Classifications

• • 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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the subject of the present invention is compounds capable of interacting with the protein PEA-15 (Enzyme Enriched Phosphoprotein in Astrocytes with a Molecular Weight of 15 kDa), their fluorescent derivatives, and the use of these compounds in screening and diagnostic methods, and pharmaceutical compositions.
• PEA-15 Enzyme Enriched Phosphoprotein in Astrocytes with a Molecular Weight of 15 kDa
• PEA-15 is a small, 130-amino acid cytoplasmic protein abundantly expressed in the brain, particularly in astrocytes, and, more weakly, ubiquitously in many other tissues.
• DED Death Effector Domain
• the genomic sequence of PEA-15 is composed of four exons and spans about 10.2 kb of genomic DNA (Wolford et al., 2000, Gene, 241: 143).
• PEA-15 is present in vivo in various forms: non-phosphorylated, mono- and bi-phosphorylated, each having a different biological activity.
• PEA-15 is a multifunctional protein capable of interacting with many partners by means of its different functional domains, and according to its degree of phosphorylation (Renault et al., Biochem, Pharmacol, 2003, 66: 1581). To date, seven partners of PEA-15 have been identified, participating in the multiple functions fulfilled by this protein, namely FADD, caspase 8, Omi / HtraA2, ERK1 / 2, Akt, Rsk2, and phospholipase D1. Through these multiple interactions, PEA appears to play a central role in many physiological and / or pathological cellular processes.
• this protein possesses, in particular, the properties of inhibiting apoptosis, of inhibiting the entry of cells into the cell cycle, of being involved in the recovery of integrin signaling inhibited by expression.
• H-Ras oncogene to inhibit cell proliferation, and to be involved in glucose transport and insulin secretion (Renault et al., Biochem Pharmacol., 2003, 66: 1581).
• deletion of PEA-15 expression in astrocytes results in an increase in sensitivity of these cells to apoptosis induced by TNF alpha (Kitsberg et al., J.
• PEA-15 expression has been observed in various tumors such as gliomas, ovarian cancer, kidney cancer, breast cancer, in hepatocellular carcinomas, lymphomas or tumors.
• melanomas Hwang et al., Genomics, 1997, 42: 540, Bera et al., Proc Natl Acad Sci USA, 1994, 91: 9789).
• PEA-15 overexpression has been observed in fibroblasts, striated muscles and adipose tissue of patients with type II diabetes (Condorelli et al., EMBO J., 1997, 17: 3858), and suppressing the expression of this protein has been shown to restore insulin secretion in response to glucose.
• EP 1 189 060 proposes the use of PEA-15 as a marker and a therapeutic target in neurodegenerative diseases. Therefore, it appears that PEA-15 could serve as a therapeutic target in many pathological conditions. However, to date, there are no easily accessible compounds capable of modulating the activity of this protein. Moreover, there are also no tools that can easily screen such compounds.
• the present invention aims to give satisfaction in these terms.
• n, p, r, R 1 , R 2 , R 3 , R 4 and A are as defined below, are capable of interacting with and modulating the activity of PEA-15.
• a compound according to the invention such as, for example, the fluorescent compound 6D6-1, detailed below, is capable of specifically interacting with a PEA-15 protein and of modulating its biological activity.
• a compound of the invention in particular fluorescent in combination with a fluorescent fusion protein GFP-PEA-15 (Green Fluorescent Protein) to develop processes implementing a fluorescence resonance energy transfer (FRET) allowing, for example, the screening of agents likely to interact with PEA-15 as well as the diagnosis and / or prognosis of disease states involving PEA-15.
• GFP-PEA-15 Green Fluorescent Protein
• the present invention relates to a compound of formula (I) below:
• n can be 0 or 1
• p can represent an integer ranging from 1 to 6, and in particular ranging from 2 to 4
• r can represent an integer ranging from 1 to 12, and in particular varying from 2 to 6, and in particular is equal to 4
• R 1 may represent a hydrogen atom, a linear or branched, saturated or unsaturated C 1 to C 20 alkyl radical, a saturated or unsaturated C 3 to C 10 cycloalkyl radical or a C 6 to C 6 aryl radical; 10 , optionally substituted with one or more halogen atoms, one or more C 1 -C 6 alkoxy radicals or one or more C 1 -C 10 alkyl radicals,
• R 2 may represent an amino acid side chain or an amino acid derivative
• -COR 3 may represent an acyl radical bearing a basic entity R 3, chosen in particular from the radicals of the following formulas: in which * symbolizes a covalent bond with the acyl radical, Y may represent N or N + R 7 , and R 6 and R 7 may represent, independently of one another, a hydrogen atom, an alkyl radical or C 1 to C 20 , saturated or unsaturated, linear or branched, a saturated or unsaturated C 3 to C 10 cycloalkyl radical, a C 6 to C 10 aryl radical, optionally substituted with one or more halogen atoms, a or more C 1 -C 6 alkoxy radical (s), or one or more C 1 -C 10 alkyl radicals,
• R 4 can represent a hydrogen atom, a linear or branched, saturated or unsaturated C 1 to C 10 alkyl radical, a saturated or unsaturated C 3 to C 10 cycloalkyl radical or a C 6 to C 6 aryl radical; 10 , optionally substituted with one or more halogen atoms, one or more C 1 -C 6 alkoxy radicals or one or more C 1 -C 10 alkyl radicals,
• A may represent a radical derived from a xanthene residue, in particular a 9-phenylxanthene residue, an acridine residue, in particular a 9-phenyl acridine residue, or a 4-bora-3a, 4a-diaza indacene residue, and its derivatives.
• the term “remainder” refers to a given molecule, the molecule in the form of a radical.
• A may represent a fluorescent marker.
• the present invention relates to a method for screening an agent capable of interacting with a PEA-15 protein, or an analogue thereof, comprising at least the steps of: (a) bringing together at least one PEA-15 protein carrying a fluorescent marker D, or one of its analogues, and at least one particularly fluorescent compound according to the invention, under conditions conducive to an interaction with said protein,
• a and D being such as to define an acceptor-donor pair of fluorescence energy, suitable for carrying out a fluorescence resonance energy transfer
• step a) measuring a first signal S 1 , characteristic of the assembly obtained in step a) by irradiation at a wavelength for exciting the fluorescence energy donor, (d) putting the assembly obtained in step a) in the presence of a medium presumed to contain at least one agent to be screened under conditions conducive to an interaction with said protein,
• step c) measuring a second signal S 2 , of the same nature as S 1 , characteristic of the assembly obtained in step c) by irradiation at a wavelength for exciting the fluorescence energy donor,
• PEA-15 protein analog is intended to denote a peptide-type compound having sequence homology with
• PEA-15 and a similar biological activity, as well as variants likely to result from the alternative splicing of the mRNA encoding this protein, such as that described by Underhill et al. (Genome Mamm., 2001, 12: 172), as well as fragments of this protein or peptide-like compounds having the ability to bind a compound of the formula according to the invention.
• biological activity is meant the biological properties of the PEA-15 protein, especially as indicated above.
• Sequence homology is intended to denote a sequence identity of at least 85%, in particular at least 90% and more particularly at least 95% of the analogue with the PEA-15 protein, and in particular the characteristic sequences of
• PEA-15 (Renault et al., Biochem Pharmacol 2003, 66: 1581), namely the DED domain, and in particular the conserved sequence RxDLF, the NES (Nuclear Export Signal) domain, the peptide sequences involved in the interaction of PEA-15 protein with its different protein partners (eg ERK1 / 2, Akt, FADD, caspase 8) and peptide sequences including phosphorylation sites by protein kinase C (PKC) or the calcium / camodulin-dependent protein kinase type II, namely respectively the motifs LTRIPSAKK (S 104) and DIRQPSEEIIK (S 116) (S: phosphorylated serine).
• PKC protein kinase C
• S 116 DIRQPSEEIIK
• the subject of the present invention is a method for screening an agent capable of interacting with a PEA-15 protein, or one of its analogues, comprising at least the steps consisting in:
• step c) bringing together the assembly obtained in step a) with an agent to be screened under conditions conducive to an interaction with said protein, (d) measuring a second signal S 2 , of the same nature as S 1 , characteristic of the set obtained in step c),
• the present invention relates to a method of diagnosis and / or prognosis of a pathological condition likely to involve PEA-15 by detection and, possibly, by quantification of PEA-15 in a sample biological system taken from an individual comprising at least the steps of:
• step a) measuring a first signal S 1 characteristic of the assembly obtained in step a) by irradiation at a wavelength for exciting the fluorescence energy donor,
• step a) bringing the assembly obtained in step a) in the presence of a presumed biological sample comprising at least one PEA-15 protein under conditions conducive to the interaction of said PEA-15 protein of the biological sample with said compound according to the invention
• step c) measuring a second signal S 2 , of the same nature as S 1 , characteristic of the assembly obtained in step c) by irradiation at a wavelength for exciting the fluorescence energy donor,
• the present invention also relates to an isolated complex comprising at least one PEA-15 protein and at least one compound of formula according to the invention.
• the present invention also relates to a kit for screening an agent capable of interacting with a PEA-15 protein, or one of its analogues, comprising: at least one PEA-bearing protein a fluorescent marker D or a purification marker, and - at least one compound according to the invention, where appropriate, A and D being such as to define a fluorescence energy acceptor-donor pair, suitable for use in implementation of a fluorescence resonance energy transfer.
• the compounds of the invention have the following general formula (I): (I)
• n may be 0 or 1
• - P may represent an integer ranging from 1 to 6, and in particular ranging from 2 to 4
• r may represent an integer ranging from 1 to 12, and in particular varying from 2 to 6, and in particular equal to 4
• R 1 may represent a hydrogen atom, a linear or branched, saturated or unsaturated C 1 to C 20 alkyl radical, a saturated or unsaturated C 3 to C 10 cycloalkyl radical or a C 6 to C 6 aryl radical; 10 substituted by one or more atom (s) halogen, one or more radical (s) alkoxy, C 1 -C 6, or one or more radical (s) C 1 to
• R 2 may represent an amino acid side chain or an amino acid derivative
• -COR 3 may represent an acyl radical bearing a basic entity R 3, chosen in particular from the radicals of the following formulas:
• Y may represent N or N + - R 7
• R 6 and R 7 may represent, independently of one another, a hydrogen atom, an alkyl radical C 1 to C 20 , saturated or unsaturated, linear or branched, a saturated or unsaturated C 3 to C 10 cycloalkyl radical, a C 6 to C 10 aryl radical, optionally substituted by one or more halogen atoms, one or more C 1 -C 6 alkoxy radicals or one or more C 1 -C 10 alkyl radicals,
• R 4 can represent a hydrogen atom, a linear or branched, saturated or unsaturated C 1 to C 10 alkyl radical, a saturated or unsaturated C 3 to C 10 cycloalkyl radical or a C 6 to C 6 aryl radical; 10 , optionally substituted with one or more halogen atoms, one or more C 1 -C 6 alkoxy radicals or one or more C 1 -C 10 alkyl radicals,
• A may represent a radical derived from a xanthene residue, in particular from a 9-phenylxanthene residue, from an acridine residue, in particular from a 9-phenyl acridine residue, or from a 4-bora-3a, 4a residue. -diaza indacene, and its derivatives.
• A may represent a fluorescent marker.
• the term “remainder” refers to a given molecule, the molecule in the form of a radical.
• derivative is intended to denote tautomeric forms, stereoisomeric forms, polymorphic forms, pharmaceutically acceptable salts and pharmaceutically acceptable solvates.
• the term “tautomeric form” is intended to mean one of the isomers whose structures differ by the position of an atom, generally hydrogen, and one or more multiple bonds and which are able to transform themselves easily and reversibly into one another.
• the term “stereoisomeric form” is intended to denote isomers of molecules of identical constitution, which differ only in one or more arrangements (s) different from their atoms in space.
• the term "pharmaceutically acceptable salts” means compounds obtained by reaction of a compound of general formula (I) with a base or an acid.
• a base suitable for the invention mention may be made of sodium hydroxide, sodium methoxide, sodium hydride, potassium t-butoxide, calcium hydroxide and magnesium hydroxide. and the like, and mixtures thereof, in solvents such as THF (tetrahydrofuran), methanol, t-butanol, dioxane, isopropanol, ethanol, their analogs, and mixtures thereof.
• solvents such as THF (tetrahydrofuran), methanol, t-butanol, dioxane, isopropanol, ethanol, their analogs, and mixtures thereof.
• Organic bases such as lysine, arginine, diethanolamine, choline, tromethamine, guanidine and their derivatives can also be used.
• acid addition salts which are suitable for the invention, mention may be made of those which may be prepared by reaction of a compound of general formula (I) with an acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, acetic acid, citric acid, maleic acid, acid salicylic acid, hydroxynaphthoic acid, ascorbic acid, palmitic acid, succinic acid, benzoic acid, benzenesulfonic acid, tartaric acid, and the like, and mixtures thereof, in solvents such as ethyl acetate, ether, alcoholic solvents, acetone,
• polymorphic form is intended to mean compounds obtained by crystallization of a compound of general formula (I) under different conditions, such as, for example, the use of various solvents, generally used for crystallization.
• Crystallization at different temperatures involves, for example, different cooling modes, for example very fast to very slow cooling involving heating or compound melting steps followed by gradual or rapid cooling.
• polymorphic forms can be determined by means of NMR spectroscopy, IR spectroscopy (infrared), DSC (Différenciai Scaning) analysis.
• alkyl radical is intended to mean a linear or branched, saturated or unsaturated hydrocarbon radical containing from 1 to 20 carbon atoms, in particular from 2 to 18 carbon atoms, in particular from 3 to to 16 carbon atoms, in particular from 4 to 12 atoms and more particularly from 6 to 10 carbon atoms, may be substituted by radicals as defined below.
• radicals such as methyl, ethyl, isopropyl, n-butyl, t-butyl, t-butylmethyl, n-propyl, pentyl, n-hexyl, 2-ethylbutyl, heptyl, octyl , nonyl, or decyl.
• cycloalkyl radical means an alkylene ring, optionally branched, saturated or unsaturated, of 3 to 10 carbon atoms, in particular C 4 to C 8 and more particularly C 6 , such cyclopropyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl.
• aryl radical means an aromatic ring comprising from 1 to 3 aromatic rings, optionally fused, of 6 to 20 carbon atoms, in particular of 10 to 14 carbon atoms, optionally comprising one or more heteroatoms chosen from O, N and S, and where appropriate being substituted by radicals as defined above and hereinafter.
• aryl radicals suitable for the implementation of the invention, it is possible to mention the phenyl radical, the benzyl radical, the phenethyl radical, the naphthyl radical, the anthryl radical, and all the aromatic rings comprising a several heteroatoms chosen from O, N and S, such as for example pyridine, thiophene, pyrrole, furan, quinoline, acridine, xanthene, 4-bora-3a, 4a diaza indacene.
• alkoxy radical means a radical -
• alkyl radical is a linear, branched or cyclic, condensed or unsaturated, saturated or unsaturated hydrocarbon radical containing from 1 to 20 carbon atoms, in particular from 2 to 18 carbon atoms, in particular from 3 to 16 carbon atoms , in particular from 4 to 12 atoms and more particularly from 6 to 10 carbon atoms.
• the hydrocarbon-based chain of the abovementioned radicals may, if appropriate, be interrupted by one or more heteroatoms chosen, for example, from O, N and S, to form, for example, a heteroalkyl radical such as an alkyl ether radical, an alkyl ester radical or a heterocyclic.
• a heteroalkyl radical such as an alkyl ether radical, an alkyl ester radical or a heterocyclic.
• heterocyclic radical is understood to mean, for example, and without limitation, a furanyl radical, a thiophenyl radical, a pyrrolyl radical, an oxazolyl radical, an isoxazolyl radical, a thiazolyl radical or a radical.
• radicals defined above may optionally be substituted with one or more halogen atoms.
• halogen atom is understood to mean an F, Cl, Br or I atom.
• the halogen atoms advantageously used in the present invention are fluorine and chlorine.
• alkylhalogenated radicals can be perfluoroalkyl radicals of general formula C n F 2 IVf 1 in which n can vary from 1 to 10, in particular from 2 to 8 and more particularly from 3 to 6.
• R 1 may especially represent a hydrogen atom, a C 1 -C 18 alkyl radical or a C 2 -C 16 alkyl radical, for example a C 6 to C 10 aryl radical, for example. examples optionally substituted with one or more halogen atoms.
• R 1 may especially represent a hydrogen atom, a methyl radical, an ethyl radical, an isopropyl radical, an n-propyl radical, a benzyl radical, a phenethyl radical, or a perfluoroalkyl radical of formula C n F 2n + ! in which n can vary from 1 to 10, in particular from 2 to 8 and more particularly from 3 to 6.
• R 1 may be a methyl radical or a benzyl radical.
• R 2 may represent a side chain of an amino acid or amino acid derivative chosen, for example, from alanine, glutamine, leucine, glycine, tryptophan, ⁇ -alanine phenylalanine, 4-chlorophenylalanine, isonipecotinic acid, 4-aminomethylbenzoic acid, 3-tetrahydroisoquinoline acid and free or benzylated histidine.
• an amino acid or amino acid derivative chosen, for example, from alanine, glutamine, leucine, glycine, tryptophan, ⁇ -alanine phenylalanine, 4-chlorophenylalanine, isonipecotinic acid, 4-aminomethylbenzoic acid, 3-tetrahydroisoquinoline acid and free or benzylated histidine.
• amino acid or amino acid derivative may, for example, be chosen from:
• R 2 may be of formula (VI) below: in which
• R 5 may represent a hydrogen atom, a linear or branched, saturated or unsaturated C 1 to C 20 alkyl radical, a radical saturated or unsaturated C 3 to C 10 cycloalkyl, a C 6 to C 10 aryl radical, optionally substituted with one or more halogen atoms.
• the alkyl or cycloalkyl radicals which may be present in the radical R 5 may also be substituted with the radicals as defined above or have their hydrocarbon chains interrupted by one or more heteroatoms as defined above.
• R 5 may represent a hydrogen atom, a C 1 -C 18 alkyl radical, a C 2 -C 16 alkyl radical or a C 6 to C 10 aryl radical, optionally substituted by one or more atomic groups. (s) halogen.
• R 5 may represent a hydrogen atom, a methyl radical, an ethyl radical, an isopropyl radical, an n-propyl radical, a benzyl radical, a phenethyl radical or a perfluoroalkyl radical of formula C n F 2 IVf 1 in which n can vary from 1 to 10, in particular from 2 to 8 and more particularly from 3 to 6.
• R 5 can be a methyl radical or a benzyl radical.
• R 2 may especially be a histidine or a histidine derivative such as a benzylated histidine.
• -COR 3 may be an acyl radical, in particular an acetyl radical, substituted with a basic entity R 3 as defined above.
• this basic entity R 3 can be a radical of formula (VII) below:
• Y may represent N or N + R 7
• - R e and R 7 may represent, independently of one another, a hydrogen atom or a C 1 alkyl radical; -C 18 , a C 2 -C 16 alkyl radical, a C 6 -C 10 aryl radical, optionally substituted by one or more halogen atom (s).
• R 6 and R 7 may represent, independently of one another, a hydrogen atom, a methyl radical, an ethyl radical, an isopropyl radical, an n-propyl radical, a benzyl radical or a phenethyl radical.
• n can vary from 1 to 10, in particular from 2 to 8 and more particularly from 3 to 6.
• R 6 and R 7 may be independently of one another a methyl radical or a benzyl radical
• the radical A may represent a radical of general formula (Va):
• R 12 * -NHSO 2 - or * -NHCO-, with * symbolizing a covalent bond with the remainder of the compound of formula (I) -
• R 13 H, HSO 3 - or COOH.
• radical A of formula (Va) may be such that R 12 may be in the ortho, meta or para position.
• R 12 * -NHSO 2 -, it can be advantageously in the ortho position.
• A may represent a radical of general formula (Vb):
• R 14 may represent a C 2 -C 4 acyl residue
• R 15 may represent a C 5 to C 7 heterocycle radical, and in particular a thiophenyl radical, and
• the radicals of formula (Va) and (Vb) may be fluorescent marker groups.
• fluorescent markers examples include rhodamine and its derivatives such as tetramethylrhodamine, rhodamine Red-X (lissamine), and Bodipy and its derivatives. , Texas Red ® and its derivatives, fluorescein and its derivatives, Alexa ® and its derivatives, and Oregon Green ® and its derivatives.
• the radical A may represent a fluorescent marker chosen from fluorescent markers of the following formula (s):
• the fluorescent marker A may be chosen from fluorescent markers derived from rhodamine, for example a derivative of sulfonylrhodamine B.
• the remainder of the compound of general formula (I) may be bonded ortho to the derivative of sulfonylrhodamine (lissamine).
• radical derived from lissamine may be represented by the radical of the following formula:
• a compound according to the invention may be of general formula (I) in which n may be equal to 0.
• a compound according to the invention can be represented, for example, by the following general formula (II):
• R 1 , R 2 , R 3 , R 4 , A and p may be, for example, as defined above.
• a compound according to the invention may be of general formula (I) in which n may be equal to 0, p may be equal to 4, R 1 may represent a methyl radical, R 2 may represent a radical of the following formula:
• R 3 may represent an acyl radical, especially acetyl, substituted by a basic entity R 3 , of the following formula:
• a compound according to the invention may have the following general formula (III):
• a compound in accordance with the invention may be a compound of general formula (III) as defined above in which the fluorescent marker A may be, for example, a sulphonylrhodamine radical as defined above, and in particular such that the remainder of the compound of formula (III) is ortho of the sulphonylrhodamine radical, R 5 may be a benzyl radical, Y may be N and R e may be a methyl radical.
• a compound in accordance with the invention is not a compound of general formula (I) as defined above in which the fluorescent marker A is a sulphonylrhodamine (lissamine) radical such that the remainder of the compound of formula (I) is in para of the sulfonylrhodamine radical.
• a compound in accordance with the invention is not a compound of general formula (III) as defined previously in which the fluorescent marker A is a sulphonylrhodamine (lissamine) radical such that the remainder of the compound of formula (III) is in para of the sulfonylrhodamine radical, and R 5 is a benzyl radical, Y is N and R 6 is a methyl radical.
• a compound according to the invention may be represented by the following formula (IV):
• Compounds according to the invention can be obtained either in the form of a library of compounds of various formulas, or in the form of isolated compounds in pure form or in a mixture of stereoisomers.
• the synthesis process can be carried out on a REM type polystyrene resin (REgenerated Michael), consisting of a hydroxymethylpoylstyrene resin functionalized with a Michael acceptor acrylic ester.
• REgenerated Michael a polystyrene resin
• the REM type resin is particularly suitable for the synthesis of tertiary amine libraries, via an initial Michael-type addition of an amine in order to fix the amine to the support followed by the synthesis of the molecule on support and finally its cleavage of the support. according to a process of quaternization of the amine and then an elimination of type
• the other residues can be introduced using conventional peptide coupling methods, using DIC / HOBt (1,3-diisopropylcarbodiimide / 1-hydroxybenzotriazole) activation.
• a radical A for example of the sulforhodamine type (lissamine, for example), can be grafted directly onto an amine function, for example the 8-NH 2 radical of a lysine or on a spacer grafted onto the 8-NH 2 radical.
• lysine such as a diamino-butane spacer, by means of a urethane linkage.
• the release of the compound (s) from the resin may be carried out after alkylation of the secondary amine in the presence, for example, of an alkyl halide such as methyl iodide, or benzyl bromide, followed by treatment with presence of a basic Amberlite ion exchange resin IRA-95.
• an alkyl halide such as methyl iodide, or benzyl bromide
• IRA-95 a basic Amberlite ion exchange resin
• a compound according to the invention can be obtained according to a solid support preparation process comprising at least the steps of: a. couple on a solid support of formula
• R 4 may be as defined above.
• R 2 may be as defined above, c. deprotecting the compound of formula (2), then coupling said deprotected compound with the compound of the following formula:
• p can be as defined above, d. deprotecting from the Fmoc group the compound of formula (3), then coupling said deprotected compound with a compound R 3 COOH to obtain a compound of formula (4) below:
• COR 3 can be as defined above, e. deprotecting the compound of formula (4), to obtain a compound of formula (5) below:
• r can be as defined above, g. reacting the compound of formula (5) or the compound of formula (6) with an electrophilic tracer, in particular with A-Cl, to obtain a compound of formula (7) below:
• R 2 , R 3 , A, n, p and r being as defined above. h. cleaving the compound of formula (7) with a compound of formula R1X, R1 being as defined above, and X may represent a halogen atom, especially I or Br, to obtain a compound of formula (I) such than previously defined.
• the present invention also relates to a method for screening an agent capable of interacting with a PEA-15 protein or one of its analogues, comprising at least the steps of:
• step a) measuring a first signal S 1 characteristic of the assembly obtained in step a) by irradiation at a wavelength for exciting the fluorescence energy donor,
• step c) bringing the assembly obtained in step a) in the presence of a medium presumed to contain at least one agent to be screened under conditions conducive to an interaction with said protein, (d) measuring a second signal S 2 , of same nature as S 1 , characteristic of the assembly obtained in step c) by irradiation at a wavelength for exciting the fluorescence energy donor,
• a and D is intended to mean that they define an acceptor-donor pair of fluorescence energy, suitable for the implementation of a resonance energy transfer.
• a pair of fluorescent markers whose emission spectrum of one (fluorescence energy donor) covers all or part of the excitation spectrum of the other (fluorescence energy acceptor).
• the excitation spectrum of the donor does not overlap, or that in very small part, the excitation spectrum of the acceptor, avoiding or reducing the appearance of false positives.
• the first and second signals may be fluorescent signals of the acceptor and / or the energy donor.
• a compound carrying a fluorescence energy acceptor capable of interacting with a fluorescent energy donor compound irradiating the assembly at a length of the donor energy spectrum Fluorescence can produce a fluorescence resonance energy transfer (FRET).
• FRET fluorescence resonance energy transfer
• fluorescence energy transfer is understood to mean a physical process, dependent on distance, by which energy is transmitted, in a non-radiative manner, from an excited chromophore, the fluorescence energy donor, to another chromophore, the fluorescence energy acceptor, by dipole-dipole interaction.
• the manifestation of such a transfer may be detected by a modulation of the fluorescent signal of the donor and / or the fluorescent signal of the acceptor, such as, for example, the decrease in the amplitude of the fluorescent signal of the fluorescence donor and / or an increase in amplitude of the fluorescent signal of the acceptor.
• the amplitude variations of the fluorescent signal of the donor may be concomitant with the amplitude variations of the fluorescent signal of the acceptor.
• one of the fluorescent signals may vary without variation of the other fluorescent signal being detected.
• the comparison of the first and second signals can make it possible to detect an amplitude modulation of a fluorescent signal.
• amplitude modulation of a fluorescent signal is intended to mean, in the context of fluorescence resonance energy transfer, any modulation of the amplitude of the fluorescence signal of the donor, of the the amplitude of the excitation spectrum or the amplitude of the donor transmission signal as defined above. Modulation of these fluorescence signals may reflect a possible interaction of the agent to be screened with a PEA-15 protein bearing a fluorescent marker D. Such an interaction being capable of causing the dissociation of a PEA-15 protein complex carrying a fluorescent marker D / compound according to the invention.
• a screening method according to the invention may comprise, in addition, a step of preparing at least one control sample in which said medium added in step c), of the method according to the invention defined above , is devoid of agent to be screened.
• control sample or samples may be prepared, according to a method according to the invention, simultaneously or independently of the implementation of such a method for the screening of an agent capable of interacting with PEA-15.
• a method according to the invention may comprise a step of comparing a signal S 3 measured from a control sample with the signals S 1 and S 2 as defined above to derive information on said agent to be screened.
• a difference between the signals thus compared may be informative of the presence, amount, and / or interaction with PEA-15 of an agent to be screened in a sample.
• the fluorescent marker D carried by the PEA-15 protein may be chosen, in a non-limiting manner, from a protein, such as a fluorescent protein, a fluorescent marker, for example chosen from a derivative of fluorescein. , a derivative of rhodamine, a derivative of Alexa ® 532, a Bodipy derivative or derivative Oregon Green ®, provided that D and a are as defined above.
• the fluorescent protein may be chosen, in a non-exhaustive manner, from the Green Fluorescent Protein, or one of its fluorescent variants, such as the Yellow Fluorescent Protein (YFP), the Cyan Fluorescent Protein (GFP) or the Red Fluorescent Protein (RFP) or DS Red or one of its variants.
• YFP Yellow Fluorescent Protein
• GFP Cyan Fluorescent Protein
• RFP Red Fluorescent Protein
• the PEA-carrying a fluorescent marker may be in particular a fusion protein, for example GFP-PEA-15.
• a fusion protein for example GFP-PEA-15.
• Such a protein may be obtained by any molecular biology technique known to those skilled in the art. , and in particular those described in "Molecular Cloning: A Laboratory
• expression vector containing a fusion protein such as, for example, GFP-PEA-15 is a matter of familiarity and practice with those skilled in the art.
• sequences coding for these proteins are for example available in databases, on the website www.ncbi.nlm.nih.gov or on the ca.expasy.org website, and also commercially.
• Expression vectors containing a nucleic acid sequence encoding GFP (or a variant thereof) or DS Red may be commercially available, particularly from companies such as Invitrogen or Clontech.
• Such vectors can be carried out in any suitable host cell, and the recovery of the fusion protein or, if appropriate, of the nucleic acid encoding such a protein, such as mRNA or cDNA, can be carried out by any suitable means known to those skilled in the art.
• a GFP-PEA-15 fusion protein has been described by KITSBERG et al. (J. Neurosci., 1999, 19: 8244).
• a screening method according to the invention can be implemented, for example, using a compound according to the invention of formula (IV).
• the PEA-carrying a fluorescent marker D can be a GFP-PEA-15 fusion protein.
• a screening method according to the invention can be carried out ex vivo or in vivo. vitro.
• a method according to the invention implemented can be carried out ex vivo from a tissue taken from a genetically modified laboratory animal so that its cells express, in a tissue-specific or non-specific manner, a protein GFP-PEA-15 melting point.
• a method according to the invention can be carried out in vitro, in particular in cellulo from intact cells, or ex cellulo, for example in a cell lysate or after separation of the elements of interest, such as the GFP-PEA fusion protein. -15.
• a screening method according to the invention can be carried out in cellulo in cells expressing the fusion protein according to the invention, either after transfection of cells, primary or in lines, using an expression vector as defined.
• a fusion protein according to the invention or a nucleic acid sequence coding for this fusion protein.
• the present invention relates to a method for screening an agent capable of interacting with a PEA-15 protein, or an analogue thereof, comprising at least the steps of:
• step a) bringing together the assembly obtained in step a) with an agent to be screened under conditions that are conducive to an interaction with said protein
• step c) measuring a second signal S2, of the same nature as Sl, characteristic of the assembly obtained in step c), (e) comparing Sl and S2 in order to draw a conclusion relating to a possible interaction of said protein PEA-15 with the agent to be screened.
• the PEA-15 protein may be bound to a carrier by means of a so-called "purification marker".
• purification marker denotes any structure that can be used for binding purposes between the PEA-15 protein and a support.
• a purification marker may be, for example and without limitation, a FLAG tag, a polyHistidine tag, or a GST (Glutathione S Transferase) protein.
• a PEA-protein bound to a purification marker as defined above may be a fusion protein obtained by any method of molecular biology known to those skilled in the art, especially as indicated above.
• a GST-PEA-15 fusion protein may be obtained according to the protocol described by Kitsberg et al. (J. Neurosci, 1999, 19: 8244).
• a support suitable for putting the invention may be, for example and in a non-exhaustive manner, a surface of a Sepharose ball or a plate for culturing cells coated with glutathione such that 96-well plates marketed by SIGMA (P3233), a nickel column, an anti-FLAG antibody bound to a protein G or protein A column, or to the surface of a Sepharose ball or to the bottom of a well of a cell culture plate.
• the PEA-15 protein can be linked to the surface of a sensor ship, for an implementation in a method for detecting a surface plasmon resonance signal, according to means known to those skilled in the art.
• the compound according to the invention may be fluorescent and the first signal S 1 and the second signal S 2 may be fluorescence signals.
• the measurement of these signals can be obtained by any spectrofluorimetry or fluorescence imaging methods known to those skilled in the art.
• the excitation and recording conditions of the fluorescence emission are to be adapted according to various factors known to those skilled in the art, such as, for example and in a non-exhaustive manner, the nature of the fluorescent marker A, the support on which is the PEA-15 protein.
• the first signal S 1 and the second signal S 2 may be the signals obtained by surface plasmon resonance, for example using a Biacore ® type apparatus, according to human protocols known art.
• the compound according to the invention may not be fluorescent.
• the PEA-15 protein can be attached to a sensor ship as previously described.
• a compound according to the invention may be contacted with said protein attached to the sensor ship.
• the interactions between the compound and the protein can be detected by surface plasmon resonance.
• a possible interaction of the PEA-15 protein with an agent to be screened resulting in the displacement of the compound of the invention previously bound can be detected by surface plasmon resonance.
• the subject of the present invention is a method for diagnosing and / or prognosing a pathological condition that may involve
• PEA-15 by detection and, optionally, quantification of the PEA-15 protein in at least one presumed biological sample comprising said protein comprising at least the steps of:
• a and D being such as to define an acceptor-fluorescence energy donor pair, suitable for carrying out fluorescence resonance energy transfer, (c) measuring a first S 1 signal characteristic of the assembly obtained in step a) by irradiation at a wavelength for exciting the fluorescence energy donor,
• step a) bringing the assembly obtained in step a) in the presence of a presumed biological sample comprising at least one PEA-15 protein under conditions conducive to the interaction of said PEA-15 protein of the biological sample with said compound according to the invention
• step c) measuring a second signal S 2 , of the same nature as S 1 , characteristic of the assembly obtained in step c) by irradiation at a wavelength for exciting the fluorescence energy donor,
• step f) comparing S 1 and S 2 in order to draw a conclusion regarding a possible presence of the PEA-15 protein in said biological sample, and possibly a conclusion as to the amount of said protein.
• a comparison of the first and second signals may allow detection of an amplitude modulation of a fluorescent signal. Such modulation may be informative of the presence, and possibly, the amount of PEA-15 protein possibly present in the sample.
• PEA-15 optionally compared with reference values obtained, either from a control sample comprising a known quantity of this protein, or from a healthy biological sample, possibly in parallel with the previous measurement, may be informative of a pathological state involving in particular PEA-15 and / or an evolution of such a state.
• a biological sample can be obtained from biological tissue or body fluid.
• a method according to the invention may comprise a step of comparing a signal S 3 measured from a control sample with the signals S 1 and S 2 as defined above to derive information from the presence and, possibly, the quantity PEA-15 in a biological sample.
• the first and second signals may be compared to one or more fluorescent signals detected from one or more control samples.
• control samples can be obtained by implementing a method according to the invention and by replacing in step c) the biological sample with sample (s) comprising a known amount of PEA-15 protein.
• control sample or samples may be prepared according to a method according to the invention simultaneously or independently of the implementation of a method according to the invention for the detection and, optionally, the quantification of the PEA-15 protein in a sample. organic. According to one embodiment, it is possible to vary the known amounts of PEA-15 of the control sample (s) or the amount of PEA-15 protein. carrying a fluorescent marker D and or fluorescent compound according to the invention so as to obtain a standard range.
• a correlation of a fluorescence signal with one of the previously defined variable quantities can thus be achieved.
• a correlation of a FRET signal can be established with known amounts of PEA-bearing protein carrying a fluorescent marker D, fluorescent compound according to the invention of PEA-15 protein.
• the PEA-15 protein carrying a fluorescent marker D may, in particular, be as defined above.
• a PEA-protein carrying a fluorescent marker D may be a GFP-PEA-15 fusion protein.
• the compound according to the invention may be as defined above, and in particular may be of formula (IV) as specified above.
• IV formula (IV) as specified above.
• Numerous variations of a diagnostic method according to the invention can be envisaged and, if appropriate, combined with characteristics of a screening method according to the invention.
• the invention relates to a method for diagnosis and / or prognosis of a pathological condition likely to involve PEA-15 implemented according to the principles indicated above for the screening method, by example, implementing a surface plasmon resonance detection, in which the agent to be screened is replaced by the biological sample.
• the PEA-15 possibly present in such a sample can bind to the compound according to the invention and thus modify the recorded signal.
• the present invention also relates to a kit for screening an agent capable of interacting with a PEA-15 protein, or of one of its analogues, or for the diagnosis and / or prognosis of a pathological condition susceptible of involve PEA-15 comprising: at least one PEA-15 protein carrying a fluorescent marker D or a purification marker, and at least one compound according to the invention, where appropriate, A and D being such that they can define an acceptor-donor pair of fluorescence energy, suitable for the implementation of a resonance energy transfer fluorescence.
• the PEA-15 protein carrying a fluorescence marker D or a purification marker may be as defined above.
• kit according to the invention when the kit according to the invention is more particularly used for the diagnosis and / or prognosis of a pathological state, it may further comprise at least one unmarked PEA-15 protein.
• the protein fusion protein when the kit according to the invention is more particularly used for the diagnosis and / or prognosis of a pathological state, it may further comprise at least one unmarked PEA-15 protein.
• the protein fusion protein when the kit according to the invention is more particularly used for the diagnosis and / or prognosis of a pathological state, it may further comprise at least one unmarked PEA-15 protein.
• the protein fusion protein the protein fusion protein
• PEA-15 with a fluorescent protein or unlabeled PEA-15 protein may be present in a kit according to the invention in the form of a nucleic acid sequence encoding said proteins, such as cDNA, mRNA , or an expression vector.
• the subject of the present invention is also a compound according to the invention for use as an active agent in a pharmaceutical composition.
• pharmaceutical composition is intended to mean a composition or substance presented as possessing curative or preventive properties with regard to human or animal diseases, as well as a substance or composition intended to be used. in order to establish a diagnosis and / or prognosis of a pathological state or not, or to restore, correct or modify the organic functions of an individual.
• the diagnostic and / or prognostic method that can be implemented by a pharmaceutical composition according to the invention can be carried out in vitro or ex vivo.
• a pharmaceutical composition according to the invention may comprise a compound according to the invention of general formula (I), or a derivative thereof such as a tautomeric form, a stereoisomeric form, a polymorphic form, a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, combination with vehicles, diluents, or excipients usually used in pharmacy.
• a compound according to the invention of general formula (I) or a derivative thereof such as a tautomeric form, a stereoisomeric form, a polymorphic form, a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, combination with vehicles, diluents, or excipients usually used in pharmacy.
• a pharmaceutical composition according to the invention may be presented in a dosage form usually used in the field, such as tablets, capsules, a powder, a syrup, a solution, a suspension.
• a pharmaceutical composition according to the invention may be presented in a dosage form suitable for administration by other routes, such as the oral route, the nasal route, the sublingual route, the topical route, the ophthalmic route, the route of rectal, etc.
• a cosmetic composition according to the invention may also be presented in a sterile form suitable for parenteral administration, such as the subcutaneous, transdermal, intramuscular, intravenous, intra-arterial, intracardiac, etc. route.
• a pharmaceutical composition according to the invention can also be presented in a freeze-dried form, combined at the time of its use with a sterile aqueous solution or not.
• the aqueous solution can be sterile if the composition according to the invention is intended for parenteral administration.
• the amount of compound according to the invention present in a pharmaceutical composition is to be adjusted, for example, according to the route of administration, the type of individual to be treated, the nature of the pathology to be treated.
• a composition according to the invention generally comprises a sufficient amount of a compound according to the invention.
• the term "sufficient quantity" is intended to mean the quantity necessary to obtain a desired effect.
• such an effect can be for example the reduction or the treatment of the symptoms presented by an alleged individual suffering from a pathology such as cancer or type II diabetes.
• the cancer may be for example glioma, kidney cancer, breast cancer, or melanoma.
• the subject of the present invention is the use of a compound according to the invention for the manufacture of a pharmaceutical composition intended for the treatment of a pathological condition likely to involve PEA-15.
• the PEA-15 protein may be implicated either by an alteration of its expression, for example, overexpression or lack of expression, or by an alteration of its biological activity, resulting, for example, in an increase in its activity.
• treatment is intended to mean reducing the severity of a disease, such as, for example, reducing symptoms or preventing these symptoms.
• a compound according to the invention can be administered before the development of the pathological state.
• the pathological conditions targeted by the present invention may be in particular those defined above, such as cancer, and in particular gliomas, kidney cancers, breast cancers, ovarian cancers, melanomas, and also diabetes mellitus. type II.
• the term "individual” is intended to mean humans, non-human primates, and laboratory animals such as rodents (for example mouse, rat, guinea pig or hamster), particularly economically valuable animals such as poultry, cattle, sheep, pigs, goats and fish, and particularly those producing products suitable for human consumption such as meat, eggs and milk. This term also refers to domestic animals such as cats and dogs.
• the present invention also relates to a pharmaceutical composition as defined above.
• the present invention also relates to an isolated complex comprising at least one PEA-15 protein carrying a fluorescent marker D or a purification marker and at least one compound of formula according to the invention, A and D being such that they can define an acceptor-donor pair of fluorescence energy, suitable for the implementation of a fluorescence resonance energy transfer.
• a complex according to the invention may comprise as a PEA-protein carrying a fluorescent marker D, a GFP-PEA-15 fusion protein and, as a compound of formula according to the invention, a compound of formula (IV), as defined above.
• FIG. 1 shows images obtained by confocal imaging of the localization of the intracellular compound of the ERK and PEA-15 proteins before and after treatment with 50 ⁇ M 6D6-1. Treatment of the cells with the 6D6-1 compound results in relocation of the ERK protein into the nucleus while the PEA-15 protein remains cytoplasmic.
• the scale bar corresponds to 40 ⁇ m.
• Figure 2 represents the mean fluorescence intensity of glutathione-coated Sepharose bead, carrying a GST-PEA-15 fusion protein, incubated in the presence of 1 and 5 ⁇ M of 6D6-1.
• the REM resin (regenerated Michael, polystyrene resin) (5 g, 4 mmol, 0.8 mmol.g .mol "1 theoretical loading) is swollen in a minimum quality of dimethylformamide (DMF).
• DMF dimethylformamide
• the resin (2.3 g, 1.5 mmol) is foamed in 20 ml of DMF and the methyl iodide (3.81 ml, 61 mmol) is added. for 24 hours, the resin is filtered, washed with 3 DMF / DCM sequences Under the same conditions a second alkylation step with methyl iodide is repeated The cleavage of the piperidine on the resin is carried out in a flask with 40 mL of DCM and in the presence of the IRA-95 resin (3.16 g, 1.5 mmol).
• reaction mixture is then diluted with 50 ml of DCM, washed twice with 10% HCl (10 ml), and then the organic phase is dried over sodium sulphate. evaporated to dryness.
• the purification and separation of the two isomers of position is obtained on silica gel (dichloromethane / methanol / acetic acid: 94/5/1) and leads to two violet powders.
• the 1-methyl-piperidin-4-His (Bzl) -NHBoc (0.03 g, 0.08 mmol) is treated with a solution of TFA / DCM (ImL / ImL) for 1H30. The solution is then evaporated to dryness under reduced pressure and the product is dried under vacuum for 18 hours. The amine thus obtained is dissolved in 0.5 ml of DMF and are successively added Fmoc-Lys (o-Lissamine) -OH (0.06 g, 0.07 mmol), PyBop (0.03 g, 0.07 mmol) and TEA (0.01 ml). 0.07 mmol). After a magnetic stirring of 4h at room temperature, the reaction is evaporated to dryness and then purified by HPLC and conducted after lyophilization to a violet powder.
• TFA / DCM ImL / ImL
• 1-methyl-piperidin-4-His (Bzl) -Lys (o-Lissamine) -NHFmoc (0.01 mmol, 0.01 mmol) is treated with 0.12 ml of piperidine in 0.5 ml of DMF for 1 hour at room temperature. ambient temperature. The solution is then directly injected on semi-preparative HPLC and leads to the deprotected product on the terminal amine with a yield of 40%.
• the amine (0 .004 g, 0.004 mmol) thus obtained is engaged in a last coupling step with 1-methyl-4-imidazole acetic acid hydrochloride (0.001 g, 0.007 mmol) in the presence of PyBop (0.003 g, 0.007 mmol). ) and DIEA (0.005 mL, 0.033 mmol) in 0.3 mL of DMSO. After stirring for 30 minutes at room temperature, the solution is directly injected on semi-preparative HPLC and after lyophilization is conducted to a violet powder.
• the 3T3 cell line expressing GFP-PEA-15 (3T3 -GFP-PEA-15 cell) was obtained as described by FORMSTECHER et al. (Dev., CeIl., 2001, 1: 239) by transfection of NIH3T3 cells with a pEGFP-PEA-15 plasmid obtained as described by KITSBERG et al. (J. Neurosci., 1999, 19: 8244).
• Neomycin resistant clones were selected and cultured in DMEM (Roche) supplemented with 10% fetal calf serum, 2mM glutamine, penicillin (5 IU / ml) and streptomycin (5mg). g / ml).
• the expression of the GFP-PEA-15 protein was verified by measuring the fluorescence of the living cells and a Western blot analysis with an anti-GFP antibody (Roche, Cat No. 1,814,460 mixture of two monoclonal antibodies made in the mouse (clone 7.1 and clone 13.1)) and an anti-PEA-15 (rabbit polyclonal antibody,
• the 3T3 -GFP-PEA-15 cells are then cultured to confluence in a HAM-F 12 medium comprising penicillin (10,000 U / ml) / streptomycin (10,000 ⁇ g / ml), 7% calf serum fetal (BIOWHITTAKER) before fluorescence resonance energy transfer (FRET) experiments.
• a HAM-F 12 medium comprising penicillin (10,000 U / ml) / streptomycin (10,000 ⁇ g / ml), 7% calf serum fetal (BIOWHITTAKER) before fluorescence resonance energy transfer (FRET) experiments.
• a stock solution of compound 6D6-1, dissolved in DMSO at a concentration of about 20 mM is diluted before use in PBS at a concentration of 10 times the final concentration.
• Compound 6D6-1 is tested at 10 ⁇ M and 10 -5 M.
• the cells are gently shaken (100 rpm) for 60 minutes before reading the fluorescence to allow the compounds to diffuse and enter the cells.
• the cells are irradiated at an excitation length of 465 nm.
• the GFP protein excited at 465 nm emits a fluorescence signal at 535 nm.
• the binding of the compound 6D6-1 to the GFP-PEA-15 protein allows a fluorescence resonance energy transfer (FRET) resulting in the emission of a fluorescence signal at 590 nm.
• FRET fluorescence resonance energy transfer
• astrocytes Primary cultures of astrocytes were prepared from cortex and striatum of mouse embryos (day 16) as described by ARAUJO et al. (J. Biol Chem., 1993, 268: 5911). Primary cultures of astrocytes were maintained for 24 hours in the absence of serum, a condition known to induce a predominantly cytoplasmic localization of ERK.
• the cells were then treated or not with 50 ⁇ M of compound 6D6-1 for 2 hours.
• ERK and PEA-15 The subcellular localization of ERK and PEA-15 was observed by confocal microscopy after labeling the cells with fluorescent antibodies.
• the cells were washed twice with PBS (Dulbecco's phosphate buffered saline, without CaCl 2 or MgCl 2 , Sigma), and fixed with 4% paraformaldehyde in PBS (pH 7.5), for 15 minutes, then washed twice with PBS comprising 0.1 M glycine, at room temperature.
• PBS Dulbecco's phosphate buffered saline, without CaCl 2 or MgCl 2 , Sigma
• Non-specific sites were blocked with PBS containing 10% normal goat serum (NGS) for one hour at room temperature.
• NGS normal goat serum
• the cells were then incubated overnight at 4 ° C with antibodies specific for PEA-15 (rabbit polyclonal antibody, Sharif et al, Neuroscience, 2004) or ERK (polyclonal rabbit antibody, Santa Cruz K-23 ( ref sc-94)), diluted in PBS containing 1.5% NGS. After three washes in PBS, the cells are incubated for one hour at room temperature with an Alexa-488 labeled anti-rabbit antibody (Molecular Probes).
• PEA-15 rabbit polyclonal antibody, Sharif et al, Neuroscience, 2004
• ERK polyclonal rabbit antibody, Santa Cruz K-23 ( ref sc-94)
• the cell nuclei were labeled with TOPRO 2 iodide according to the manufacturer's specifications (HOECHST).
• the coverslips were mounted on glass slides in a medium
• FLUOROMOUNT Southern Biotechnology
• TCS SP2, LEICA confocal microscopy
• the excitation wavelengths were 488 nm for Alexa 488 and 633 nm for TOPRO, the emission wavelengths were 510-525 nm for Alexa-488 and 647 nm for TOPRO.
• the GST-PEA-15 fusion protein was obtained according to the protocol described by Kitsberg et al. (J. Neurosci, 1999, 19: 8244).
• the GST-PEA-15 fusion proteins were recovered after bacteria and incubated in the presence of glutathione-coated Sepharose bead.
• the beads thus obtained are incubated in the presence of 1 or 5 ⁇ M of 6D6-1, in 20 mM Tris-HCl buffer pH 7.4; 100 mM NaCl; 1 mM MgCl 2 ; Triton X-100 1%). After a series of three washings, the beads are dried on a cellulose membrane and the fluorescence is quantified by means of a phospholmager (Biorad), by measuring the fluorescence of lissamine by excitation at 543 nm and measuring the emission at 590 nm.
• a phospholmager Biorad
• results are the average of three independent experiments.
• the results indicate that the average fluorescence intensity depends on the concentration of the compound and thus suggest a specific interaction between the 6D6-1 compound and the PEA-15 protein.

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