohen, P.T.W (1994) Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv. Prot. Phosphatases VoI 8, 371-276).
  • PPP stands for phospho-protein-phosphatase
  • PPM stands for protein phosphatase Mg- dependent.
  • PP2C is a 42 kDa monomer with broad substrate specificity and is dependent on divalent cations (mainly Mn 2+ or Mg 2+ ) for its activity.
  • PP2C proteins share a conserved N-terminal region with an invariant DGH motif, which contains an aspartate residue involved in cation binding (PROSITE PDOC00792).
  • PP2Cs protein phosphatases, since they constitute a separate gene family and are monomeric enzymes.
  • the predominant isotypes in mammalian cells are PP2C alpha (PPM1A) and PP2C beta (PPM1 B), which are separate gene products of 43-48 kDa (identity 77%) (Cohen, P.T.W (1994)
  • the PP2C family in addition includes PP2C gamma (also called FIN13), PP2C delta and WIP1 (Guthridge, M. A., Bellosta, P. , Tavoloni, N. and Basilico, C (1997) FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression. MoI. Cell. Biol. VoI: 17, 5485-5498 and Tong, Y., Quirion, R. and Shen S.-
  • Wip1 a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc. Natl. Acad. Sci. USA VoI: 94, 6048-6053).
  • AMP-activated protein kinase (Davies, S.P., Helps, N. R., Cohen, P.T.W. and Hardie, D. G. (1995) 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bone protein phosphatase-1AC. FEBS Lett. VoI: 377, 421-425);
  • MAPK mitogen-activated protein kinase
  • Cystic fibrosis transmembrane conductance regulator (Travis, S. M., Berger, H. A. and Welsh, M.J. (1997) Protein phosphatase type 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. VoI: 94, 1105511060); and
  • Cyclic - dependent kinases CDK2 and CDK6 (Cheng, A., Kaldis P. and Solomon M.J. (2000) Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms. J. Biol. Chem. VoI: 275, 34744- 34749).
  • P2C protein phosphatase 2C
  • BAD (BAD stands for Bcl-2/Bcl-XL-antagonist, causing cell death) represents a point of convergence of several different signal-transduction pathways which are activated by survival factors inhibiting apoptosis in mammalian cells.
  • BAD is a pro-apoptotic protein which binds to the anti-apoptotic proteins Bcl-2 and BcI-XL (Yang, E., Zha, J., Jockel, J. Bioise, L.H., Thompson, CB. and Korsemeyer, S.J. (1995) BAD, a heterodimeric partner for BcI-XL and Bcl-2 displaces Bax and promotes cell death. Cell VoI: 80, 285- 291).
  • the intention is to provide modulators which can be used in the specific treatment of apoptosis (especially of EC cells) thereby providing a treatment or prevention for cardiovascular diseases, especially arteriosclerosis.
  • a serine-/threonine protein phosphatase type 2C (PP2C) inhibitor which comprises at least a derivative of a fatty acid of the group consisting of - fatty acids
  • Said derivative of a fatty acid has a sufficient fipophilicity to inhibit fatty acid induced activation of PP2C and a free carboxyl group.
  • the inhibitors according to the present invention are able to bind to PP2C thereby blocking the binding pocket for fatty acids that otherwise activate PP2C such as e.g. oleic acid.
  • Said derivative of a fatty acid is preferably a fatty acid, more preferably one selected from the group consisting of saturated fatty acids (SFA), unsaturated fatty acids, straight and branched fatty acids.
  • SFA saturated fatty acids
  • unsaturated fatty acids straight and branched fatty acids.
  • Branches fatty acids may carry C1 - C5 alkyl groups. Preferred are small alkyl groups such as C1 to C3 alkyl group. Apparently, such branched fatty acids block the binding pocket sterically.
  • said fatty acid derivative may comprise at least one polyethylene moiety.
  • the polyethylene moiety may be within the fatty acid chain or at the end of it.
  • said fatty acid is a saturated fatty acid (SFA).
  • SFA saturated fatty acid
  • the saturated fatty acid (SFA) used according to the present invention has preferably a chain length of more than 16 C-atoms.
  • SFAs can be used wherein the chain length is between C17.0 and C30.0, preferably between C17: and C25:0, even more preferred between C17:0 and C23.0.
  • the SFA could be selected from the group consisting of heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0) and behenic acid (C22:0).
  • said derivative of a fatty acid is either a neutral or a phospho lipid.
  • Suitable examples are triglycerides.
  • the triglycerides preferably comprise at least one of the fatty acids mentioned above.
  • said inhibitory derivative is an ester.
  • Said ester preferably comprises at least one of the fatty acids mentioned above.
  • a triglyceride or an ester offers the opportunity to develop a pharmaceutical which comprises the active inhibitory substance in form of a prodrug.
  • Such prodrugs are metabolized to release the actual active ingredient in the body through enzymatic cleavage.
  • said derivative of a fatty acid carries a substituent.
  • Said substituent is preferably a halogen substituent such as a fluorine.
  • the inhibitors provided with the present invention have several major advantages.
  • BAD a proapoptotic member of the Bcl-2 family
  • BAD a proapoptotic member of the Bcl-2 family
  • BAD promotes apoptosis by heterodimerization with the antiapoptotic oncogenes Bcl-2 and BcI-X 1 .
  • lmmuncytochemistry revealed that PP2C and its substrate BAD are co-localized within the cytosol of HUVECs. Further the co- localization of these two proteins in macrophages was demonstrated (Fig. 1 ).
  • SFAs which are not able to activate PP2C in vitro (Fig. 3) were able to inhibit the oleic acid-induced activation of PP2C and thus act as specific inhibitors of fatty acid mediated PP2C activation.
  • SFAs have a free carboxyl group and are lipophil. Apparently, they displace oleic acid from the active center of the PP2C phosphatase without activating the enzyme and consequently avoid the oleic acid-induced activation by blocking the binding site necessary for activation.
  • Saturated fatty acids (C14:0 - C22:0) carrying a free carboxyl group and being lipophil thus comprise one preferred embodiment according to the present invention.
  • other derivatives of fatty acids can act as inhibitors of PP2C. Their characteristics were described above in detail.
  • SFA saturated fatty acids
  • the vascular endothelium is inceimpulsly exposed to free fatty acids (FFAs) because the lipoprotein lipase (LPL) that is associated with the luminal surface of ECs hydrolyzes triglycerides of circulating lipoproteins (Stins et al., 1992). It was demonstrated that physiologic fatty acids from human lipoproteins activate PP2C in vitro and induce apoptosis in cultured human umbilical endothelial cells (HUVECs)
  • RNA interference (RNAi) mechanisms were employed to demonstrate the causal relationship between fatty acid- induced activation of PP2C and induction of apoptosis by fatty acids in ECs (Schwarz et al., 2006).
  • Macrophages produce a variety of biologically active products including proteases, growth factors, apo E (Nathan, 1987;
  • LPL-mediated hydrolysis of triglycerides plays not only a role in the uptake of lipids in ECs but also in macrophages (Floren et al., 1981 ; Lindquist et al., 1983).
  • the present invention provides inhibitors of FFA and especially oleic acid-induced activation of PP2C. It was demonstrated by experiments that the inhibitors according to the present invention can protect both cell types (EC and macrophages) from the damaging effect of FFAs as exemplified by oleic acid.
  • the inhibitors are SFAs with a sufficient lipophilicity to bind to and to inhibit activation of PP2C by FFAs.
  • they Preferably, they have a chain length longer than 16 C-atoms. It was shown that they are able to inhibit the oleic acid-induced activation of PP2C in vitro. Furthermore, it could also be exhibited that these SFAs abolish the damaging effect of oleic acid in cultured HUVECs and macrophages and thus prove their therapeutic potential.
  • Saturated fatty acids form straight chains and, as a result, can be packed together very tightly, allowing living organisms to store chemical energy very densly.
  • the fatty tissues of animals contain large amounts of long-chain saturated fatty acids.
  • fatty acids In IUPAC nomenclature, fatty acids have an -oic acid suffix.
  • SFAs with a insufficient lipophilicity e.g. with a chain length shorter than 16 C-atoms, like e.g. myristic acid (C14.0), pentadecanoic acid (C15.O), and palmitic acid (C16:0), could not influence the oleic acid-induced activation of PP2C, wherein
  • SFAs with a chain length longer than 16 C-atoms like e.g. heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), and behenic acid (C22:0), could do so by inhibiting the activation of PP2C (Fig. 4).
  • compounds with a sufficient lipophilicity allowing interaction with the binding pocket of PP2C e.g. molecules having a chain length longer than 16 C-atoms
  • these molecules according to the present invention cause no activation of the PP2C enzyme.
  • they avoid the binding of oleic acid (as an example of an activated FFA), thus inhibiting the oleic acid-induced activation by preventing binding of these activators.
  • These molecules thus basically antagonise the activators.
  • Double staining with the DNA fluorochrome Hoechst 33258 and Nile blue revealed that the different SFAs and oleic acid were similarly taken up by HUVECs and preferably heptadecanoic acid (C17:0) and stearic acid (C18:0) could slow down the damaging effect of oleic acid (Fig. 9). This could be a competitive displacement from oleic acid from the respective center of PP2C, which slows down the oleic-induced programmed cell death of ECs and macrophages.
  • inhibition of the fatty acid-induced mediated activation of PP2C by the inhibitors of the present invention is a new and promising therapeutic strategy for preventing apoptosis in EC and/or macrophage cells.
  • the experimental results demonstrate that it is possible to specifically inhibit the fatty acid-induced apoptosis of ECs and macrophages with the inhibitors according to the invention and provide new insights to avoid the fatty acid-induced progression of atherogenesis/arteriosclerosis.
  • a pharmaceutical composition comprising at least one inhibitor as described above as a physiologically active substance and a pharmaceutically acceptable carrier, wherein said inhibitor possesses sufficient lipophilicity to bind to and inhibit PP2C in particular inhibiting the FFA induced activation.
  • Also part of the present invention is the use of an inhibitor according to the present invention for the manufacture of a medicament for the treatment or prevention of fatty acid induced apoptosis in cells, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group.
  • the inhibitors according to the present invention may be used for the manufacture of a medicament for the treatment or prevention of cardiovascular diseases, especially for the treatment or prevention of atherogenesis and/or arteriosclerosis, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group
  • Fig. 1 shows a Western blots for detection of PP2C and BAD in macrophages. The localization of PP2Cbeta and BAD in macrophages is shown.
  • A shows Western blots for detection of PP2Cbeta and BAD. Macrophage proteins (30 ⁇ g/lane) were electrophoresed on 15% SDS-polyacrylamide gels and after transfer to nitrocellulose immunoblotted with an antibody specific for PP2Cbeta diluted 1:1000 (A, left panel) or with an antibody against BAD diluted 1:1000 (A, right panel).
  • B-E Depicted are fluorescence laser scanning photographs showing subcellular co-localization of PP2Cbeta and BAD. Macrophages cell culture and immunocytochemistry were performed as described in Section 2.
  • B Incubation with the antibodies against
  • Fig. 2 shows two bar graphs, wherein in item (A) the effect of SFAs on PP2Cbeta activity and in item (B) the effect of oleic acid in combination with different SFAs on
  • PP2Cbeta activity are shown.
  • (A) shows the effect of SFAs on PP2Cbeta activity. Dephosphorylation of [ 32 P]casein by PP2C was measured upon addition of various fatty acids. Oleic acid stimulated the activity 9-fold, wherein SFAs with different chain lengths failed to activate the enzyme. Fatty acids added were oleic acid (18:1 cis- ⁇ 9), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid
  • Short chain SFAs ( ⁇ 17:0) could not affect the oleic acid-induced activation of PP2C, wherein SFAs with a chain length longer than 16 C-atoms could inhibit activity.
  • Dephosphorylation of [ 32 P]casein was determined with 98 ng PP2Cbeta per incubation. Activity in the absence of fatty acids (1) was 6.8 nmol Pj/min/mg.
  • Fig. 3 shows two bar graphs, wherein in item (A) the effect of SFAs with different chain length on ECs and in item (B) the effect of different SFAs (14:0 - 18:0) on macrophages are shown.
  • (A) illustrates the effect of SFAs with different chain length on ECs.
  • HUVECs were incubated with 200 ⁇ M of different SFAs.
  • Control cells were treated with vehicle only (0,2% DMSO). Apoptotic damage was revealed by Hoechst
  • Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. The cells showing fragmented nuclei and condensed chromatin were counted as apoptotic cells. Apoptotic cells are expressed as a percentage of total number of cells ⁇ S.D. ***p ⁇ 0,001.
  • Fig. 4 shows two bar graphs, wherein in item (A) effect of heptadecanoic acid and stearic acid in view of the protection of ECs from oleic acid-induced apoptosis an in item (B) the effect of heptadecanoic acid and stearic acid in the protection of macrophages from oleic acid-induced apoptosis are shown.
  • (A) depicts that heptadecanoic acid and stearic acid protect ECs from oleic acid-induced apoptosis. Double treatment of HUVECs with oleic acid (18:1 cis- ⁇ 9) and different short chain
  • SFAs myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (C16:0)
  • heptadecanoic acid (17:0) and stearic acid (18:0) could slow down the oleic acid-induced apoptosis of ECs.
  • the percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Control cells received vehicle only (0,4% DMSO). Apoptotic cells are expressed as percentage of total number of cells ⁇ S.D. * ** p ⁇ 0,001 , compared with vehicle-treated control.
  • Heptadecanoic acid and stearic acid protect macrophages from oleic acid-induced apoptosis.
  • Double treatment of macrophages with oleic acid (18:1 cis- ⁇ 9) and different short chain SFAs could not abolish the toxic effect of oleic acid, wherein heptadecanoic acid (17:0) and stearic acid (18:0) could inhibit the oleic acid-induced apoptosis of ECs.
  • the percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Control cells received vehicle only (0,4% DMSO). Apoptotic cells are expressed as percentage of total number of cells ⁇ S.D. * * * * * p ⁇ 0,001, compared with vehicle-treated control.
  • Fig. 5 shows a Nile blue staining of the cells, wherein the uptake of SFAs and with or without oleic acid is demonstrated.
  • ECs were incubated with vehicle (0.4% DMSO) oleic acid (18:1), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) for 6 h and 24 h at a concentration of
  • Double treatment with oleic acid revealed, that myristic acid, pentadecanoic acid (15:0), palmitic acid (16:0) are not able to protect HUVECs from oleic acid-induced apoptosis, cognisable on the apoptotic morphology of ECs.
  • heptadecanoic acid (17:0) and stearic acid (18:0) slow down the toxicity of oleic acid and protect ECs.
  • Apoptotic damage was revealed by Hoechst 33258 staining as described in Section 2.
  • Fig. 6 shows further examples of inhibitors.
  • HUVECs and cell growth medium containing 2% heat-inactivated fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 ⁇ g/ml hydrocortisone, 1 ng/ml fibroblast factor and antibiotics were purchased from Promocell (Heidelberg, Germany);
  • THP-1 cells were obtained as a gift from lnstitut fur Atheroskleroseforschung M ⁇ nster, Germany; THP-1 monocytes cell medium containing RPMI-1640, FSC 10%, penicillin 100U/ml, streptomycin 100 ⁇ g/ml, L-glutamine 2 mM, sodiumpyruvate 100 mM, phorbol-12-myristat-13-acetat (PMA) and mercaptoethanol were received from Sigma Aldrich (Taufkirchen, Germany).
  • Hoechst 33258 Nile blue and fatty acids (myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, arachidic acid and behenic acid), bovine serum albumin (BSA) were obtained from Sigma Aldrich (Taufkirchen, Germany). Nile blue staining was performed with a confocal laser scanning microscope Zeiss LM510 (Jena, Germany) and analysis of apoptotic cells was accomplished with the fluorescent microscope Ax/overt 100 Zeiss (Jena, Germany).
  • ECL enhanced chemoluminescence
  • Western blotting detection reagents and secondary antibody rabbit IgG, peroxidase linked, from donkey were from Amersham Pharmacia Biotech (Freiburg, Germany).
  • Antibodies against BAD were purchased from New England Biolabs GmbH (Frankfurt, Germany) and recombinant antibody against PP2Cbeta was prepared as described (Klumpp et al., 2000).
  • Biotinylated secondary anti rabbit antibody and secondary Texas-red anti mouse antibody were obtained from Alexis (Gr ⁇ nberg, Germany).
  • PP2Cbeta assays contained 30 mM Tris- HCI, pH 7.0, 0.1% 2-mercaptoethanol, 0.6 mg/ml BSA, 1 ⁇ M [ 32 P] casein (5 x 10 4 cpm), and 10-100 ng PP2Cbeta in a final volume of 30 ⁇ l.
  • PP2Cbeta is characterized by its dependence on divalent cations for activity. If not otherwise indicated, 1 mM magnesium acetate was present.
  • HUVECs were cultured in endothelial cell growth medium to 90% confluence. In all experiments cells were used at passages 3-6 and morphology was controlled prior to treatment. The cells were seeded at a density of 500000 cells per 9.6 cm 2 dishes.THP-
  • monocytes were cultured in suspension at a density of 4 million cells/ 5 ml for four days.
  • monocytes received 100 ng/ml phorbol-12- myristic-13-acetate and 50 ⁇ M 2-mercaptoethanol (Dory et al., 1993).
  • monocytes differentiated to macrophages and became adherent. Experiments were only accomplished with adherent and confluent macrophages (density > 1 million cells/9.6 cm 2 dishes). Both cell types were cultured at 37°C in an incubator supplemented with 5% CO 2 and 95% air.
  • HUVECs and macrophages were incubated for an additional 48 h. Cell cultures were then treated for 24h with oleic acid (200 ⁇ M) to induce apoptosis, with different concentrations of c) or with combinations of both.
  • the FFAs were dissolved in dimethylsulfoxid (DMSO), and DMSO was used as control. The final concentration of DMSO in the culture medium did not exceed 0.5%.
  • Fatty acid-treated cells were washed with phosphate buffered saline (PBS) 1 fixed for 30 min with paraformaldehyde and then incubated for 30 min with the DNA fluorochrome Hoechst 33258 (10 ⁇ g/ml) in methanol at room temperature in the dark. After washing with PBS, the nuclear morphology of HUVECs and macrophages was analyzed under a fluorescent microscope at an excitation wave length of 350 nm and an emission wavelength of 450 nm. Cells showing shrunken or fragmented nuclei or chromatin condensation were counted as apoptotic cells. All values were expressed as means ⁇ S.D.
  • Nile blue was used to reveal uptake of FFAs in ECs. After fixation with paraformaldehyde for 30 min, HUVECs were washed with PBS, stained with Nile blue solution (10 ⁇ g/ml) for 2 h and analyzed under a confocal laser scanning microscope at a wavelength of 488/525 nm. 1.6. Protein determination
  • the concentration of proteins was determined by BCA- or Lowry-assay (Lowry et al., 1951) using BSA as a standard.
  • BAD and PP2Cbeta were analyzed on 15% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature with 5% non-fat milk powder in TBS-T (Tris buffered saline with Tween) (10 mM Tris-HCI pH 7.6, 150 mM NaCI, 0.1% Tween 20), incubated overnight at 4°C in TBS-T containing 5% BSA and primary antibody (anti-BAD and anti-PP2Cbeta 1 :1000, respectively), followed by incubation in 5% non-fat milk powder in TBS-T for 1 h with peroxidase- conjugated anti-rabbit IgG (1 :2500). Blots were developed with ECL reagent.
  • TBS-T Tris buffered saline with Tween
  • PP2Cbeta activated by unsaturated fatty acids is one of the phosphatases that acts on
  • Oleic acid (18:1 cis-A Q ) stimulates the activity of PP2Cbeta at least 10-fold, wherein the trans- and methylester derivatives do not (Klumpp et al., 1998).
  • the structural requirements for activation of PP2Cbeta strikingly correlate with the induction of apoptosis in endothelial cells (Hufnagel et al., 2005).
  • To evaluate this relation to macrophages they were treated with 18:1 compounds and tested for survival. Exposure of macrophages to oleic acid (250 ⁇ M) for 24 h caused cell death (Fig. 2). In contrast, the corresponding trans derivative and the oleic acid methylester both were not harmful when administered in the same concentration range as oleic acid (Fig. 2).
  • Double-treatment of PP2C with oleic acid and SFAs showed interesting new results: SFAs with a chain length shorter than 16 C-atoms, like myristic acid (C14:0), pentadecanoic acid (C15:0) and palmitic acid (C16:0), were not able to inhibit the activation-capacity of oleic acid, wherein SFAs with a chain length of more than 16 C-atoms, like heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20.0) and behenic acid (C22:0) inhibited the oleic acid induced activation of PP2C (Fig. 4) in vitro.
  • SFAs with a chain length shorter than 16 C-atoms like myristic acid (C14:0), pentadecanoic acid (C15:0) and palmitic acid (C16:0)
  • Myristic acid (C14.0), pentadecanoic acid (C15:0) and palmitic acid (C16:0) were not able to abolish the toxic affect of oleic acid, wherein heptadecanoic acid (C17:0) and stearic acid (C18:0) significantly inhibited the oleic acid-induced apoptosis in both cell types and are therefore protective (Fig. 7 and 8).
  • Arachidic acid (C20:0) and behenic acid (C22:0) were almost insoluble in the endothelial cell growth medium and therefore could not be tested.
  • HUVECs were treated with Nile blue, a dye to explore the fate of lipids and to possibly perceive the location within ECs.
  • Control cells received 0,5% DMSO as control for lipid solvent and subsequent staining resulted in a weak fluorescence reflecting endogenous lipids.
  • addition of myristic acid to demonstrate the fatty acid-uptake.

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Abstract

Activity of serine-/threonine protein Phosphatase type 2C (PP2C) is known to be stimulated by certain unsaturated fatty acids and this enzyme dephosphorylates BAD1 thus acting on apoptosis. The invention provides saturated fatty acid (SFA) as new inhibitors for the activity of the PP2C. Therefore, the invention offers a new therapeutic strategy for the treatment and prevention of arteriosclerosis.

Description

INHIBITORS FOR PP2C
This invention relates to serine-/threonine protein phosphatase type 2C (PP2C) inhibitors comprising at least a derivative of a fatty acid. Furthermore, the invention relates to a pharmaceutical composition comprising respective inhibitors and their use in therapy.
Reversible protein phosphorylation is an ubiquitous strategy used to control many of the intracellular events in eukaryotic cells. It is estimated that more than ten percent of proteins active in a typical mammalian cell are phosphorylated. Kinases catalyze the transfer of high-energy phosphate groups from adenosine triphosphate (ATP) to target proteins on the hydroxyamino acid residues serine, threonine, or tyrosine. Phosphatases, in contrast, remove these phosphate groups.
Extracellular signals including hormones, neurotransmitters, and growth and differentiation factors can activate kinases, which can occur as cell surface receptors or as the activator of the final effector protein, but can also occur along the signal transduction pathway. Cascades of kinases are, as well as kinases sensitive to second messenger molecules. This system allows for the amplification of weak signals (low abundance growth factor molecules, for example), as well as the synthesis of many weak signals into an all-or-nothing response. Phosphatases, then, are essential in determining the extent of phosphorylation in the cell and, together with kinases, regulate key cellular processes such as metabolic enzyme activity, proliferation, cell growth and differentiation, cell adhesion, cell cycle progression and apoptosis.
Apoptosis, or programmed cell death, is involved in fundamental biological processes. It also plays a fundamental role in pathological processes like atherogenesis (Geng et al., 1997; Kockx et al., 2000; Kockx et al., 1998). More than 25 years ago it was proposed that arteriosclerosis arises as a response of the vascular wall to endothelial injury. Evidence accumulated in the last decade showed that this injury could be due to endothelial apoptosis (Rόssig et al., 2001; Choy et al., 2001; Stefanec et al., 2000). Endothel cell (EC) apoptosis may result in increased permeability of the endothelial monolayer through the loss of EC number. This loss of integrity could facilitate the migration and deposition of lipids, like oxidized LDL, or monocytes in the subintimal space, further damaging vascular development and propagating plaque development. Since apoptotic cells are procoagulant, atherogenesis may also depend on increased coagulation of platelets resulting from EC apoptosis (McDonald et al., 1999). Therefore, enhanced apoptosis of endothelial cells (ECs) and macrophages is an important pathophysiologic mechanism involved in atherogenesis.
Apoptosis is an energy dependent process that may be regulable at several levels. However, identifying the role of the regulators of the apoptotic pathway may lead to a greater understanding as to how cellular interactions contribute to atherogenesis, as well as providing new insights into potential therapeutic targets for the treatment of arteriosclerosis. Reversible phosphorylation is the most common post-translational modification of proteins. By phosphorylation almost all aspects of cellular life from classical metabolic pathways and even cell death are regulated. As mentioned before, many cellular constituents are switched "on" or "off" by phosphorylation and dephosphorylation. These processes are catalyzed by various specific protein kinases and phosphatases.
Protein phosphatases are generally characterized as either serine/threonine or tyrosine specific, based on their preferred phospho-amino acid substrate. However, some phosphatases (DSPs, for dual specificity phosphatases) can act on phosphorylated tyrosine, serine, or threonine residues.
Serine/threonine protein phosphatases are subdivided phylogenetic into the PPP family (comprising PP1 , PP2A, PP2B) and the PPM family (Mg<2+>- dependent) (Cohen, P.T.W (1994) Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv. Prot. Phosphatases VoI 8, 371-276). PPP stands for phospho-protein-phosphatase and PPM stands for protein phosphatase Mg- dependent.
Protein phosphatase type 2C (PP2C) is one of the four major serine / threonine protein phosphatases (PP1 , PP2A, PP2B = calcineurin, PP2C) found in eukaryotic cells and classified according to biochemical criteria (Ingebritsen, T.S. and Cohen, P. (1983) Protein phosphatases: Properties and role in cellular regulation. Science VoI: 221 , 331- 337 and Wera, S. and Hemmings, B.A. (1995) Serine/threonine protein phosphatases. Biochem. J. VoI: 311 , 17-29). PP2C relates to the main enzyme subtype of PPM. PP2C is a 42 kDa monomer with broad substrate specificity and is dependent on divalent cations (mainly Mn2+ or Mg2+) for its activity. PP2C proteins share a conserved N-terminal region with an invariant DGH motif, which contains an aspartate residue involved in cation binding (PROSITE PDOC00792).
The dephosphorylation activity of PP2C absolutely requires the presence of Mn<2+> or Mg<2+> ions (McGowan, CH. and Cohen, P. (1988) Protein phosphatase-2C from rabbit skeletal muscle and liver: A Mg<2+>-dependent enzyme. Meth. Enzymol. VoI 159, 416-426). Dephosphorylation of [<32>P]casein in the presence of 20 mM Mg<2+> is considered the standard technique to determine PP2C activity in vitro (McGowan,
CH. and Cohen, P. (1988) Protein phosphatase-2C from rabbit skeletal muscle and liver. A Mg<2+>-dependent enzyme. Meth. Enzymol. Vo1 159, 416-426).
Molecular cloning has defined PP2Cs as distinct protein phosphatases, since they constitute a separate gene family and are monomeric enzymes. The predominant isotypes in mammalian cells are PP2C alpha (PPM1A) and PP2C beta (PPM1 B), which are separate gene products of 43-48 kDa (identity 77%) (Cohen, P.T.W (1994)
Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv. Prot. Phosphatases VoI: 8, 371-276 and Mann, DJ. , Campbell, D. G., McGowan, CH. and Cohen, P.T.W. (1992) Mammalian protein serine/threonine phosphatase type 2C: cDNA cloning and comparative analysis of amino acid sequences. Biochem. Biophys. Acta VoI: 1130, 100-104).
The PP2C family in addition includes PP2C gamma (also called FIN13), PP2C delta and WIP1 (Guthridge, M. A., Bellosta, P. , Tavoloni, N. and Basilico, C (1997) FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression. MoI. Cell. Biol. VoI: 17, 5485-5498 and Tong, Y., Quirion, R. and Shen S.-
H. (1998) Cloning and characterization of a novel mammalian PP2C isozyme. J. Biol.
Chem. VoI: 273, 35282-35290 and Fiscella, M., Zhang, H. L. , Fan, S., Sakaguchi, K., Shen, S., Mercer, W. E., vande Woude, G. F., O'Connor P. M., and Appella, E. (1997)
Wip1 , a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc. Natl. Acad. Sci. USA VoI: 94, 6048-6053).
The list of potential physiological functions proposed for PP2C alpha and PP2C beta comprises a wide range. A growing list of substrates has been described to be specifically dephosphorylated by PP2C alpha and PP2C beta indicating an involvement in various metabolic pathways:
(i) Hydroxymethylglutaryl-CoA reductase (Ball, K. L., Dale, S., Weekes J. and Hardie, D.G. (1994) Biochemical characterization of tow forms of 3-hydrodroxy-3- methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia). Eur. J. Biochem. VoI: 219, 743-750);
(ii) AMP-activated protein kinase (Davies, S.P., Helps, N. R., Cohen, P.T.W. and Hardie, D. G. (1995) 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bone protein phosphatase-1AC. FEBS Lett. VoI: 377, 421-425);
(iii) Ca<2+>-calmodulin-dependent protein kinase Il (Fukunaga, K., Kobayashi, T.,
Tamura, S. and Miyamoto, E. (1993) Dephosphorylation of autophosphorylated Ca<2+>/Calmodulin-dependent protein kinase Il by protein phosphatase 2C. J. Biol. Chem. VoI: 268, 133-137);
(iv) P38 mitogen-activated protein kinase (MAPK) plus MAPK4 and MAPK6 (Terasawa,
T., Kobayashi, T., Murakami, T., Ohnishi, M., Shunsuke, K., Tanaka, O., Kondo, H., Yamamoto, H., Takeuchi, T. and Tamura, S. (1993) Molecular cloning of a novel isotype of Mg<2+>-dependent protein phosphatase beta (Type 2C beta ) enriched in brain and heart. Arch. Biochem. Biophys. VoI: 307, 342-349.13);
(v) Cystic fibrosis transmembrane conductance regulator (Travis, S. M., Berger, H. A. and Welsh, M.J. (1997) Protein phosphatase type 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. VoI: 94, 1105511060); and
(vi) Cyclic - dependent kinases CDK2 and CDK6 (Cheng, A., Kaldis P. and Solomon M.J. (2000) Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms. J. Biol. Chem. VoI: 275, 34744- 34749).
As mentioned before it was recently found that protein phosphatase 2C (PP2C) is implicated in the regulation of apoptosis. It was found that BAD, a proapoptotic member of the Bcl-2 family, is a substrate of PP2C, indicating that PP2C is a responsible factor triggering the pro-apoptotic function of BAD (Klumpp et al., 2003).
BAD (BAD stands for Bcl-2/Bcl-XL-antagonist, causing cell death) represents a point of convergence of several different signal-transduction pathways which are activated by survival factors inhibiting apoptosis in mammalian cells. BAD is a pro-apoptotic protein which binds to the anti-apoptotic proteins Bcl-2 and BcI-XL (Yang, E., Zha, J., Jockel, J. Bioise, L.H., Thompson, CB. and Korsemeyer, S.J. (1995) BAD, a heterodimeric partner for BcI-XL and Bcl-2 displaces Bax and promotes cell death. Cell VoI: 80, 285- 291). This interaction is thought to neutralize the anti-apoptotic effects of Bcl-2/Bcl-XL and may represent one of the mechanisms by which BAD promotes apoptosis. Phosphorylation of BAD results in its interaction with 14-3-3 proteins instead of Bcl-2 or BcI-XL, leading to liberation of these anti-apoptotic proteins which can then interact with Bax to inhibit apoptosis (Zha, J., Harada, H., Yang, E., Jokel, J. and Korsemeyer,
S.J. (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BcI-XL. Cell VoI: 87, 618-628). Two sites on BAD, Ser<112> and Ser<136> have been reported to be phosphorylated in vivo. The dephosphorylation of those sites was shown to be carried out by PP 1 and PP2B (Ayllon, V., Martinez-A., C, Garcia, A., Cayla, X. and Rebollo, A. (2000) Protein phosphatase 1 alpha is a ras-activated bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J. VoI: 19, 2237-2246 and Wang, H. G., Pathan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki. F., McKeon, F., Bobo, T., Franke, T.F. and Reed, J. C. (1999) Ca<2+>-induced apoptosis through calcineurin dephosphorylation of BAD. Science VoI: 284, 339-343). In addition, PP2A also has also been reported to act on phospho-BAD (Deng, X., Ito, T., Carr, B., Mumby, M. and May, W.S. (1998) Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A. J. Biol. Chem. VoI: 273, 34157-34163). The major site on BAD phosphorylated by PKA in vitro just recently discovered was Ser<155>. The phosphorylation of Ser<155> triggers the dissociation of BAD from Bcl-2 and BcI-XL and promotes its interaction with 14-3-3 proteins (Lizcano, J. M., Morrice, N. and Cohen, P. (2000) Regulation of BAD by cAMP- dependent protein kinase is mediated via phosphorylation of a novel site, Ser<155>. Biochem. J. VoI: 349, 547-557).
Previous studies suggested that PP2C is an additional player in the development and progress of apoptosis (see above) and is especially involved in apoptotic processes of endothelial cells. Hence, it appeared as an interesting point to regulate apoptosis and especially to provide a treatment or a prophylaxis for arteriosclerosis. However, until now, there is no such prophylaxis/treatment available as no specific inhibitor for PP2C is available.
Accordingly, it is an object of the present invention to provide an inhibitor for PP2C.
The intention is to provide modulators which can be used in the specific treatment of apoptosis (especially of EC cells) thereby providing a treatment or prevention for cardiovascular diseases, especially arteriosclerosis.
This object is solved according to the present invention by a serine-/threonine protein phosphatase type 2C (PP2C) inhibitor, which comprises at least a derivative of a fatty acid of the group consisting of - fatty acids
- glycerol lipids
- sphingolipids.
Said derivative of a fatty acid has a sufficient fipophilicity to inhibit fatty acid induced activation of PP2C and a free carboxyl group. Apparently the inhibitors according to the present invention are able to bind to PP2C thereby blocking the binding pocket for fatty acids that otherwise activate PP2C such as e.g. oleic acid.
Said derivative of a fatty acid is preferably a fatty acid, more preferably one selected from the group consisting of saturated fatty acids (SFA), unsaturated fatty acids, straight and branched fatty acids.
Branches fatty acids may carry C1 - C5 alkyl groups. Preferred are small alkyl groups such as C1 to C3 alkyl group. Apparently, such branched fatty acids block the binding pocket sterically.
Furthermore, said fatty acid derivative may comprise at least one polyethylene moiety. The polyethylene moiety may be within the fatty acid chain or at the end of it.
According to one embodiment, said fatty acid is a saturated fatty acid (SFA). The saturated fatty acid (SFA) used according to the present invention has preferably a chain length of more than 16 C-atoms. E.g. SFAs can be used wherein the chain length is between C17.0 and C30.0, preferably between C17: and C25:0, even more preferred between C17:0 and C23.0. For example, the SFA could be selected from the group consisting of heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0) and behenic acid (C22:0).
According to a further embodiment said derivative of a fatty acid is either a neutral or a phospho lipid. Suitable examples are triglycerides. The triglycerides preferably comprise at least one of the fatty acids mentioned above.
According to a further embodiment, said inhibitory derivative is an ester. Said ester preferably comprises at least one of the fatty acids mentioned above.
The use of a triglyceride or an ester as described offers the opportunity to develop a pharmaceutical which comprises the active inhibitory substance in form of a prodrug. Such prodrugs are metabolized to release the actual active ingredient in the body through enzymatic cleavage. To provide new protein phosphatase inhibitors and control mechanisms of apoptose satisfies a need in the art by providing compounds that inhibit PP2C which are useful in the diagnosis, prevention, and treatment of disorders caused by apoptosis, including atherogenesis, arteriosclerosis and in the assessment of function of PP2C in vitro and in vivo. As outlined before, despite intensive searches, no inhibitors of PP2C were known in the prior art.
According to a further embodiment said derivative of a fatty acid carries a substituent. Said substituent is preferably a halogen substituent such as a fluorine.
Also provided are the above described inhibitors in form of a salt.
The inhibitors provided with the present invention have several major advantages.
First of all, they are able to selectively inhibit the activation of PP2C by certain activating unsaturated fatty acids like oleic acid. Such activators of PP2C usually have to fulfill special structural requirements as they have to be lipophilic, oxidizable, acidic, and must be cis-configu rated (Klumpp et al., 1998). The results of the present experiments on activation and on specific inhibition by the inhibitors according to the invention can be summarised as follows:
The essential chemical and structural features of compounds essential for activation of PP2Cbeta are as follows:
(i) at least one double bond at a special position;
(ii) (ii) a minimum chain length of 15 C-atoms;
(iii) (iii) a free negatively charged group and
(iv) (Hi) c/s-configuration.
Selected compounds fulfilling these requirements are capable of stimulating PP2Cbeta activity and simultaneously causing apoptosis in ECs. In addition it was demonstrated that BAD, a proapoptotic member of the Bcl-2 family, is a substrate of PP2Cbeta, indicating that PP2Cbeta can trigger the pro-apoptotic function of BAD. In its dephosphorylated state, BAD promotes apoptosis by heterodimerization with the antiapoptotic oncogenes Bcl-2 and BcI-X1.. lmmuncytochemistry revealed that PP2C and its substrate BAD are co-localized within the cytosol of HUVECs. Further the co- localization of these two proteins in macrophages was demonstrated (Fig. 1 ).
Extensive studies with a variety of fatty acids further demonstrated a striking correlation between activation of PP2C and induction of apoptosis in ECs. Also in macrophages the described correlation was verified: SFAs (C14:0 - C22:0), trans fatty acids like eladaic acid (18:1 trans-Δ9) or methylesterderivatives like oleic acid methylester (18:1 cis-Δ9 ester) do not fulfill the structural chemical requirements for PP2C-activation and could also not damage cultured macrophages (Fig. 2 and 6).
However, the inventors found that e.g. SFAs which are not able to activate PP2C in vitro (Fig. 3) were able to inhibit the oleic acid-induced activation of PP2C and thus act as specific inhibitors of fatty acid mediated PP2C activation. These SFAs have a free carboxyl group and are lipophil. Apparently, they displace oleic acid from the active center of the PP2C phosphatase without activating the enzyme and consequently avoid the oleic acid-induced activation by blocking the binding site necessary for activation.
Saturated fatty acids (C14:0 - C22:0) carrying a free carboxyl group and being lipophil thus comprise one preferred embodiment according to the present invention. However, as described above, also other derivatives of fatty acids can act as inhibitors of PP2C. Their characteristics were described above in detail.
Saturated fatty acids (SFA) do not contain double bonds or other functional groups along the lipophilic chain part interacting with the PP2C molecule. However, also unpolar substituents such as the alkylgroup may be present in the molecule (see above).
The analysed mechanism of specific fatty acid activation and inhibition of PP2C in EC cells enables a targeted therapy approach for treatment and prophylaxis of atherogenesis and even arteriosclerosis due to the following reasons:
The vascular endothelium is incessantly exposed to free fatty acids (FFAs) because the lipoprotein lipase (LPL) that is associated with the luminal surface of ECs hydrolyzes triglycerides of circulating lipoproteins (Stins et al., 1992). It was demonstrated that physiologic fatty acids from human lipoproteins activate PP2C in vitro and induce apoptosis in cultured human umbilical endothelial cells (HUVECs)
(Hufnagel et al., 2005). These data suggest PP2C as an additional player in the complex system of atherogenesis/arteriosclerosis. RNA interference (RNAi) mechanisms were employed to demonstrate the causal relationship between fatty acid- induced activation of PP2C and induction of apoptosis by fatty acids in ECs (Schwarz et al., 2006).
The obtained results provide evidence that the phosphatase PP2C in fact causes oleic acid-induced apoptosis in ECs. Therefore, the inhibition of FFAs-mediated PP2C activation is a new therapeutic strategy to prevent the loss of endothelial integrity or rather atherogenesis and/or to treat arteriosclerosis. This therapy strategy is made available by the present invention as inhibitors of PP2C are provided that specifically inhibit the FFA mediated activation of PP2C. Such a selective inhibition is favourable as an unspecific inhibition of apoptotic processes might lead to uncontrollable side- effects.
Another majority of apoptotic cells in atherosclerotic lesions are macrophages. In late lesional events, in which clearance of apoptotic cells is probably meanly, apoptosis leads to secondary necrosis, and necrosis in turn almost certainly promotes plaque destabilization or rupture and this process can ultimately induce vascular obliteration (Tabas, 2004; Ball et al., 1995; Libby et al., 1996). Macrophages produce a variety of biologically active products including proteases, growth factors, apo E (Nathan, 1987;
Kayden et al., 1985) and they are also able to secrete active LPL (Mahoney et al., 1982; Khoo et al., 1981 ). LPL-mediated hydrolysis of triglycerides plays not only a role in the uptake of lipids in ECs but also in macrophages (Floren et al., 1981 ; Lindquist et al., 1983).
Like ECs, macrophages in atherosclerotic plaque are also exposed to high levels of fat. The liberated FFAs by LPL could theoretically activate PP2C within the cytosol, thus acting on apoptosis and this process finally lead to plaque destabilization. As already demonstrated in ECs, immunocytochemistry revealed PP2C and its substrate BAD within the cytosol of macrophages (please refer to Fig. 1). Furthermore it could be observed that in macrophages the same striking correlation between PP2C-activation and induction of apoptosis like in ECs (please refer to Fig. 2).
The present invention provides inhibitors of FFA and especially oleic acid-induced activation of PP2C. It was demonstrated by experiments that the inhibitors according to the present invention can protect both cell types (EC and macrophages) from the damaging effect of FFAs as exemplified by oleic acid.
According to one embodiment of the present invention the inhibitors are SFAs with a sufficient lipophilicity to bind to and to inhibit activation of PP2C by FFAs. Preferably, they have a chain length longer than 16 C-atoms. It was shown that they are able to inhibit the oleic acid-induced activation of PP2C in vitro. Furthermore, it could also be exhibited that these SFAs abolish the damaging effect of oleic acid in cultured HUVECs and macrophages and thus prove their therapeutic potential. These results provide a targeted inhibition of fatty acid-induced apoptosis in cells such as EC and macrophages and hence provide a new strategy to prevent atherogenesis and to treat/prevent arteriosclerosis.
Saturated fatty acids form straight chains and, as a result, can be packed together very tightly, allowing living organisms to store chemical energy very densly. The fatty tissues of animals contain large amounts of long-chain saturated fatty acids. In IUPAC nomenclature, fatty acids have an -oic acid suffix.
For example, the effect of SFAs with different chain length (C14:0 - C22:0) in combination with oleic acid on PP2C activity was tested in the experiments described below.
Surprisingly, SFAs with a insufficient lipophilicity e.g. with a chain length shorter than 16 C-atoms, like e.g. myristic acid (C14.0), pentadecanoic acid (C15.O), and palmitic acid (C16:0), could not influence the oleic acid-induced activation of PP2C, wherein
SFAs with a chain length longer than 16 C-atoms, like e.g. heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), and behenic acid (C22:0), could do so by inhibiting the activation of PP2C (Fig. 4).
Therefore, compounds with a sufficient lipophilicity allowing interaction with the binding pocket of PP2C (e.g. molecules having a chain length longer than 16 C-atoms) and with a free carboxyl group are able to interact with the binding pocket of PP2C wherein the activating FFAs bind. However, these molecules according to the present invention cause no activation of the PP2C enzyme. However, they avoid the binding of oleic acid (as an example of an activated FFA), thus inhibiting the oleic acid-induced activation by preventing binding of these activators. These molecules thus basically antagonise the activators.
Exposure of HUVECs and macrophages to myristic acid (C14:0), pentadecanoic acid (C15:0) and palmitic acid (C16:0) in combination with oleic acid caused apoptosis. In contrast, treatment with e.g. heptadecanoic acid (C17:0) or stearic acid (C18:0) in combination with oleic acid in an equimolar (Fig. 7 and 8) or higher concentration (data not shown) abolished the oleic acid-induced cell death. These data proves the inhibitory effect of the molecules according to the present invention.
Double staining with the DNA fluorochrome Hoechst 33258 and Nile blue revealed that the different SFAs and oleic acid were similarly taken up by HUVECs and preferably heptadecanoic acid (C17:0) and stearic acid (C18:0) could slow down the damaging effect of oleic acid (Fig. 9). This could be a competitive displacement from oleic acid from the respective center of PP2C, which slows down the oleic-induced programmed cell death of ECs and macrophages. The striking correlation observed between inhibition of oleic acid-induced activation of PP2C in vitro and inhibition of oleic acid- induced apoptosis in both described cell types points to a hitherto unknown mechanism of inhibition of the fatty acid-induced activation of PP2C. In conclusion, apoptosis of ECs and macrophages mediated by PP2C activation is a crucial step in atherogenesis/arteriosclerosis and plaque destabilisation. Unsaturated physiologic FFAs like oleic acid increased PP2C activity dramatically, thus acting on apoptosis by dephosphorylation of the proapoptotic Bcl-2-protein BAD in various cell types (Hufnagel et al., 2005; Schwarz et al., 2006; Zhu et al., 2005). Since most of the fatty acids are transported by lipoproteins and have to pass the endothelium to be absorbed by the body, this mechanism of fatty acid- induced apoptosis should be a crucial factor for atherogenesis in organs with high energy consumption like the heart and hence for the development arteriosclerosis.
This could be an explanation why the coronary arteries are mostly involved in arteriosclerosis and why patients with diabetes mellitus exhibit macroangiopathys. Further this hypothesis could explain why lipid loaded macrophages in atherosclerotic plaque undergo apoptosis and finally induce plaque instability and rupture.
Therefore, inhibition of the fatty acid-induced mediated activation of PP2C by the inhibitors of the present invention is a new and promising therapeutic strategy for preventing apoptosis in EC and/or macrophage cells. The experimental results demonstrate that it is possible to specifically inhibit the fatty acid-induced apoptosis of ECs and macrophages with the inhibitors according to the invention and provide new insights to avoid the fatty acid-induced progression of atherogenesis/arteriosclerosis.
Furthermore, a pharmaceutical composition is provided comprising at least one inhibitor as described above as a physiologically active substance and a pharmaceutically acceptable carrier, wherein said inhibitor possesses sufficient lipophilicity to bind to and inhibit PP2C in particular inhibiting the FFA induced activation.
Also part of the present invention is the use of an inhibitor according to the present invention for the manufacture of a medicament for the treatment or prevention of fatty acid induced apoptosis in cells, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group.
The inhibitors according to the present invention may be used for the manufacture of a medicament for the treatment or prevention of cardiovascular diseases, especially for the treatment or prevention of atherogenesis and/or arteriosclerosis, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group
The following figs, illustrate exemplary results of preferred embodiments according to the present invention: Fig. 1 shows a Western blots for detection of PP2C and BAD in macrophages. The localization of PP2Cbeta and BAD in macrophages is shown. (A) shows Western blots for detection of PP2Cbeta and BAD. Macrophage proteins (30 μg/lane) were electrophoresed on 15% SDS-polyacrylamide gels and after transfer to nitrocellulose immunoblotted with an antibody specific for PP2Cbeta diluted 1:1000 (A, left panel) or with an antibody against BAD diluted 1:1000 (A, right panel). (B-E) Depicted are fluorescence laser scanning photographs showing subcellular co-localization of PP2Cbeta and BAD. Macrophages cell culture and immunocytochemistry were performed as described in Section 2. (B) Incubation with the antibodies against
PP2Cbeta. (1:100), and (C) directed against BAD (1 :350). (D) Merge. (E) Control omitting primary antibodies against PP2Cbeta and BAD.
Fig. 2: shows two bar graphs, wherein in item (A) the effect of SFAs on PP2Cbeta activity and in item (B) the effect of oleic acid in combination with different SFAs on
PP2Cbeta activity are shown. (A) shows the effect of SFAs on PP2Cbeta activity. Dephosphorylation of [32P]casein by PP2C was measured upon addition of various fatty acids. Oleic acid stimulated the activity 9-fold, wherein SFAs with different chain lengths failed to activate the enzyme. Fatty acids added were oleic acid (18:1 cis-Δ9), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid
(17:0), stearic acid (18:0), arachidic acid (20:0) and behenic acid (22:0). Dephosphorylation of [32P]casein was determined with 98 ng PP2Cbeta per incubation. Activity in the absence of fatty acids (1) was 6.8 nmol P/min/mg. (B) shows the effect of oleic acid in combination with different SFAs on PP2Cbeta activity. Phosphatase activity was measured with oleic acid and oleic acid in combination with different SFAs.
Short chain SFAs (<17:0) could not affect the oleic acid-induced activation of PP2C, wherein SFAs with a chain length longer than 16 C-atoms could inhibit activity. Dephosphorylation of [32P]casein was determined with 98 ng PP2Cbeta per incubation. Activity in the absence of fatty acids (1) was 6.8 nmol Pj/min/mg.
Fig. 3: shows two bar graphs, wherein in item (A) the effect of SFAs with different chain length on ECs and in item (B) the effect of different SFAs (14:0 - 18:0) on macrophages are shown. (A) illustrates the effect of SFAs with different chain length on ECs. HUVECs were incubated with 200 μM of different SFAs. Control cells were treated with vehicle only (0,2% DMSO). Apoptotic damage was revealed by Hoechst
33258 staining as described before. Apoptotic cells are expressed as percentage of total number of cells ± S.D. ***p<0,001, compared with vehicle-treated control. (B) demonstrates the effect of different SFAs (14:0 - 18:0) on macrophages. Cells were incubated with myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) at a concentration of 200 μM.
Morphology was analyzed under a fluorescence microscope after nuclear staining with Hoechst 33258. The cells showing fragmented nuclei and condensed chromatin were counted as apoptotic cells. Apoptotic cells are expressed as a percentage of total number of cells ± S.D. ***p<0,001.
Fig. 4 shows two bar graphs, wherein in item (A) effect of heptadecanoic acid and stearic acid in view of the protection of ECs from oleic acid-induced apoptosis an in item (B) the effect of heptadecanoic acid and stearic acid in the protection of macrophages from oleic acid-induced apoptosis are shown. (A) depicts that heptadecanoic acid and stearic acid protect ECs from oleic acid-induced apoptosis. Double treatment of HUVECs with oleic acid (18:1 cis-Δ9) and different short chain
SFAs (myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (C16:0)) could not abolish the toxic effect of oleic acid, wherein heptadecanoic acid (17:0) and stearic acid (18:0) could slow down the oleic acid-induced apoptosis of ECs. The percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Control cells received vehicle only (0,4% DMSO). Apoptotic cells are expressed as percentage of total number of cells ± S.D. ***p<0,001 , compared with vehicle-treated control. (B) Heptadecanoic acid and stearic acid protect macrophages from oleic acid-induced apoptosis. Double treatment of macrophages with oleic acid (18:1 cis-Δ9) and different short chain SFAs (myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0)) could not abolish the toxic effect of oleic acid, wherein heptadecanoic acid (17:0) and stearic acid (18:0) could inhibit the oleic acid-induced apoptosis of ECs. The percentage of apoptotic cells was determined by nuclear staining with Hoechst 33258. Control cells received vehicle only (0,4% DMSO). Apoptotic cells are expressed as percentage of total number of cells ± S.D. ***p<0,001, compared with vehicle-treated control.
Fig. 5 shows a Nile blue staining of the cells, wherein the uptake of SFAs and with or without oleic acid is demonstrated. ECs were incubated with vehicle (0.4% DMSO) oleic acid (18:1), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0) and stearic acid (18:0) for 6 h and 24 h at a concentration of
200 μM, respectively. Double treatment with oleic acid revealed, that myristic acid, pentadecanoic acid (15:0), palmitic acid (16:0) are not able to protect HUVECs from oleic acid-induced apoptosis, cognisable on the apoptotic morphology of ECs. In contrast, heptadecanoic acid (17:0) and stearic acid (18:0) slow down the toxicity of oleic acid and protect ECs. Apoptotic damage was revealed by Hoechst 33258 staining as described in Section 2.
Fig. 6 shows further examples of inhibitors. Examples
The concept of the present invention was proven inter alia by the following experiments:
1. Materials and Methods
1.1 Materials, reagents and equipment
HUVECs and cell growth medium containing 2% heat-inactivated fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, 1 ng/ml fibroblast factor and antibiotics were purchased from Promocell (Heidelberg, Germany);
THP-1 cells were obtained as a gift from lnstitut fur Atheroskleroseforschung Mϋnster, Germany; THP-1 monocytes cell medium containing RPMI-1640, FSC 10%, penicillin 100U/ml, streptomycin 100 μg/ml, L-glutamine 2 mM, sodiumpyruvate 100 mM, phorbol-12-myristat-13-acetat (PMA) and mercaptoethanol were received from Sigma Aldrich (Taufkirchen, Germany).
Hoechst 33258, Nile blue and fatty acids (myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, arachidic acid and behenic acid), bovine serum albumin (BSA) were obtained from Sigma Aldrich (Taufkirchen, Germany). Nile blue staining was performed with a confocal laser scanning microscope Zeiss LM510 (Jena, Germany) and analysis of apoptotic cells was accomplished with the fluorescent microscope Ax/overt 100 Zeiss (Jena, Germany).
ECL (enhanced chemoluminescence) Western blotting detection reagents and secondary antibody rabbit IgG, peroxidase linked, from donkey, were from Amersham Pharmacia Biotech (Freiburg, Germany). Antibodies against BAD were purchased from New England Biolabs GmbH (Frankfurt, Germany) and recombinant antibody against PP2Cbeta was prepared as described (Klumpp et al., 2000). Biotinylated secondary anti rabbit antibody and secondary Texas-red anti mouse antibody were obtained from Alexis (Grϋnberg, Germany).
1.2. Measurement of PP2Cbeta activity Activity of PP2Cbeta was measured using [32P]-labelled casein as a substrate
(Mc Gwoan et al. 1988, Klumpp et al., 1998). PP2Cbeta assays contained 30 mM Tris- HCI, pH 7.0, 0.1% 2-mercaptoethanol, 0.6 mg/ml BSA, 1 μM [32P] casein (5 x 104 cpm), and 10-100 ng PP2Cbeta in a final volume of 30 μl. PP2Cbeta is characterized by its dependence on divalent cations for activity. If not otherwise indicated, 1 mM magnesium acetate was present. Reactions were terminated after 10 min at 37°C adding trichloroacetic acid, the samples were centrifuged, and the supernatant was analyzed for [32P]-phosphate content. For determination of PP2Cbeta activity in HUVECs (5-10 g protein per assay), 100 nM okadaic acid was added to inhibit phosphatases type-1 and type-2A. Assays were additionally run in the absence and in the presence of magnesium ions (1 mM EDTA vs. 1 mM Mg2+) to verify identity of PP2Cbeta.
1.3. Human umbilical vein endothelial cell and macrophages cell cultures
HUVECs were cultured in endothelial cell growth medium to 90% confluence. In all experiments cells were used at passages 3-6 and morphology was controlled prior to treatment. The cells were seeded at a density of 500000 cells per 9.6 cm2 dishes.THP-
1 monocytes were cultured in suspension at a density of 4 million cells/ 5 ml for four days. For differentiation to macrophages, monocytes received 100 ng/ml phorbol-12- myristic-13-acetate and 50 μM 2-mercaptoethanol (Dory et al., 1993). After 72 h culture, monocytes differentiated to macrophages and became adherent. Experiments were only accomplished with adherent and confluent macrophages (density > 1 million cells/9.6 cm2 dishes). Both cell types were cultured at 37°C in an incubator supplemented with 5% CO2 and 95% air.
1.4. Fatty acid treatments of HUVECs and macrophages After the replacement of culture medium, HUVECs and macrophages were incubated for an additional 48 h. Cell cultures were then treated for 24h with oleic acid (200 μM) to induce apoptosis, with different concentrations of c) or with combinations of both. The FFAs were dissolved in dimethylsulfoxid (DMSO), and DMSO was used as control. The final concentration of DMSO in the culture medium did not exceed 0.5%.
1.5. Nuclear staining with Hoechst and Nile blue
Fatty acid-treated cells were washed with phosphate buffered saline (PBS)1 fixed for 30 min with paraformaldehyde and then incubated for 30 min with the DNA fluorochrome Hoechst 33258 (10 μg/ml) in methanol at room temperature in the dark. After washing with PBS, the nuclear morphology of HUVECs and macrophages was analyzed under a fluorescent microscope at an excitation wave length of 350 nm and an emission wavelength of 450 nm. Cells showing shrunken or fragmented nuclei or chromatin condensation were counted as apoptotic cells. All values were expressed as means ± S.D.
Nile blue was used to reveal uptake of FFAs in ECs. After fixation with paraformaldehyde for 30 min, HUVECs were washed with PBS, stained with Nile blue solution (10 μg/ml) for 2 h and analyzed under a confocal laser scanning microscope at a wavelength of 488/525 nm. 1.6. Protein determination
The concentration of proteins was determined by BCA- or Lowry-assay (Lowry et al., 1951) using BSA as a standard.
1.7. Western blots and immunocytochemistry
BAD and PP2Cbeta were analyzed on 15% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature with 5% non-fat milk powder in TBS-T (Tris buffered saline with Tween) (10 mM Tris-HCI pH 7.6, 150 mM NaCI, 0.1% Tween 20), incubated overnight at 4°C in TBS-T containing 5% BSA and primary antibody (anti-BAD and anti-PP2Cbeta 1 :1000, respectively), followed by incubation in 5% non-fat milk powder in TBS-T for 1 h with peroxidase- conjugated anti-rabbit IgG (1 :2500). Blots were developed with ECL reagent.
For immunocytochemistry, macrophages were fixed with paraformaldehyde for 30 min at room temperature. Cell membrane was permeabilized by incubation with 0.2% Triton
X-100 for 5 min. Unspecific binding was blocked by 5% goat serum in PBS for 30 min. Cells were washed three times with PBS and subsequently incubated with polyclonal anti-PP2Cbeta antibody (1 :100) overnight at 4°C. Macrophages were washed with PBS, exposed to a biotinylated secondary antibody (1 :200) for 1 h at room temperature and then incubated with fluorescein isothiocyanate Avidin for 1 h in darkness. For the detection of BAD, the cells were blocked with 5% horse serum in PBS and incubated with BAD-antibody (1 :200) at 4°C overnight. After washing with PBS, cells were exposed to Texas Red anti-mouse secondary antibody (1 :200) for 1 h in dark. Controls were performed by omitting the primary antibody.
1.8. Statistics
All values are given as means ± S. D.. One way analysis of variance (ANOVA) followed by Scheffe's test was applied. PP2Cbeta-assays were run in duplicate, respectively, and independently repeated at least twice.
2. Results
2.1. Co-localization of PP2Cbeta and BAD in macrophages
The phosphorylation status of BAD is crucial to life and death (Zha et al., 1996). PP2Cbeta activated by unsaturated fatty acids is one of the phosphatases that acts on
BAD (Klumpp et al., 2003). In previous work, we could demonstrate the co-localization of these two proteins in HUVECs (Hufnagel et al., 2005). Therefore, we first set out to search for PP2Cbeta and BAD in macrophages. An antibody specific for PP2Cbeta and BAD showed the presence of these proteins also in macrophages (Fig. 1A). More detailed studies using immunocytochemistry localized PP2Cbeta and BAD within the cytosol of macrophages (Fig. 1 B; 1C). Like in ECs, the demonstrated co-localization can be interpreted as a prerequisite for molecular interactions among PP2Cbeta and BAD in macrophages.
2.2. Effects of oleic acid and derivatives in macrophages
Oleic acid (18:1 cis-AQ) stimulates the activity of PP2Cbeta at least 10-fold, wherein the trans- and methylester derivatives do not (Klumpp et al., 1998). Overall, the structural requirements for activation of PP2Cbeta strikingly correlate with the induction of apoptosis in endothelial cells (Hufnagel et al., 2005). To evaluate this relation to macrophages, they were treated with 18:1 compounds and tested for survival. Exposure of macrophages to oleic acid (250 μM) for 24 h caused cell death (Fig. 2). In contrast, the corresponding trans derivative and the oleic acid methylester both were not harmful when administered in the same concentration range as oleic acid (Fig. 2).
2.3. Saturated fatty acids with a particular chain length inhibit the oleic acid- induced activation of PP2Cbeta in vitro.
In previous work we set out to search for regulatory components of PP2C (Klumpp et al., 1998). We found out that the activity of PP2C increases at a physiological concentration of 0.5-1 mM free Mg2+ after the addition of certain unsaturated fatty acids, like oleic acid, 10-12 fold. The result of our studies demonstrated that activators of PP2C must fulfill special structural requirements, as a minimum chain length of 15 C- atoms, cis-configuration and a free carboxyl group. For example SFAs are incapable of stimulating the activity of PP2C (Klumpp et al., 1998).
For instance, specific inhibitors of PP2C are not available and targeting subunits are unknown. To further study the regulation of PP2C, we wanted to elucidate if SFAs with different chain length are able to abolish the oleic acid-induced activation of PP2C. Treatment of PP2C with SFAs with chain lengths from C-14:0 until C-22:0 showed no activation of the enzyme (Fig.3). Double-treatment of PP2C with oleic acid and SFAs showed interesting new results: SFAs with a chain length shorter than 16 C-atoms, like myristic acid (C14:0), pentadecanoic acid (C15:0) and palmitic acid (C16:0), were not able to inhibit the activation-capacity of oleic acid, wherein SFAs with a chain length of more than 16 C-atoms, like heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20.0) and behenic acid (C22:0) inhibited the oleic acid induced activation of PP2C (Fig. 4) in vitro. 2.4. Effects of saturated fatty acids on oleic acid-induced apoptosis in HUVECs and macrophages
In further experiments we wanted to analyze if these "long-chain" SFAs are also able to abolish the oleic acid-induced apoptosis in ECs (Hufnagel et al., 2005) and macrophages. First of all, the different cell types were treated with different SFAs
(C14:0- C22:0). Overall, the structural requirements for activation of PP2C correlated strikingly with the induction of apoptosis in ECs and macrophages, and the tested SFAs were not able to influence the viability of both cell types (Fig. 5 and 6). In parallel to the described in vitro results, we attempted in the next experimental step to elucidate how double-treatment of ECs and macrophages with SFAs and oleic acid influences the oleic acid-induced apoptosis. Myristic acid (C14.0), pentadecanoic acid (C15:0) and palmitic acid (C16:0) were not able to abolish the toxic affect of oleic acid, wherein heptadecanoic acid (C17:0) and stearic acid (C18:0) significantly inhibited the oleic acid-induced apoptosis in both cell types and are therefore protective (Fig. 7 and 8). Arachidic acid (C20:0) and behenic acid (C22:0) were almost insoluble in the endothelial cell growth medium and therefore could not be tested.
2.5. Fatty acid-uptake by HUVECs
To demonstrate the fatty acid-uptake, HUVECs were treated with Nile blue, a dye to explore the fate of lipids and to possibly perceive the location within ECs. Control cells received 0,5% DMSO as control for lipid solvent and subsequent staining resulted in a weak fluorescence reflecting endogenous lipids. In contrast, addition of myristic acid
(C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), heptadecanoic acid (C17:0) and stearic acid (C18.0) resulted in a bright fluorescence. Double treatment of both cell types with the same lipids and oleic acid yielded only cell debris in the presence of
SFAs with a chain length from C14:0 up to C16:0. Heptadecanoic acid and stearic acid protect the ECs and could slow down the oleic acid-induced apoptosis (Fig. 9).
Finally we could demonstrate that inhibition of oleic acid-induced stimulation of PP2C activity by SFAs with a chain length longer than 16 C-atoms correlated with the inhibition of oleic acid-induced apoptosis in HUVECs and macrophages.

Claims

Claims
1. A serine-/threonine protein phosphatase type 2C (PP2C) inhibitor which comprises at least one derivative of a fatty acid of the group consisting of - fatty acids
- glycerol lipids
- sphingolipids
2. Inhibitor according to claim 1 , wherein said derivative of a fatty acid has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group.
3. Inhibitor according to claim 1 , wherein said derivative of a fatty acid is a fatty acid, preferably one selected from the group consisting of saturated fatty acids (SFA), unsaturated fatty acids, straight and branched fatty acids.
4. Inhibitor according to claims 1 to 3, comprising at least one polyethylene moiety.
5. Inhibitor according to claim 3, wherein said fatty acid is a saturated fatty acid
(SFA).
6. Inhibitor according to claim 5, wherein the saturated fatty acid has a chain length of more than 16 C-atoms.
7. Inhibitor according to claim 5 or 6, wherein the SFA has a chain length of C17:0 to C30:0, preferably C17: to C25:0.
8. Inhibitor according to claim 5 or 6, wherein it is selected from the group consisting of heptadecanoic acid, stearic acid, arachidic acid and behenic acid.
9. Inhibitor according to claim 1 or 2, wherein said derivative of a fatty acid is either a neutral or a phospho lipid.
10. Inhibitor according to claim 9, which is a triglyceride.
11. Inhibitor according to claim 9, wherein the triglyceride comprises at least one of the fatty acids mentioned in the above claims.
12. Inhibitor according to at least one of the above claims 1 to 11 , wherein said derivative of a fatty acid is substituted preferably with a halogen substituent.
13. Inhibitor according to at least one of the above claims which is present in form of a salt or an ester.
14. A pharmaceutical composition comprising at least one inhibitor according to at least one of the claims 1 to 13 as a physiologically active substance or a prodrug thereof and a pharmaceutically acceptable carrier, wherein said inhibitor has a sufficient lipophilicity to bind to and inhibit PP2C.
15. The pharmaceutical composition according to claim 14, wherein the inhibitor is present in form of a prodrug which releases the physiologically active substance upon conversion in the body.
16. The pharmaceutical composition according to claim 15, wherein said produg is a triglyceride or an ester and wherein said triglyceride or said ester comprises a fatty acid according to at least one of the claims 3 to 8.
17. Use of an inhibitor according to at least one of the claims 1 to 13 for the manufacture of a medicament for the treatment or prevention of fatty acid induced apoptosis in cells, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group.
18. Use according to claim 17, wherein said inhibitor is used for the manufacture of a medicament for the treatment or prevention of cardiovascular diseases, especially for the treatment or prevention of atherogenesis and/or arteriosclerosis, wherein said inhibitor has a sufficient lipophilicity to inhibit fatty acid induced activation of PP2C and comprises a free carboxyl group.
19. Use of a composition comprising according to any of claims 14 to 16 for the manufacture of a medicament for the treatment or prevention of cardiovascular diseases, especially for the treatment or prevention of atherogenesis and/or arteriosclerosis.
20. Use of the inhibitor according to any of claims 1 to 13 in an in vitro screening with PP2C and for testing apoptosis.
21. Use of the inhibitor according to claims 20 for testing the inhibition of the oleic acid-induced activation of PP2C in an in vitro screening.
22. Use of a compound according to any of claims 1 to 13 as an inhibitor for PP2C to inhibit fatty acid induced activation of PP2C.
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