Modulation of beta-catenin/tcf-activated transcription

FIELD: chemistry.

SUBSTANCE: invention relates to a method of modulating expression of a target gene induced by β-catenin using an agent which increases linkage of p300 with β-catenin and reduces linkage of CBP with β-catenin, involving bringing a composition containing β-catenin, CBP and p300, where β-catenin is more likely linked to CBP than p300, into contact with an agent in an amount which is effective for changing the probability of linking β-catenin to CBP compared to p300, where the said agent is a compound with a structure selected from formula (I), or its stereoisomers: where A represents -(C=O)-, B represents -(CHR4)-, D represents -(C=O)-, E represents -(ZR6)-, G represents -(XR7)n-> W represents (C=O)NH-, X represents nitrogen or CH, Z represents CH, n = 0 or 1. Values of substitutes R1 and R2 are indicated in the formula of invention. The invention also relates to a composition for modulating expression of a target gene induced by β-catenin.

EFFECT: novel compounds have useful biological properties.

9 cl, 7 tbl, 30 dwg, 7 ex

 

The LEVEL of TECHNOLOGY

The technical field to which the invention relates.

The invention relates to compounds and methods for modulation of transcription of activated β-catenin/TCF, for example, selective inhibition of genes that are targeted by the path of the Wnt/β-catenin.

Description of the prior art,

The path of the Wnt/β-catenin initiates a cascade of signal transmission, critical for normal development and for the initiation and progression of cancer (Wodarz et al., “Mechanisms of Wnt signaling in development,” Annu. Rev. Cell Dev. Biol. 14:59-88 (1998); Morin, P.J. “Beta-catenin signaling and cancer,” Bioessays 21:1021-30 (1999); Moon et al., “The promise and perils of Wnt signaling through beta-catenin,” Science 296:1644-46 (2002); Oving et al., “Molecular causes of colon cancer,” Eur. J. Clin. Invest. 32:448-57 (2002)). The hallmark of this path is that it activates the transcriptional role of the multifunctional protein β-catenin. In normal cells, a large part of β-catenin is found in the cell membrane associated with Katherina, where it plays an important role in the adhesion of cells. Another pool of β-catenin was detected in the cytoplasm and nucleus, where it regulates transcription (Gottardi et al., “Adhesion signaling: how beta-catenin interacts with its partners,” Curr. Biol. 11:R792-4 (2001)). In his various roles as a mediator of cell adhesion in the plasma membrane and as an activator of transcription of β-catenin interacts with many proteins, most of which, however is the lack of significant sequence homology, competing for the same Armadillo-repeats of β-catenin. Crystal structure together with mutational studies mapped the binding sites of β-catenin several proteins in different Armadillo-repeat (Gottardi et al., “Adhesion signaling: how beta-catenin interacts with its partners,” Curr. Biol. 11:R792-4 (2001); Huber et al., “The structure of The beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin,” Cell 105:391-402 (2001)).

The cytoplasmic pool of β-catenin is regulated through phosphorylation “destructive complex”, which includes glikogensintetazy-kinase-3β (GSK-3β), caseinline-1α (SC-1α), a protein skeleton of the molecule, axin, and the tumor suppressor protein adenomatous family of intestinal polyposis (APC), among other proteins (Behrens J., “Control of beta-catenin signaling in tumor development,” Ann. N.Y. Acad. Sci. 910:21-33 (2000); discussion 33-5). In the absence of a Wnt signal transmission, phosphorylation marks cytoplasmic β-catenin for the directed SCF complex (Skp1-Cullin-F-box) ubiquitination and proteosomal degradation. Activation of the Wnt-pathway inactivates the function of GSK-3β, preventing the phosphorylation of β-catenin, allowing through this accumulation of β-catenin in the cytoplasm and then move into the nucleus where it forms a transcriptionally active complex and triggers the expression of its gene targets. A key step in the activation of target genes is the formation of a complex between β-catene the om and family members factor T cells (TCF)/lymphoid enhancer factor (LEF-1) transcription factors (Brantjes et al., “TCF: Lady Justice casting the final verdict on the outcome of Wnt signaling,” Biol. Chem. 383:255-61 (2002)). To generate a transcriptionally active complex of β-catenin recruits coactivator transcription CREB-binding protein (CBP) or its closely related homolog R (Hecht et al., “The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates,” EMBO J. 19:1839-50 (2000); Takemaru et al., “The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression,” J. Cell Biol. 149:249-54 (2000)), as well as other components of the basic transcription apparatus.

The exact mechanism by which the complex of β-catenin/TCF activates the transcription of Wnt-responsive genes has not been elucidated, but the domains of β-catenin involved in activation of transcription, were mapped in NH2- and COOH-ends (Staal et al., “Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin,” EMBO J. 3:63-68 (2002)). COOH-terminal region of β-catenin consists of approximately 100 amino acids, and it has been shown that it interacts with the TATA-binding protein (TBP) (Hecht et al., “Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro,” J. Biol. Chem. 274:18017-25 (1999)). At the confluence with LEF-1 COOH-end is sufficient to stimulate TRANS-activation (Vleminsky et al., “The C-terminal transactivation domain of beta-catenin is necessary and sufficient for signaling by the LEF-1/beta-catenin complex in Xenopus laevis,” Mech. Dev. 81:65-74 (1999)). NH2-terminal part of β-catenin consists of approximately 130 amino acids, containing sites fosfauriliruet the Oia GSK-3β, required for proteasomal degradation.

The path of the Wnt/β-catenin in normal regulates the expression of several genes involved in the stimulation of proliferation and differentiation. However, >85% of cases of colon cancer is one of the destructive components of the complex, APC, and/or β-catenin is mutated, leading to increased nuclear β-catenin and constitutive activation of target genes (Fearnhead et al., “Genetics of colorectal cancer: hereditary aspects and overview of colorectal tumorigenesis,” Br. Med. Bull. 64:27-43 (2002)). Many of these genes, including cyclin D1 (Shtutman et al., The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway,” Proc. Natl. Acad. Sci. USA 96:5522-27 (1999); Tetsu et al., “Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells,” Nature 398:442-26 (1999)) and c-myc (He et al., “Identification of C-MYC as a target of the APC pathway,” Science 281:1509-12 (1998)), which play critical roles in the growth, proliferation and differentiation of cells, together with the genes required for invasive growth, such as matrilysin (Crawford et al., “The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors,” Oncogene 18:2883-91 (1999)), fibronectin (Gradl et al., “The Wnt/Wg signal transducer beta-catenin controls fibronectin expression,” Mol. Cell. Biol. 19:5576-87 (1999)), CD44 (Wielenga et al., “Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway,” Am. J. Pathol. 154:515-23 (1999)), µPAR (Mann et al., Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas”, Proc. Natl. Acad. Sci. USA 96:1603-08 (1999)), and activated properly.

Assuming that most cases of colon cancer include SEB is the activation of the transmission signal, β-catenin, and the fact that multiple mutations leading to this activation, there is a clear need for medicines that weaken the nuclear function of β-catenin. This invention provides agents that counteract the β-catenin/TCF-mediated transcription, and provides related advantages as described in detail below.

The INVENTION

Briefly, this invention provides agents that are antagonists of β-catenin/TCF-mediated transcription, and methods of their use. In one aspect, the invention provides methods whereby a subpopulation of β-catenin/TCF-sensitive genes specifically negatively regulated, while in a related aspect, the invention provides compounds applicable in this way. In another aspect, the invention provides methods whereby binding between the SVR and β-catenin is destroyed, but the binding between structurally related coactivator R and β-catenin is not destroyed, while in a related aspect, the invention provides compounds applicable in this way. In another aspect, the invention provides methods whereby genes that are stimulated by CBP, but not R selectively activated at that time what I like in a related aspect, the invention provides compounds, applied in this way. In addition, this invention provides methods whereby genes that are stimulated R, but not CBP, selectively activated, while in a related aspect, the invention provides compounds applicable in this way. In another aspect, the invention provides methods in which carcinoma cells treated with a chemical agent to stop development in the G1-phase of the cell cycle, where prolonged treatment with this chemical agent induces apoptosis, which is not detected in normal cells of the colon (colonocytes). These carcinoma cells can be, for example, cells of the colon, breast, prostate, etc.

For example, in one aspect, the invention provides a method of modulating β-catenin-induced gene expression. The method comprises contacting the composition with an agent, where the composition comprises β-catenin, CBP and R, and β-catenin has a probability of binding to the SVR in comparison with R. This agent is present in the composition in amounts effective to change the probability of binding of β-catenin with CBP in comparison with R. In other words, in the absence of that agent β-catenin is associated with SVR and R in different degree than the degree of binding observed is the presence of this agent. For example, depending on its chemical structure, this agent can produce the following: to increase the binding of CBP with β-catenin, an optional reducing binding R with β-catenin; or increase the binding R with β-catenin in the optional reduction of the binding of CBP with β-catenin. This method can be performed in vivo or ex vivo. In one aspect of this method is performed ex vivo and this song contains stem cells. In another aspect of this method is performed in vivo, and this composition is in the mammal. The method of this invention can be used to treat various medical conditions. For example, in various aspects of the present invention: this mammal may suffer from cancer, and this number is an effective treatment for cancer; this composition is in a cage, and this agent increases the likelihood that the cell will differentiate; this composition is in a cage, and this agent increases the likelihood that the cell will proliferate.

In another aspect, the invention provides a composition comprising β-catenin, CBP, R and agent. β-catenin has a high probability of binding to the SVR in comparison with R, and this agent is present in the composition in amounts effective to change the probability of binding of β-catenin with CBP in with what Anenii with R. In other words, in the absence of that agent β-catenin is associated with SVR and R in different degree than the degree of binding observed in the presence of this agent. For example, depending on its chemical structure, this agent can produce the following: to increase the binding of CBP with β-catenin, an optional reducing binding R with β-catenin; or increase the binding R with β-catenin in the optional reduction of the binding of CBP with β-catenin. This composition can be in vivo or ex vivo. In one aspect, the composition is ex vivo, and this composition additionally contains stem cells. In another aspect, the composition is in vivo, and this song is a mammal, such as a mouse.

In another aspect, the invention provides a method of modulating the activity of Wnt-way involving: (a) the contacting of the components of the Wnt-pathway, which activates Wnt-way, to provide an activated Wnt-way; and (b) contacting the activated Wnt-way with a chemical agent, which is fully or substantially interferes with binding between R and catenin, but causes a slight inhibition or does not cause inhibition of binding between SVR and catenin. Optional, Wnt-pathway is located within the cell. Optionally, this method is performed ex vivo. Optional is entrusted, compound that activates Wnt-path selected from LiCl, or an inhibitor of GSK.

In another aspect, the invention provides a method of modulating cell proliferation, providing: (a) providing a population of cells under conditions where a portion of this population will proliferate and the part of this population will be differentiated; and (b) adding a chemical agent to this population, where the agent causes an increase in the portion of cells that proliferate concerning the part of cells that are differentiated. In various optional embodiments, the implementation of this method: this compound inhibits the binding between R and catenin; this method is provided for adding an agent to this population, which activates Wnt-path; this population of cells is a population of stem cells; this method is performed ex vivo; this method further provides for the addition of an agent that induces differentiation of a population of cells, where, for example, cells in this population are differentiated with the formation of blood cells, or cells in this population are differentiated with the formation of nerve cells.

In another aspect, the invention provides a method for maintaining stem cells in an undifferentiated state, providing for the engagement of this stem cell with an agent is m, which inhibits cell differentiation or stimulates cell proliferation in an amount effective to maintain stem cells in an undifferentiated state. In some embodiments, the implementation, the agent used in this method, selectively inhibits the interaction of β-catenin/R regarding the interaction of β-catenin/CBP.

Briefly, in other aspects the invention provides:

the method of selective inhibition of the interaction of β-catenin/CBP regarding the interaction of β-catenin/R, and this method includes the introduction of the compound in the composition, where the composition comprises β-catenin, CBP and R, and this compound selectively inhibits the interaction of β-catenin/CBP regarding the interaction of β-catenin/R;

the method of selective inhibition of the interaction of β-catenin/R regarding the interaction of β-catenin/CBP, and this method includes the introduction of the compound in the composition, where the composition comprises β-catenin, CBP and R, and this compound selectively inhibits the interaction of β-catenin/R regarding the interaction of β-catenin/CBP;

method of strengthening the movement of β-catenin from the nucleus to the cytosol, and this method provides for the introduction of compounds into the cell, where the cell contains a nucleus and the cytosol, and the nucleus contains β-catenin, and is connected to the e entails the transfer of β-catenin from the nucleus to the cytosol;

the method of selective inhibition of the expression of genes that are targeted by the Wnt/β-catenin-way, and this method provides for the introduction of compounds into the composition containing the genes that are targeted by the Wnt/β-catenin-way, and this connection causes a change in the expression of genes that are targeted by the Wnt/β-catenin-path.

In the methods and compositions of the present invention, the chemical agent is optionally selected from compounds of formula (I):

where a denotes -(CHR3)- or -(C=O)-, means -(CHR4)- or -(C=O)-, D is -(CHR5)- or -(C=O)-, E denotes -(ZR6)- or -(C=O)-, G represents -(XR7)n-, -(CHR7)-(NR8)-, -(C=O)-(XR9)- or -(C=O)-, W denotes-Y(C=O)-, -(C=O)NH-, -(SO2)- or is absent, Y denotes oxygen or sulfur, X and Z represent independently nitrogen or CH, n=0 or 1; and R1, R2, R3, R4, R5, R6, R7, R8and R9are the same or different and independently selected from the side chains of amino acid-derived portion of the side chains of the amino acids or the rest of the molecule, and their stereoisomers.

In some embodiments, implementation, R1, R2, R3, R4, R5, R6, R7, R8and R9formula (I) independently selected from the group consisting of amines2-5of alkyl, guanidino2-5/sub> of alkyl, C1-4alkylguanine2-5of alkyl, dis1-4alkylguanine-C2-5of alkyl, amidino2-5of alkyl, C1-4alkylamino2-5of alkyl, dis1-4alkylamides2-5of alkyl, C1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), bis-phenylmethyl, substituted bis-phenylmethyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl, alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyridyl1-4of alkyl, substituted pyridyl1-4the alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyrimidyl1-4of alkyl, substituted pyrimidyl1-4the alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy or nitro, carboxy, cyano, Sulfuryl or hydroxyl), triazine-2-yl-C1-4of alkyl, substituted triazine-2-yl-C1-4the alkyl (where the triazine substituents are independently selected from the aqueous or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), imidazo1-4of alkyl, substituted imidazoles1-4the alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), imidazolines1-4of alkyl, N-amidinopropane-N-C0-4of alkyl, hydroxys2-5of alkyl, C1-5alkylamino2-5of alkyl, C1-5dialkylamino2-5of alkyl, N-amidinopropane1-4the alkyl and 4-aminocyclohexane0-2the alkyl.

In some embodiments, implementation, And indicates -(CHR3)-, Denotes -(C=O)-, D is -(CHR5)-, E denotes -(C=O)-, G represents -(XR7)n-and this compound has the following General formula (II):

where R1, R2, R3, R5, R7, W, X and n are defined in formula (I) values.

In some embodiments, implementation, And denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G represents -(is=O)-(XR 9), and this compound has the following General formula (III):

where R1, R2, R4, R6R9W and X are as defined in formula (I) values, Z represents nitrogen or CH (when Z represents CH, then X denotes a nitrogen).

In some embodiments, implementation, And denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G represents -(XR7)n-and this compound has the following General formula (IV):

where R1, R2, R4, R6, R7, W, X and n are defined in formula (I) values, and Z represents nitrogen or CH, provided that when Z represents a nitrogen, then n is zero, and when Z represents CH, then X denotes nitrogen and n is not zero.

In some embodiments, implementation, this compound has the following General formula (VI):

where Radenotes a bicyclic aryl group having 8 to 11 ring members, which may have 1-3 heteroatoms selected from nitrogen, oxygen or sulfur, and Rbdenotes a monocyclic aryl group having 5-7 ring members, which may have 1-2 heteroatoms selected from nitrogen, oxygen or sulfur, and an aryl ring in this compound may have one or more substituents selected from the group Castiadas of halogen, hydroxy, cyano, lower alkyl groups and lower alkoxygroup. Optional, Rameans naftalina, hyalinella or athinodorou group, and Rbdenotes phenyl, pyridyl or piperidyl, all of which can be substituted by one or more substituents selected from the group consisting of halide, hydroxy, cyano, lower alkyl groups and lower alkoxygroup. In some embodiments, implementation, Radenotes naphthyl, and Rbdenotes phenyl which may be substituted by one or more substituents selected from the group consisting of halide, hydroxy, cyano, lower alkyl groups and lower alkoxygroup.

In some embodiments, implementation, this compound selected from COMPOUNDS 1, 3, 4, and 5.

In other aspects, the invention provides methods of screening, i.e. the ways in which you can be identified biologically active compounds and/or can be evaluated for their effectiveness. For example, this invention provides a way of identifying with a small molecule inhibitor of the interaction of β-catenin:CBP, providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with molecules containing CBP 1-111; (b) contacting the mixture of stage (a) part of the molecule, containing β-catenin; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule that contains β-catenin, stage (b)with a portion of the molecule containing the CBP 1-111, stage (a); and (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the CBP 1-111, with part of the molecule that contains β-catenin, a small molecule stage (a) as an inhibitor of the interaction of β-catenin:CBP.

Optionally, the above method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (d) with a mixture containing (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing R 1-111 with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified part of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

This invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:CBP, predusmatriva the stage: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with part of the molecule, containing β-catenin; (b) contacting the mixture of stage (a) part of the molecule containing CBP 1-111; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule containing the CBP 1-111, stage (b), with part of the molecule that contains β-catenin, stage (a); and (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the β-catenin with a portion of the molecule containing the CBP 1-111 this small molecule stage (a) as an inhibitor of the interaction of β-catenin:CBP.

Optionally, the above method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (d) with a mixture containing (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing R 1-111, with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

This invention also provides an authentication method having the th small molecule inhibitor of the interaction of β-catenin:CBP, providing stage (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with part of the molecule that contains (1) β-catenin associated with CBP 1-111; (b) determine, through analysis, to dissociate whether the specified molecule stage (a) CBP 1-111 from β-catenin; and (C) identifying, after determining that the specified small molecule stages (a) to dissociate the binding of β-catenin from SVR 1-110, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:CBP.

Optionally, the above method may further comprise a stage (d) contacting identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (C) with a mixture containing (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (e) determine, through analysis, not inhibits whether the specified molecule stage (d) linking the specified portion of the molecule containing R 1-111, with β-catenin; and (f) confirm, after determining that the specified small molecule stage (d) did not inhibit the binding of the specified portion of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

This invention also provides a method of identifying with a small molecule inhibitor interaction is the major β-catenin:R, providing stage: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R with molecules containing R 1-111; (b) contacting the mixture of stage (a) part of the molecule containing the β-catenin; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule that contains β-catenin, stage (b)with a portion of the molecule containing R 1-111, stage (a); and (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing R 1-111, with part of the molecule that contains β-catenin, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:R.

Optionally, the above method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (d) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing the CBP 1-111, with the specified β-catenin that this mA is the first molecule is a selective inhibitor of the interaction of β-catenin:R.

This invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:R providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R part of the molecule containing the β-catenin; (b) contacting the mixture of stage (a) part of the molecule containing R 1-111; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule containing R 1-111, stage (b), with part of the molecule that contains β-catenin, stage (a); (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the β-catenin with a portion of the molecule containing R 1-111, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:R.

Optionally, the above method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (d) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (g) confirmation, after determining that the little mole who Kula stage (e) is not inhibits the binding of the specified portion of the molecule, containing CBP 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:R.

This invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:R providing stage (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R with part of the molecule that contains (1) β-catenin associated with R 1-111; (b) determine, through analysis, to dissociate whether the specified molecule stage (a) R 1-111 from β-catenin; and (C) identifying, after determining that the specified small molecule stages (a) to dissociate the binding of β-catenin from R 1-110, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:R.

Optionally, the above method may further comprise a stage (d) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (C) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (e) determine, through analysis, not inhibits whether the specified molecule stage (d) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (f) confirm, after determining that the specified small molecule stage (d) did not inhibit the binding of indicated which part of the molecule, containing CBP 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:R.

In other aspects, the invention provides nucleic acid sequences and peptides, where these sequences are applicable as, for example, therapeutic agents, or, for example, in methods of screening. Thus, in various exemplary aspects, the invention provides:

essentially purified and the sequence of nucleic acids containing SEQ ID NO:1, or a sequence having at least 80% identity relative to SEQ ID NO:1, provided that said sequence does not encode a protein CBP;

essentially purified and the sequence of nucleic acids containing a fragment of SEQ ID NO:1, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein CBP;

essentially cleared and the selected peptide containing SEQ ID NO:2, or a peptide having at least 80% identity relative to SEQ ID NO:2, provided that said peptide is not a protein CBP;

essentially cleared and the selected peptide containing a fragment of SEQ ID NO:2, or a sequence having at least 80% of identichnost is relative to the specified slice provided that the peptide is not a protein CBP;

essentially purified and the sequence of the nucleic acids consisting essentially of SEQ ID NO:1, or a sequence having at least 80% identity relative to SEQ ID NO:1, provided that said sequence does not encode a protein CBP;

essentially purified and the sequence of the nucleic acids consisting essentially of a fragment of SEQ ID NO:1, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein CBP;

essentially cleared and the selected peptide consisting essentially of SEQ ID NO:2, or a peptide having at least 80% identity relative to SEQ ID NO:2, provided that said peptide is not a protein CBP;

essentially cleared and the selected peptide consisting essentially of a fragment of SEQ ID NO:2, or a sequence having at least 80% identity with respect to the specified fragment, provided that the peptide is not a protein CBP;

essentially purified and the sequence of the nucleic acids consisting of SEQ ID NO:1, or a sequence having at least 80% identity relative to SEQ ID NO:1, provided that the specified sequence n which encodes a protein CBP;

essentially purified and the sequence of nucleic acid, consisting of a fragment of SEQ ID NO:1, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein CBP;

essentially cleared and the selected peptide consisting of SEQ ID NO:2, or a peptide having at least 80% identity relative to SEQ ID NO:2, provided that said peptide is not a protein CBP;

essentially cleared and the selected peptide consisting of a fragment of SEQ ID NO:2, or a sequence having at least 80% identity with respect to the specified fragment, provided that the peptide is not a protein CBP;

essentially purified and the sequence of nucleic acids containing SEQ ID NO:3, or a sequence having at least 80% identity relative to SEQ ID NO:3, provided that said sequence does not encode a protein R;

essentially purified and the sequence of nucleic acids containing a fragment of SEQ ID NO:3, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein R;

essentially cleared and the selected peptide containing SEQ ID NO4, or a peptide having at least 80% identity relative to SEQ ID NO:4, provided that the peptide is not a protein R;

essentially cleared and the selected peptide containing a fragment of SEQ ID NO:4, or a sequence having at least 80% identity with respect to the specified fragment, provided that the peptide is not a protein R;

essentially purified and the sequence of the nucleic acids consisting essentially of SEQ ID NO:3, or a sequence having at least 80% identity relative to SEQ ID NO:3, provided that said sequence does not encode a protein R;

essentially purified and the sequence of the nucleic acids consisting essentially of a fragment of SEQ ID NO:3, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein R;

essentially cleared and the selected peptide consisting essentially of SEQ ID NO:4, or a peptide having at least 80% identity relative to SEQ ID NO:4, provided that the peptide is not a protein R;

essentially cleared and the selected peptide consisting essentially of a fragment of SEQ ID NO:4, or a sequence having at least 80% identity with respect to the specified is ragment, provided that the peptide is not a protein R;

essentially purified and the sequence of the nucleic acids consisting of SEQ ID NO:3, or a sequence having at least 80% identity relative to SEQ ID NO:3, provided that said sequence does not encode a protein R;

essentially purified and the sequence of nucleic acid, consisting of a fragment of SEQ ID NO:3, or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein R;

essentially cleared and the selected peptide consisting of SEQ ID NO:4, or a peptide having at least 80% identity relative to SEQ ID NO:4, provided that the peptide is not a protein R;

essentially cleared and the selected peptide consisting of a fragment of SEQ ID NO:4, or a sequence having at least 80% identity with respect to the specified fragment, provided that the peptide is not a protein R.

These and related aspects of the present invention is described in more detail below.

BRIEF DESCRIPTION of DRAWINGS

Figure 1. COMPOUND 1 inhibits the transcription of β-catenin/TCF.

A(i) and(ii). The structure of COMPOUND 1, COMPOUND 2, COMPOUND 3, COMPOUND 4 and COMPOUND 5.

In(i) and(ii). The CONNECTION IS GIVING 1 selectively inhibits the design of β-catenin/TCF-reporter gene with IC 505 μm. Cells SW480 (105), (figure 1B(i)), transfusional design of β-catenin/TCF-luciferase. Cells were treated with COMPOUND 1 (1-64 μm). 24 hours after treatment has been lysates and subjected to dual luciferase analysis. These data are presented in a different form in figure 1B(ii).

C. COMPOUND 1 does not affect reporter construct NFAT. Stable transfetsirovannyh Jurkat cells, right panel, with reporter construct NFAT (nuclear factor of activated T-cells)stimulated with PMA (turbolover ether) (10 ng/ml)/ionomycin (1 μg/ml) and was treated with COMPOUND 1 (0,781-50 μm). 24 hours after treatment has been lysates and subjected to dual luciferase analysis. All experiments were performed in duplicate and values built in the form of average ± standard deviation.

D. SVR is the molecular target of the COMPOUND 1. Nuclear extracts of SW480 cells were incubated with beads streptavidin-agarose, covered with COMPOUND 2 (25 μm). Pellets were washed 3 times and the eluate was subjected to gel electrophoresis and Western blot turns using anti-CBP antibodies. The arrow indicates the band corresponding in size and immunoreactivity SVR.

E.14C-labeled COMPOUND 1 is associated with SVR. COMPOUND 1 was obtained by inclusion of14C-labeled tyrosine. The SW480 cells were transfusional vectors that Express youdemi full-β-catenin Xenopus laevis (2,2 g) or full-SVR mouse (1.1 µg). 50 µg of nuclear lysate (set NE-PER, Pierce) was treated with either 20 μm14C-labeled COMPOUND 1 (7,16 × 104pulse/min) with DMSO (0.5%), and either 100 μm and 200 μm cold (non-radioactive) CONNECTION 1. Lysates were absoluely using columns 1 ml G-25 (Pharmacia) to remove unbound14C-labeled COMPOUNDS 1 and measured the inclusion of14C-labeled COMPOUND 1.

Figure 2. The first 111 amino acids of CBP, but not R specifically bind CONNECTION 1.

A. Schematic representation of the RAF wild-type and deletion constructs SVR.

Century CBP (1-111) contains the minimum domain binding COMPOUND 1. Shows the levels of expression of deletion constructs SVR in SW480 cells (upper panel). 10 μg of total protein was subjected to gel electrophoresis and subjected to Western blot turns using anti-His antibodies. Lysates of whole SW480 cells expressing the deletion constructs of SVR was associated with streptavidin-agarose pellets coated with 100 μm COMPOUND 2. Related fractions were subjected to gel electrophoresis and Western blot turns using anti-His antibodies (bottom panel). Arrows indicate structures that remained bound to the coated CONNECTION 2 granules.

C. an Excess of COMPOUNDS 1 competitive removes CBP (1-111), but not R (1-111). Deletion constructs CBP, CBP (1-111), SVR (1-211) and SVR (1-351) and deletion intercept the products R, R (1-111), R (1-211) and R (1-351) was transfusional in SW480 cells. Lysates of whole cells were incubated with streptavidin-agarose pellets or with streptavidin-agarose pellets coated with 100 μm COMPOUND 2 (bottom panel). Linking structures CBP (1-111, 1-211 and 1-351) and structures R (1-111, 1-211 and 1-351) with granules stimulated by an excess of COMPOUND 1 (150 μm).

D. CBP (1-111) associated with the CONNECTION 1-independent phosphorylation. CBP (1-111) or R (1-111) expressed in E. coli and purified using Ni-NTA-agarose (colored Kumasi blue gel). Shown CBP (1-111) (top right). 1 and 3 µl of the purified proteins were subjected to Western blot turns using anti-His antibodies. Arrows indicate the recombinant proteins, recognizable by their respective antibodies. Increasing amounts of purified CBP (1-111) (0.5, 1 and 3 µl) and R (1-111) (1, 3, and 5 µl) were incubated with streptavidin-agarose pellets coated with 100 μm of COMPOUND 2. These granules were washed and erwerbende proteins were subjected to gel electrophoresis followed by Western blot turns using anti-His antibodies. Arrows indicate proteins on PVDF membranes. Interaction of specific binding stimulated using an excess of COMPOUND 1 (300 μm).

E. Design SVR Δ1-111 + NLS is not able to save the transcription of β-catenin/TCF, inhibited by COMPOUND 1. Cells W480 was transfusional (0,1-1) µg expressing vectors, expressing either full-SVR or SVR (Δ1-111 + NLS). 24 hours after transfection cells were treated with COMPOUND 1 (25 μm) or control (0.5% DMSO). 24 hours after treatment has been lysates and subjected to their dual luciferase analysis.

Figure 3. CONNECTION 1 destroys the complex β-catenin/CBP, but not complex R/β-catenin.

A. Plasmids full-β-catenin Xenopus laevis (of 1.1, 2.2 and 3.3 µg) or mouse CBP (or 0.14, and 0.28, 0.55, which, of 1.1 and 2.2 µg) was cotranslationally in SW480 cells with a construct containing a reporter gene, β-catenin/TCF (1.1 µg). An empty vector pcDNA3 was used to equalize the amount of DNA used in each reaction. Dual luciferase assays were performed 24 hours after treatment with COMPOUND 1. All experiments were performed in duplicate and values were expressed as average ± standard deviation.

C. CONNECTION 1 competes with β-catenin for SVR. The SW480 cells were treated with 5 or 10 μm COMPOUND 1 or control (0.5% DMSO). 24 hours after processing, the lysates were incubated with beads coated with either a control antibody or anti-CBP or anti-R-antibodies. Coimmunoprecipitation proteins were subjected to gel electrophoresis and Western blot turns using anti-β-catenin antibodies.

C. CBP (1-111) is the minimum area of interaction with β-catenin. Cells SW480, tra is sizeranne next deletion SVR, exposed thus using anti-β-catenin antibodies deposited in the form of a coating on Protein a-agarose pellets. Immune complexes were washed and subjected to gel electrophoresis followed by Western blot turns using anti-His antibodies. Arrows indicate structures that remained associated with these granules.

D. CONNECTION 1 competes with β-catenin for construction CBP, but not for design R. As constructions of CBP (1-111, 1-211 and 1-351)and design R (1-111, 1-211 and 1-351) was transfusional in SW480 cells (bottom panel). After 48 hours after transfection received lysates of whole cells and subjected them thus using Protein a-agarose-anti-β-catenin antibodies described above (middle panel). Immune complexes were washed and subjected to gel electrophoresis and Western blot turns using anti-His antibodies. Arrows indicate associated proteins (upper panel). For competitive assays of immune complexes in these granules were stimulated with 50 μm COMPOUND 1.

That is, the Mapping of sequences of CBP and R with consensus motifs for the binding of β-catenin (SEQ ID NO:47-55). Consensus sequences are enclosed in “boxes”. Mapping was performed using the BLAST program for protein sequences in the NCBI database.

F. Mapping posledovatelno is it CBP 1-111 (SVR M1, SEQ ID NO:2) and R 1-111 (R M1, SEQ ID NO:4).

Figure 4. COMPOUND 1 reduces nuclear β-catenin.

A. Microscopy immunofluorescence assay of β-catenin. Cells in logarithmic phase SW480 (left) and NST (right) were fixed and stained against β-catenin in red (upper panel a), or against the FIS green (upper panel). The Central panel is superimposed β-catenin and CBP in SW480 cells (left) and cells NST (right). The bottom panel shows the redistribution of β-catenin in the cytoplasm of cells SW480 (left) and cytoplasmic membrane of cells NST (right) after treatment with COMPOUND 1 (25 μm) for 24 hours.

C. Western blot analysis of β-catenin. 25 μg of total protein from cell extracts of SW480 cells with treatment for 24 hours with or without treatment 24 hours COMPOUND 1 used for detection of cytosolic and nuclear β-catenin.

Figure 5. COMPOUND 1 inhibits the expression of cycline D1.

A. Levels cycline D1 reduced by treatment with COMPOUND 1. Western blot analysis was performed with 25 μg of lysates of whole SW480 cells treated for 4, 8 or 24 hours or 25 μm of COMPOUND 1, or control (0.5% DMSO). The Western blot turns were performed using anti-cyclin D1 antibodies (Santa Cruz Biotechnology Inc.).

C. In vivo activity of the promoters of cyclin D1 and c-myc SVR and R. ChIP (see materials and methods) shows the SVR and R-occupation of promote the c-myc after treatment with COMPOUND 1. ChIP analyses were performed on the promoter of c-myc from SW480 cells, which were treated for 8 hours with COMPOUND 1 (25 μm) or control (0.5% DMSO). Used SVR-specific antibodies (AU-26, a kind gift from Dr. David Livingston, Harvard University, Boston, MA) to assess lessons promoter SVR or R in the presence of treatment with COMPOUND 1 or control (DMSO).

Figure 6.

A. CONNECTION 1 delay cells in the phase of G1. FACS analysis was performed on the SW480 cells (lower panel) and NST (top panel), treated for 24 hours with COMPOUND 1 (25 μm) (right) or control (0.5% DMSO) (left). 5.5 x 105cells were fixed and stained with iodide of propecia (PI).

C. COMPOUND 1 selectively activates caspase cell lines carcinoma of the colon. Cells SW480 and NST (105) (left chart), together with normal cells of the colon, CCD18Co (right chart) were treated with control (0.5% DMSO) or COMPOUND 1 (25 μm). 24 hours after treatment cells were literally and measured the enzymatic activity of caspase 3/7. Relative fluorescence units (RFU) was calculated by subtracting the standardized values-blind experiment (control, no cells) from the values of the treated samples (CONNECTION 1 or control) and built in the form of a diagram.

Figure 7. COMPOUND 1 reduces the growth of colonies in soft agar dependent on dose.

A. took cialsis concentration of 5-fluorouracil (5-FU) (0.5 to 32 μm) and COMPOUND 1 (0.25 to 5 μm) was added to the SW480 cells (5000 cells per well) in triplicate wells. Cells were washed and suspended in the medium to grow on soft agar. The number of colonies after 8 days (colonies with a diameter of more than 60 μm) believed and put it on the schedule depending on the concentration of the compounds. Shown are average ± SE of three replicates. The number of colonies of control in the absence of a connection was 1637±71.

C. Schematic representation of the actions of COMPOUND 1. Without the intention of binding theory, the applicants suggest that the complex of β-catenin/TCF regulates the expression of his following in the process of target genes dependent on coactivation way. More specifically, it is assumed that the nuclear complex of β-catenin/TCF differentially associated with SVR or R and this trimeric complex triggers the expression of subpopulations sensitive to the complex β-catenin/TCF genes. COMPOUND 1 specifically and selectively focused on the complex β-catenin/TCF/CBP, but not on the complex β-catenin/TCF/R and negatively regulates the expression of such genes like cyclin D1 and axin2 and hnkd, which depend on the SVR. Treatment with COMPOUND 1, through blockade of the interaction of β-catenin/CBP, increases the amount of complexes of β-catenin/TCF available for gene expression, the promoter of which can use any coactivator (c-myc) or preferably use R as coactivator (c-jun and fra-1).

Figure 8 provides a General schema the synthesis for the production of chemical agents, applicable in the practice of this invention.

Figure 9 provides a General scheme of synthesis for the production of chemical agents applied in the practice of this invention.

Figure 10. Analysis of the metabolism of COMPOUND 3.

A. Diod Array trace (recorded using a diode matrix) (upper panel) and total ion current (bottom panel) for COMPOUND 3 and its metabolites in the rat.

Century Diod Array trace (recorded using a diode matrix) (upper panel) and total ion current (bottom panel) for COMPOUND 3 and its metabolites in man.

DETAILED description of the INVENTION

This invention provides agents that counteract the β-catenin/TCF-mediated transcription, and related methods. In one aspect, the invention provides methods whereby a subpopulation of β-catenin/TCF-sensitive genes specifically negatively regulated, while in a related aspect, the invention provides compounds applicable in this way. In another aspect, the invention provides methods whereby binding between the SVR and β-catenin is destroyed, but the binding between structurally related coactivator R and β-catenin is not destroyed, while in a related aspect, the invention provides compounds applicable in this way. In other the second aspect, the invention provides methods, by which genes that are stimulated by CBP, but not R selectively activated, while in a related aspect, the invention provides compounds applicable in this way. In addition, this invention provides methods by which genes that are stimulated R, but not CBP, selectively activated, while in a related aspect, the invention provides compounds applicable in this way. In another aspect, the invention provides methods in which carcinoma cells treated with a chemical agent to stop development in the G1-phase of the cell cycle, where prolonged treatment with this chemical agent induces apoptosis, which is not detected in normal cells of the colon (colonocytes).

More specific details of these methods and agents are provided below. However, before describing these details provided the following definitions to facilitate understanding of this description the reader.

Definition

SEQ ID NO:1 is the nucleic acid sequence: tgaggaatca acagccgcca tcttgtcgcg gacccgaccg gggcttcgag cgcgatctac tcggccccgc cggtcccggg ccccacaacc gcccgcgctc gctcctctcc ctcgcagccg gcagggcccc cgacccccgt ccgggccctc gccggcccgg ccgcccgtgc ccggggctgt tttcgcgagc aggtgaaaat ggctgagaac ttgctggacg gaccgcccaa ccccaaaaga gccaaactca gctcgcccgg tttctcggcg aatgacagca cagattttgg atcattgttt gacttggaaa atgatcttcc tgatgagctg.

SEQ ID NO:2 is the amino acid placentas is lacking: MAENLLDGPPNPKRAKLSSPGFSANDSTDFGSLFDLENDLPDELIPNGGELGLLNSGNLVPDAASKHKQLSELLRGGSGSSINPGIGNVSASSPVQQGLGGQAQGQPNSAN.

SEQ ID NO:3 is the nucleic acid sequence: ccttgtttgt gtgctaggct gggggggaga gagggcgaga gagagcgggc gagagtgggc aagcaggacg ccgggctgag tgctaactgc gggacgcaga gagtgcggag gggagtcggg tcggagagag gcggcagggg ccagaacagt ggcagggggc ccggggcgca cgggctgagg cgacccccag ccccctcccg tccgcacaca cccccaccgc ggtccagcag ccgggccggc gtcgacgcta ggggggacca ttacataacc cgcgccccgg ccgtcttctc ccgccgccgc ggcgcccgaa ctgagcccgg ggcgggcgct ccagcactgg.

SEQ ID NO:4 is the amino acid sequence: MAENVVEPGPPSAKRPKLSSPALSASASDGTDFGSLFDLEHDLPDELINSTELGLTNGGDINQLQTSLGMVQDAASKHKQLSELLRSGSSPNLNMGVGGPGQVMASQAQQSSPGLGL.

Inhibitor small molecule: the term “small molecule” refers to a chemical compound having a formula weight less than about 5000 g/mol. This compound can be organic or inorganic, can be synthetic or natural origin and can be classified as, for example, peptide, oligonucleotide, peptide mimetic, an oligonucleotide mimetic, oligosaccharide, oligosaccharide mimetic, analog or derivative of a natural product or purely synthetic compound, which may include, for example, one or more acyclic, cyclic, carbocyclic, heterocyclic, polycyclic and/or aromatic groups. The term “inhibitor” refers to compounds which inhibit, to a statistically significant degree, the binding between the two polypeptides described herein, for example, β-catenin and CBP or β-catenin and R. In other words, in the presence of this is on inhibitor binding between two polypeptides is reduced to a statistically significant degree compared to the binding, in the absence of this inhibitor. Preferably, this inhibition is sufficient to achieve action on the cell properties, for example, a therapeutic response in the subject that received the inhibitor small molecule.

Interaction of β-catenin:CBP: each of β-catenin and CBP is a well-known polypeptide. See, for example, Morin, P.J., Bioessays 21:1021-30 (1999) and Hecht et al., EMBO J. 19:1839-50 (2000). The interaction between β-catenin and CBP were documented and measured. See, for example, Takemaru et al., J. Cell. Biol. 149:249-54 (2000). The term interaction of β-catenin:CBP refers to the binding that occurs between these two proteins.

Hypothetical: before the small molecule was tested for activity, for example, as a modulator activated β-catenin/TCF transcription, this small molecule is considered as a presumptive modulator. When this small molecule demonstrates modulatory activity, then it can be called a modulator activated β-catenin/TCF transcription. Similarly, before a small molecule was tested as an inhibitor of protein-protein interactions, for example, the interaction of β-catenin:CBP, this small molecule can be called hypothetical inhibitor of the interaction of β-catenin:CBP.

Communication: when two substances, for example, helices the second connection, protein, oligonucleotide, etc. placed both in the liquid medium, e.g. water or buffer, and do not have restrictions attached to them moving in this environment, then these two substances are in contact with each other. In addition, when two substances are placed next to each other, so these two substances in contact with each other, then these substances are in contact with each other. In running tests (assays), two substances are in contact with each other, when they, for example, both placed in the test environment.

β-catenin refers to a protein that is well known in this field, see, for example, Morin, P.J., Bioessays 21:1021-30 (1999); Gottardi et al., Curr. Biol. 11:R792-4 (2001); Huber et al., Cell 105:391-402 (2001). β-catenin was identified as a mediator of cell adhesion in the plasma membrane, and as an activator of transcription.

The term “test” (analysis) refers to the process or procedure, during which various substances (e.g. chemicals, enzymes and so on) are in contact with each other under the chosen conditions, which will, or will not, lead to the emergence of detected events. The definition is detected whether this event gives information about different substances (substance) and/or the selected conditions (the condition).

The term “inhibits binding”, “inhibits interaction”, “inhibits the formation of the complex and the like, each of which refers to the to function effectively reduced, to a statistically significant degree, strength, or extent or measure binding between two proteins. Strong binding can occur when, for example, two proteins are significantly more stable in the form of a complex in comparison with them not being in complex forms. Studies of protein-protein binding, both qualitative and quantitative, are well known in this field. Examples related to the binding of β-catenin, as described in: Brantjes et al., Biol. Chem. 383:255-61 (2002, regarding the binding of β-catenin with members of the family factors T-cells (TC)); Gottardi et al., Curr. Biol. 11:R792-4 (2001), in describing the structural elements of β-catenin, which interact with binding partners) and Takemaru et al., J. Cell Biol. 149:249-54 (2000, in the description of the interaction of β-catenin with CBP).

The term “protein CBP” refers to a protein, which is also known as CREB-binding protein, where CREB is an acronym for “the binding of C-AMP-responsive element. This protein is well known in this field, see, for example, Takemaru et al., J. Cell Biol. 149:249-54 (2000) and U.S. Patent No. 6063583.

CBP 1-111 refers to the first 111 amino acids of the protein CBP identified from the N-terminal SVR. Amino acids 1-111 for SVR separated from the person listed above as SEQ ID NO:2. The corresponding nucleic acid sequence presented above as SEQ ID NO:1. Amino acids 1-11 for SVR, isolated from mice, presented as SEQ ID NO:5 as follows: MAENLLDGPPNPKRAKLSSPGFSANDNTDFGSLFDLENDLPDELI PNGELSLLNSGNLVPDAASKHKQLSELLRGGSGSSINPGIGNVSASSPVQQGLGGQAQGQPNS TN. The corresponding nucleic acid sequence of the mouse are represented as SEQ ID NO:6 as follows: atggccgaga acttgctgga cggaccgccc aaccccaaac gagccaaact cagctcgccc ggcttctccg cgaatgacaa cacagatttt ggatcattgt ttgacttgga aaatgacctt cctgatgagc tgatccccaa tggagaatta agccttttaa acagtgggaa ccttgttcca gatgctgcgt ccaaacataa acaactgtca gagcttctta gaggaggcag cggctctagc atcaacccag ggataggcaa tgtgagtgcc agcagccctg tgcaacaggg ccttggtggc caggctcagg ggcagccgaa cagtacaaac.

R 1-111 refers to the first 111 amino acids of the protein R identified from the N-Terminus R. Amino acids 1-111 for R separated from the person identified above as SEQ ID NO:4. The corresponding nucleic acid sequence that encodes the peptide presented above as SEQ ID NO:3.

It is known that β-catenin natural interacts, i.e. forms a complex (complexes) with a large number of different proteins, including HR and SVR (see, e.g., Hecht et al., EMBO J. 19:1839-50 (2000). In the presence of multiple different potential binding partners of β-catenin to contact these potential partners to varying degrees, depending on the strength of binding between β-catenin and potential binding partner. Selective inhibition of binding of β-catenin occurs when the degree of binding between β-catenin and at least one of these is binding partners (the first binding partner) is reduced relative to the degree of binding between β-catenin and at least another partner of the binding partners (the second binding partner). This is a relative reduction of binding can be observed in the form of reduced binding between β-catenin and the first partner of the binding without affecting the binding between β-catenin and the second binding partner; or it can be seen in the form of reduced binding between β-catenin and the first partner of the binding with increased binding between β-catenin and the second binding partner; or it can be seen in the form of reduced binding between β-catenin and the first binding partner together with reduced binding between β-catenin and the second binding partner up until the decrease in the binding between β-catenin and the first a partner link is higher than the decrease in binding between β-catenin and the second partner of the binding.

The term “protein R” refers to a protein that is well known in this field. See, for example, Gusterson, R.J. et al., J. Biol. Chem. 2003 Feb 28; 278(9):6838-47; 'an and Roeder, J Biol Chem. 2003 Jan 17; 278(3):1504-10; Rebel, V.I. et al., Proc. Natl. Acad. Sci. USA 2002 Nov 12; 99(23):14789-94 and U.S. Patent No. 5658784, and cited in these references.

By “essentially purified” mean that these nucleic acid or polypeptide is separated from other nucleic acids or polypeptides, and are essentially not containing other nucleic acids or polypeptides, i.e. nucleic acid or polypeptide are the two who is the primary and active ingredient. The term “isolated” refers in this context to the molecule, separated essentially from all other molecules normally associated with it in its natural state. Essentially purified and isolated molecule is the predominant molecule present in the drug. Essentially purified molecule may be more than 60%, preferably 75%, more preferably 90% free, and most preferably 95% free from other molecules (excluding the solvent)present in the natural mixture. The term “isolated” does not include molecules that are present in their native state.

The expression “the probability of binding to the SVR in comparison with R” refers to the probability that a molecule of β-catenin, which is associated with SVR in comparison with R. This probability can be expressed and/or measured by the ratio of the number of molecules of β-catenin that are associated with SVR, to the number of molecules of β-catenin that are associated with R under particular conditions. Similarly, an agent that alters the probability of binding of β-catenin with CBP in comparison with R”, called a connection, which changes the above relation in the presence of this compound in the reaction mixture in comparison with the ratio observed when this compound is not present in this reaction mixture.

The expression “likely is here, that cell will differentiate, but do not proliferate” refers to the probability that the cell will differentiate, but do not proliferate. This probability can be expressed and/or measured by the ratio of the number of cells that are differentiated, to the number of cells that proliferate under particular conditions. The agent that increases the likelihood that the cell will differentiate, but do not proliferate”, referred to as a compound that increases the number of cells that are differentiated, to the number of cells that proliferate in the presence of this compound in comparison with the same ratio in the absence of this connection. Similarly, an agent that increases the likelihood that the cell will proliferate, and not to differentiate”, referred to as a compound that increases the number of cells that proliferate, the number of cells that differentiated in the presence of this compound in comparison with the same ratio in the absence of this connection.

The term “Wnt path” refers to the cascade of signal transmission, which can be initiated by binding of Wnt proteins (secreted glycoprotein) with frizzled seven-transmembrane receptors. This path is known and described in the field and is p is admetos numerous articles and reviews (see, for example, Huelsken and Behrens, J. Cell Sci. 115:3977-8, 2002; Wodarz et al., Annu. Rev. Cell Dev. Biol. 14:59-88 (1998); Morin, P.J., Bioessays 21:1021-30 (1999); Moon et al., Science 296:1644-46 (2002); Oving et al., Eur. J. Clin. Invest. 32:448-57 (2002); Sakanaka et al., Recent Prog. Horm. Res. 55:225-36, 2000).

The phrase “activity of the Wnt pathway” refers to the activity of at least one component of this path. For example, the activity of the Wnt pathway, in some embodiments, implementation, may relate to activity of β-catenin in the induction of the expression of target genes. Many components of the Wnt pathway known in the field and include, but are not limited to, Cerberus (Cer), FrzB, Dickkopf (DKK), LRP, heterotrimeric G-protein, Dsh, caseinline Iα (CKIα), GSK3β, βTrCP, ACP, Axin, CBP, p300, β-catenin, TCF, Froucho etc.

The compound that activates the Wnt path, called a connection, which leads to induced β-catenin expression of target genes when it is present in the system having the Wnt path. Many of the target genes, the expression of which is induced by β-catenin, known in the field and include, but are not limited to, Conductin, Myc, Twin, Cyclin D1, Nkd, Ubx, En-2, PPARδ, Xbra, ID2, Siamois, Xnr3, MMP7, TCF-1, survivin, etc. Such genes may be called “genes target path Wnt/β-catenin”.

The expression “selectively inhibiting the expression of target genes of the pathway Wnt/β-catenin” refers to the inhibition of the expression of a subpopulation of target genes of the pathway Wnt/β-catenin, but not to inhibition of the expression on the natives of target genes of the pathway Wnt/β-catenin. While not wishing to be bound to any particular mechanism, the authors of this invention are arguing that selective inhibition of gene expression may be accompanied by the destruction of the interaction between β-catenin and some, but not all, of its potential binding partners.

Agents

In one aspect, the invention provides agents that can be used in the above-described methods. Except for COMPOUNDS 1, other agents applicable in the methods of the present invention, can be identified by screening compounds of General formula (I):

where a denotes -(CHR3)- or -(C=O)-, means -(CHR4)- or -(C=O)-, D is -(CHR5)- or -(C=O)-, E denotes -(ZR6)- or -(C=O)-, G represents -(XR7)n-, -(CHR7)-(NR8)-, -(C=O)-(XR9)- or -(C=O)-, W denotes-Y(C=O)-, -(C=O)NH-, -(SO2)- or is absent, Y denotes oxygen or sulfur, X and Z represent independently nitrogen or CH, n=0 or 1; and R1, R2, R3, R4, R5, R6, R7, R8and R9are the same or different and independently selected from the side chains of amino acid-derived portion of the side chain of an amino acid or a residue of a molecule, and their stereoisomers.

In one embodiment, R1, R2, R3, R4, 5, R6, R7, R8and R9independently selected from the group consisting of amines2-5of alkyl, guanidino2-5of alkyl, C1-4alkylguanine2-5of alkyl, dis1-4alkylguanine-C2-5of alkyl, amidino2-5of alkyl, C1-4alkylamides2-5of alkyl, dis1-4alkylamides2-5of alkyl, C1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), bis-phenylmethyl is, substituted bis-phenylmethyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyridyl1-4of alkyl, substituted pyridyl1-4the alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), pyrimidyl1-4of alkyl, substituted pyrimidyl1-4the alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano of Sulfuryl or hydroxyl), the triazine-2-yl-C1-4of alkyl, substituted triazine-2-yl-C1-4the alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), imidazo1-4of alkyl, substituted imidazoles1-4the alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, imidazolyl,1-4alkylamino,1-4dialkylamino, halogen, perfors1-4of alkyl, C1-4of alkyl, C1-3alkoxy, nitro, carboxy, cyano, Sulfuryl or hydroxyl), imidazolines1-4of alkyl, N-amidinopropane-N-C0-4of alkyl, hydroxys2-5of alkyl, C1-5alkylamino2-5of alkyl, C1-5dialkylamino2-5of alkyl, N-amidinopropane1-4the alkyl and 4-aminocyclohexane0-2the alkyl.

In one embodiment, R1, R2, R6in E and R7, R8and R9in G are the same or different and represent the remainder of this compound, and R3in A, R4in or R5D is selected from the side chain of the amino acid or its derivative.

In another embodiment, R3in A, R5 in D, R6in E and R7, R8and R9in G are the same or different and represent the remainder of this connection, while one or more of, and in one aspect, all of the, R1, R2and R4in the present In a side chain amino acids. In this case, the term “residue of a compound” means any part of, the agent, connection, media molecule, linker, amino acid, peptide or protein covalently attached to the structure mimetica facing conformation in the provisions of R3, R5, R6, R7, R8and/or R9. This term also includes part of the side chains of amino acids and their derivatives.

In this context, the term “residue of this compound” means any part of, the agent, connection, media molecule, atom, linker, amino acid, peptide or protein covalently attached to the structure mimetica facing conformation. This term also includes part of the side chains of amino acids and their derivatives. In one aspect of the present invention, any one or more of the provisions of R1, R2, R3, R4, R5, R6, R7, R8and/or R9you can imagine the rest of this connection. In one aspect of the present invention, one or more of R1, R2and R4part of the side-chain amino acids and and its derivative.

In this context, the term “side chain of the amino acid” is any part of the side chain of the amino acids present in naturally occurring proteins, including parts of the side chains of the amino acids identified in table A. Other natural occurring part of the side chains of the amino acids of this invention include (but are not limited to) part of the side chains of amino acids 3.5-dibromononane, 3,5-diiodotyrosine, hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. In addition, glycosylated chains side chains of amino acids can also be used in the practice of this invention, including (but not only) glycosylated threonine, serine and asparagine.

Table a
Part of the side-chain amino acidsAmino acid
-NGlycine
-CH3Alanine
-CH(CH3)2Valine
-CH2CH(CH3)2Leucine
-CH(CH3)CH2CH3 Isoleucine
-(CH2)4NH3+Lysine
-(CH2)3NHC(NH2)NH2+Arginine
Histidine
-CH2COO-Aspartic acid
-CH2CH2COO-Glutamic acid
-CH2CONH2Asparagine
-CH2CH2CONH2Glutamine
Phenylalanine
Tyrosine
Tryptophan
-CH2SHCysteine
-CH2CH2SCH3Methionine
-CH2OHSerine
-CH(OH)CH3Threonine
Proline
Hydroxyproline

In addition to the naturally occurring parts of the side chains of amino acids, part of the side chains of the amino acids of this invention also include various derivatives thereof. In this context, “derived” part of the side chain of amino acids includes modifications and/or variations of parts of the side chains of naturally occurring amino acids. For example, parts of the side chains of the amino acids alanine, valine, leucine, isoleucine and phenylalanine can be generally classified as lower alkyl, aryl or arylalkyl part of the molecule. Derived parts of the side chains of the amino acids include other having a straight chain or branched cyclic or acyclic, substituted or unsubstituted, saturated or unsaturated lower alkyl, aryl or arylalkyl part of the molecule.

In this context, a “lower alkyl parts contain 1 to 12 carbon atoms, “lower aryl part containing 6-12 carbon atoms and lower kalkilya part containing 7-12 carbon atoms. Thus, in one embodiment, the OS is enforced, derived side-chain amino acids selected from C1-12of alkyl, C6-12aryl and C7-12arylalkyl, and in a more preferred embodiment, from C1-7of alkyl, C6-10aryl and C7-11arylalkyl.

Derivative side chains of the amino acids of the present invention optionally include substituted derivatives of lower alkyl, aryl and arylalkyl parts, where the Deputy is selected from (but are not limited to) one or more of the following chemical groups: -OH, -OR, -COOH, -COOR, -CONH2, -NH2, -Other, -NRR, -SH, -SR, -SO2R, -SO2H, -SOR and halogen (including F, Cl, Br and I), where in each case R is independently chosen from having a straight chain or branched, cyclic or acyclic, substituted or unsubstituted, saturated or unsaturated lower alkyl, aryl or Uralkalij parts. In addition, cyclic lower alkyl, aryl and arylalkyl part of the molecules of the present invention include naphthalene, and heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrrolin, pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole. Derivative side chains of amino acids optionally include heterouncinata alkyl part, lower alkyl and kalkilya parts, including (but not only) alkyl - and arachidonate and silanes.

A representative part of the molecules R1, R2, R3, R4, R5, R6, R7, R8and R9specifically include (but are not limited to,- OH, -OR, -COR, -COOR, -CONH2, -CONR, -CONRR, -NH2, -Other, -NRR, -SO2R-COSR, where in each case R is above a certain value.

In the following embodiment, except that they are a part of the side chain of the amino acid or its derivative (or the remainder of this connection in the case of R1, R2, R3, R5, R6, R7, R8and R9), R1, R2, R3, R4, R5, R6, R7, R8and R9can be a linker that facilitates the binding of this compound to the other part of the molecule or with another connection. For example, the compounds of this invention can be associated with one or more known compounds, such as Biotin, for use in diagnostic analysis or screening analysis. In addition, R1, R2, R3, R4, R5, R6, R7, R8and R9can be a linker attaching the connection to solid media (such as media used in solid-phase peptide synthesis). In this embodiment, linking to another part of the molecule or other compound or solid carrier is preferably in Polo is the situation R 1, R2, R7or R8or R9and more preferably at R1or R2.

In the embodiment, where a denotes -(CHR3)-, Denotes -(C=O)-, D is -(CHR5)-, E denotes -(C=O)-, G represents -(XR7)n-this mimetic compound of the present invention with facing conformation has the following formula (II):

where R1, R2, R3, R5, R7, W, X and n have the above specified values. In a preferred embodiment, R1, R2and R7represent the remainder of this compound, and R3or R5selected from the side chains of amino acids.

In the embodiment, where a denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G represents -(C=O)-(XR9), it is the mimetic connection with facing conformation has the following General formula (III):

where R1, R2, R4, R6R9W and X are as defined above values, Z represents nitrogen or CH (when Z represents CH, then X denotes a nitrogen). In a preferred embodiment, R1, R2, R6and R9represent the remainder of this compound, and R4selected from the side chains of amino acids.

In more specific the embodiment, where a denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G represents -(XR7)nthis is the mimetic connection with facing conformation of this invention has the following formula (IV):

where R1, R2, R4, R6, R7, W, X and n are as defined above values, Z represents nitrogen or CH (when Z represents nitrogen, then n is zero, and when Z represents CH, then X denotes nitrogen and n is not zero). In a preferred embodiment, R1, R2, R6and R7imagine the rest of this connection, and R7selected from the side chains of amino acids. In one aspect, R6or R7selected from the side chains of amino acids, when Z and X both represent CH.

These compounds can be obtained by using appropriate initial components of molecules (hereinafter referred to as “components”). Briefly, in mimetic structures with reversed conformation having the formula (I), the first and second components are associated with the formation of a combined first-second intermediate product, if necessary, third and/or fourth components associated with the formation of a combined third-fourth intermediate (or, if available commercial and, can be used only third intermediate product), then the combined first-second intermediate product and the third-fourth intermediate (or third intermediate product) connected to receive the first-second-third-fourth intermediate (or first-second-third intermediate product), which cyclist obtaining mimetic structures facing conformation of this invention. Alternatively, these mimetic structure with facing conformation of the formula (I) can be obtained by the sequential binding of the individual components or stepped manner in solution or solid phase synthesis, as it is usually practiced in solid-phase peptide synthesis.

Specific components and their Assembly to obtain the compounds of the present invention is illustrated in the figure 8. For example, the first constituent component may have the following formula S1:

where R2is above a certain value, and R is a protective group suitable for use in peptide synthesis, where this protective group can be attached to the polymeric carrier to create the possibilities of solid-phase synthesis. Suitable R groups include alkyl groups and, in predpochtitelnye implementation R denotes a methyl group. In figure 8 one of the groups R is a polymer (solid) media specified as “Pol” in this figure. These first components can be easily synthesized by reductive amination H2N-R2with SN(OR)2-CHO or a substitution reaction between the H2N-R2and SN(OR)2-CH2-LG (where LG denotes a leaving group, e.g. halogen (Hal)).

“The second constituent component may have the following formula S2:

where P denotes aminosidine group suitable for use in peptide synthesis, L1indicates the activation group of the hydroxyl or carboxyl and R4is above a certain value. Preferred protective groups include tert-butyldimethylsilyl (TBDMS), tert-butyloxycarbonyl (VOS), methoxycarbonyl (MOS), N-fluorenylmethoxycarbonyl (FMOC) and allyloxycarbonyl (Alloc). N-protected amino acids are commercially available; for example, FMOC-amino acids are available from a variety of sources. In order for this second constituent component is reactive with the first component, L1is the activation group carboxyl, and the conversion of carboxyl groups in the activated carboxyl group can be easily achieved by ways of activating carboxyl groups known in the field. Suitable activated group of carboxylic acids include halides, where L1denotes a halide such as chloride or bromide, anhydrides of carboxylic acids, where L1denotes an acyl group such as acetyl, reactive esters, such as esters of N-hydroxysuccinimide and complex pentafluorophenyl esters, and other activated intermediates such as active intermediate product formed in the reaction mix using a carbodiimide such as dicyclohexylcarbodiimide (DCC). Thus, commercially available N-protected amino acids can be converted into a carboxyl activated form method known to a specialist with expertise in this field.

If acidproducing amino acids that serves as a second component, such compounds can be obtained from the corresponding amino acids by the reaction described Zaloom et al. (J. Org. Chem. 46:5173-76, 1981).

Alternatively, the first component constituting the present invention may have the following formula S1':

where R is defined above value and L2denotes a leaving group such as halogen atom or Casilina group, and the second component constituting the present invention may have the following formula S2':

where R2, R4and R have the above specified values.

“The third constituent component of the present invention may have the following formula S3:

where G, E, L1and L2have the above specified values. Suitable third components are commercially available from various sources or may be obtained by methods well known in organic chemistry.

In figure 8 the compound of formula (1) is -(C=O)- AND -(CHR4for, -(C=O)- D, and -(CR6)- that is, the compounds of formula (1)in which the carbonyl group is in position In the group R is in position B, i.e. compounds in which a denotes a (CHR3a denotes -(C=O)-, can be obtained in a manner analogous to the method shown in figure 8, as illustrated in figure 9. Figure 9 also illustrates the addition of a fourth component to the intermediate product from the first-second and third components, not the accession of the fourth component to the third component component before the reaction with the first-second intermediate component. In addition, figure 9 illustrates the formation of compounds of the present invention, in which D denotes (CHR5)- (and -(C=O)-, as in figure 8) and E denotes -(C=O)- (and -(CHR6)-as in figure 8). N the end, figure 9 illustrates the formation of compounds in which G represents NR7.

Thus, as illustrated above, the mimetic connection facing conformation of the formula (I) can be synthesized by the reaction of the first component with the second component to receive the combined first-second intermediate, followed by reaction of the combined first-second intermediate product with a third component in series with obtaining the combined first-second-third-fourth intermediate product and then the cyclization of this intermediate product with the receipt of mimetic patterns with reversed conformation.

Mimetic structures facing conformation of the formula (III) and (IV) can be obtained by methods similar to the modular synthesis of the components described above, but with suitable modifications in relation to components.

For example, the compounds applicable in this invention, can be described by the General formula:

where R1denotes a bicyclic aryl ring having 8 to 11 ring members, which may have 1-3 heteroatoms selected from nitrogen, oxygen and sulfur, and R2denotes a monocyclic aryl ring having 5-7 ring members, which can be the t having 1-2 heteroatom, selected from nitrogen, oxygen or sulfur, and any aryl ring in this compound may have one or more substituents selected from the group consisting of halide, hydroxy, cyano, lower alkyl groups or lower alkoxygroup.

Preferably, R1means naftalina, hyalinella or athinodorou group, and R2denotes phenyl, pyridyl or piperidyl. More preferably, R1denotes naphthyl, and R2denotes phenyl.

In another preferred embodiment, this connection has the following (6S,10R)-configuration:

where R1and R2have the above specified values. In another preferred embodiment, the compound of General formula (I) has a chemical structure shown in figure 1A, where this connection is called here the CONNECTION 1. Compounds having General formula (I)can be obtained as described in U.S. Patent No. 6184223, assigned Molecumetics Ltd. The preceding discussion is presented in respect of the activity of COMPOUND 1, however, other compounds of formula (I) can be subjected to screening for activity in these ways.

Additional examples of agents that are applicable in this invention may be found in PCT Publication No. WO 03/031448, U.S. Publication No. US20040072831, Bids US rooms 10/803179 and 10/82692, both of which are entitled “Mimetics with facing conformation and associated method”.

Molecules of nucleic acids

In one aspect, the invention provides various molecules of nucleic acids encoding polypeptides that are applicable in screening for agents that selectively inhibit the interaction between β-catenin and CBP in comparison with the interaction between β-catenin and R.

In some embodiments, implementation, this invention provides for an essentially purified and isolated nucleic acid molecule containing SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity relative to SEQ ID NO:1 (or SEQ ID NO:6), provided that said sequence does not encode a full-sized protein CBP human (or mouse). In this context, the percent identity of two nucleic acids is determined using the BLAST programs (Altschul et al., (J. Mol. Biol. 215:403-10, 1990) with their default settings. These programs implement the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-8, 1990), modified as described in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-7, 1993). The BLAST program available, for example, on a web site http://www.ncbi.nlm.nih.gov. In a preferred embodiment, the nucleic acid sequence encodes a peptide that binds to β-catenin.

In some the older versions of the implementation, this nucleic acid molecule encodes the amino acid sequence which contains not more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250 or 300 consecutive amino acid residues present in the naturally occurring sequence SVR (for example, CBP person or SVR mouse). In some embodiments, implementation, this molecule nucleic acid contains SEQ ID NO:1 or SEQ ID NO:6.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence containing a fragment of SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a protein CBP. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120, or at least 150, or at least 180, or at least 210, or at least 240, at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180 or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this fragment in the sequence of the nucleus is the Idov has at least 85%, or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:1 (or SEQ ID NO:6). In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting essentially of SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80% identity relative to SEQ ID NO:1 (or SEQ ID NO:6). In various optional embodiments, implementation, this sequence has at least 85%or at least 90%or at least 95% identity relative to SEQ ID NO:1 (or SEQ ID NO:6). In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting essentially of a fragment of SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120 or less is th least 150, or at least 180, or at least 210, or at least 240, at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180, or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this fragment in the sequence of nucleotides is at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:1 (or SEQ ID NO:6). In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting of SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80% identity relative to SEQ ID NO:1 (or SEQ ID NO:6). In various optional embodiments, implementation, this sequence has at least 85%or at least 90%or at least 95% identity relative to SEQ ID NO:1 (or SEQ ID NO:6). In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention both the accounts essentially purified and selected nucleotide sequence, consisting of a fragment of SEQ ID NO:1 (or SEQ ID NO:6 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120, or at least 150, or at least 180, or at least 210, or at least 240, at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180 or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this fragment in the sequence of nucleotides is at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:1 (or SEQ ID NO:6). In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and isolated nucleic acid molecule containing SEQ ID NO:3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity relative to SEQ ID NO:3, provided that said sequence does not encode a full-sized white is to R person. In some embodiments, implementation, this sequence of nucleic acid encodes the amino acid sequence which contains not more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250 or 300 consecutive amino acid residues present in the naturally occurring sequence R (for example, R person or R mouse). In some embodiments, implementation, this molecule nucleic acid contains SEQ ID NO:3.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence containing a fragment of SEQ ID NO:3 or a sequence having at least 80% identity with respect to the specified fragment, with the proviso that said sequence does not encode a full-sized protein R. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120, or at least 150, or at least 180, or at least 210, or at least 240, at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180 or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this frag is UNT in the sequence of nucleotides is at least 85%, or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:3. In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting essentially of SEQ ID NO:3 or a sequence having at least 80% identity relative to SEQ ID NO:3. In various optional embodiments, implementation, this sequence has at least 85%or at least 90%or at least 95% identity relative to SEQ ID NO:3. In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting essentially of a fragment of SEQ ID NO:3 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120, or at least 150, or at least 180, or at least 210, the sludge is at least 240, or at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180, or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this fragment in the sequence of nucleotides is at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:3. In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting of SEQ ID NO:3 or a sequence having at least 80% identity relative to SEQ ID NO:3. In various optional embodiments, implementation, this sequence has at least 85%or at least 90%or at least 95% identity relative to SEQ ID NO:3. In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

In some embodiments, implementation, this invention provides for an essentially purified and selected nucleotide sequence consisting of a fragment of SEQ ID NO:3 or is posledovatelnosti, having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 30, or at least 60, or at least 90, or at least 120, or at least 150, or at least 180, or at least 210, or at least 240, at least 270, or at least 300 nucleotides, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 300 or 270, or 240, or 210, or 180 or 150, or 120, or 90, or 60 nucleotides. Independently, and also not necessarily, this fragment in the sequence of nucleotides is at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:3. In a preferred embodiment, the nucleotide sequence encodes a peptide that binds to β-catenin.

Molecules of nucleic acids according to this invention can be obtained by cleavage of the nucleic acid molecules encoding full-sized proteins CBP and R restriction enzymes (restrictable) or other nucleases. Molecules of nucleic acids encoding a full-sized proteins CBP or R, can be isolated from genomic DNA or cDNA, in accordance with methods known in the art (see Sambrook and Rssel, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2001). The probes are nucleic acids corresponding to the region SEQ ID NO:1, 3 or 6, can be used for screening libraries or genomic DNA, or cDNA. Oligonucleotide suitable for screening of the genomic library or cDNA library usually has a length of 20-40 nucleotides and may be labeled with a different molecules that facilitate detection (e.g., a radionuclide, an enzyme label, a protein label, fluorescent label or Biotin). Genomic library or cDNA library can be constructed in a variety of suitable vectors, including plasmids, bacteriophage, yeast artificial chromosome and komenich vectors. Alternatively, these libraries can be purchased from commercial sources (e.g., Clontech, Palo Alto, CA).

To obtain nucleic acid molecules of the present invention can be used in other ways. One preferred method is by performing polymerase chain reaction for amplifying desired regions of nucleic acid molecules encoding proteins CBP and IR (for example, regions encoding the polypeptide containing the first 111 amino acid residues SVR or R). Detailed methods PCR amplification can be found, for example, in Ausubel et al., Current Protocols in Molecular biology, Greene Publishing Associates and Wiley-Interscience, NY, 1995.

Another method according to the teachings of these molecules nucleic acids is performing expression cloning using a polypeptide probe, able to bind CBP 1-111 or R 1-111. This probe can contain antibody against CBP 1-111, R 1-111 or fragment. Methods expression cloning described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular biology, Greene Publishing Associates and Wiley-Interscience, NY 1995 and Blackwood and Eisenman, Methods in Enzymology 254: 229-40, 1995.

Polynucleotide of the present invention can also be obtained using the methods of synthetic chemistry using the sequences described here. The degeneracy of the genetic code allows much of the nucleotide sequence that encodes the amino acid sequence represented in SEQ ID NO:1, 3 or 6. All such nucleotide sequences are within the scope of this invention.

Nucleic acid sequences encoding fragments of CBP or R, can be merged with different heterologous sequences, such as sequences encoding affinity tags (e.g., GST and His-tag), and a sequence encoding a secretion signal. The merge sequence that encodes affinity tag that facilitates purification of the encoded polypeptide, allowing affinity purification through this merged labels. The merge sequence that encodes a secretion signal, also facilitates purification of the encoded polypep the IDA, because it allows you to remove the polypeptide from the cell lysate, periplasmatic space, phloem or from the environment for cultivation or fermentation medium. The secretion signals, suitable for use are widely available and well known in this field (e.g., Heijne, J. Mol. Biol. 184:99-105, 1985).

As described above, the nucleic acid molecule of the present invention may also contain variants (including alleles) of natural molecules of the nucleic acid represented in SEQ ID NO:1, 3 or 6. Such options include natural variants (e.g., degenerate forms, polymorphisms, splicing variants or mutants and variants, obtained by genetic engineering, well-known in this field.

Polypeptide sequence

In one aspect, the invention provides different polypeptide molecules that are applicable in screening for agents that selectively inhibit the interaction between β-catenin and CBP in comparison with the interaction between β-catenin and R.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide containing SEQ ID NO:2 (or SEQ ID NO:5), or a peptide having at least 80%, 85%, 90%, 95%, 98% or 99% identity relative to SEQ ID NO:2 (or SEQ ID NO:5), provided that the peptide is not a full-length protein CBP (EmOC is emer, SVR human or mouse). In this context, the percent identity of two of the peptides are determined using the programs BLAST from Altschul et al., (J. Mol. Biol. 215:403-10, 1990) with their default settings. These programs implement the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-8, 1990), modified as described in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-7, 1993). The BLAST program available, for example, on a web site http://www.ncbi.nlm.nih.gov. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, polypeptide molecule of the present invention contains an amino acid sequence which contains not more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250 or 300 consecutive amino acid residues present in the naturally occurring sequence SVR (for example, CBP person or SVR mouse). In some embodiments, implementation, this polypeptide molecule includes SEQ ID NO:2 or SEQ ID NO:5.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide containing a fragment of SEQ ID NO:2 or a sequence having at least 80% identity with respect to the specified fragment, provided that this peptide is not a full-length protein CBP. In various optional embodiments, implementation, this fragment has at least 10 or at least 20, or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:2. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting essentially of SEQ ID NO:2 or a peptide having at least 80% identity relative to SEQ ID NO:2. In various optional embodiments, implementation, this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:2. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting essentially of a fragment of SEQ ID NO:2 or a sequence having at IU is greater least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 10 or at least 20 or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:2. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting of SEQ ID NO:2 or a peptide having at least 80% identity relative to SEQ ID NO:2, provided that said peptide is not a protein CBP. In various optional embodiments, implementation, this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:2. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments done what I this invention provides essentially cleared and the selected peptide consisting of a fragment of SEQ ID NO:2 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 10 or at least 20 or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:2. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide containing SEQ ID NO:4, or a peptide having at least 80%, 85%, 90%, 95%, 98% or 99% identity relative to SEQ ID NO:4, provided that the peptide is not a full-length protein R person. In some embodiments, the implementation of this polypeptide with the contains amino acid sequence, which contains not more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250 or 300 consecutive amino acid residues present in the naturally occurring sequence R (for example, R person or R mouse). In some embodiments, implementation, this polypeptide contains SEQ ID NO:4.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide containing a fragment of SEQ ID NO:4 or a sequence having at least 80% identity with respect to the specified fragment, provided that this peptide is not a full-length protein R. In various optional embodiments, implementation, this fragment has at least 10 or at least 20 or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:4. In a preferred embodiment, the peptide with azeveda with β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting essentially of SEQ ID NO:4 or a peptide having at least 80% identity relative to SEQ ID NO:4. In various optional embodiments, implementation, this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:4. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting essentially of a fragment of SEQ ID NO:4 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 10 or at least 20 or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 5%, or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:4. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting of SEQ ID NO:4 or a peptide having at least 80% identity relative to SEQ ID NO:4, provided that the peptide is not a protein CBP. In various optional embodiments, implementation, this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:4. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, implementation, this invention provides essentially cleared and the selected peptide consisting essentially of a fragment of SEQ ID NO:4 or a sequence having at least 80% identity with respect to the specified fragment. In various optional embodiments, implementation, this fragment has at least 10 or at least 20 or at least 30 or at least 40, or at least 50, or at least 60 or at least 70 or at least 80, or at least 90 amino acid residues, whereas independently this fragment has a length (when is possible, on the basis of the minimum length of this fragment) 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20 amino acid residues. Independently, and also not necessarily, this fragment of this peptide has at least 85%or at least 90%or at least 95% identity relative to the fragment of SEQ ID NO:4. In a preferred embodiment, the peptide binds to β-catenin.

In some embodiments, the implementation, the polypeptides of the present invention contain conservative amino acid substitution, i.e. replacement of similarly charged or uncharged amino acids of SEQ ID NO:2 or 4. Conservative amino acid substitution includes replacement of one amino acid by another amino acid family of amino acid with a structurally related side chains. The naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Not naturally occurring amino acids can also be used for the formation of polypeptides of the present invention.

Yes what a great invention also provides fused proteins CBP or R, containing fragments of CBP or R or their homologues, merged with amino acid sequences that contain one or more heterologous polypeptides. Such heterologous polypeptides may correspond to the naturally occurring polypeptides from any source, or may be constructed recombinante amino acid sequences. Slit proteins are applicable for purification, generation of antibodies against the amino acid sequences and for use in various test systems. Proteins commonly used in the construction of fused proteins include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP) and chloramphenicolchloramphenicol (SAT). In addition, in the construction of fused proteins can be used epitope tags, including his-tag (His) tags, FLAG tags, labels influenza hemagglutinin (ON), Myc-tag, VSV-G-tag and thioredoxin (Trx) tags. Other fused designs include maltatoday protein (MBP), S-label, merge Lex A DNA binding domain (DBD), fusion of GAL4 - DNA binding domain and merge herpes simplex virus (HSV) protein VR.

The polypeptides of this invention can be obtained by choosing the mi methods, known in this field. For example, a fragment of SVR (or R), containing SEQ ID NO:2 (or SEQ ID NO:4), can be isolated by biochemical methods, such as affinity chromatography. Affine matrices, which can be used for polypeptide SVR or R, can be a solid phase having attached thereto a monoclonal or polyclonal anti-SVR - or anti-R antibodies raised against SEQ ID NO:2 or 4 or a fragment. Alternatively, the polypeptides known that they bind CBP or R (for example, β-catenin), can be used as affinity matrices for separation of polypeptides SVR or R or fragment.

Other biochemical methods for the selection of SVR, R or their fragments include preparative gel electrophoresis, gel filtration, affinity chromatography, ion-exchange and reversed-phase chromatography, chromatofocusing, isoelectric focusing and density gradients of sucrose or glycerol (Deutscher, Methods in Enzymology: Guide to Protein Purification, Vol. 182, Academic Press, Inc., San Diego, Chapter 38, 1990; Balch et al., Methods in Enzymology, Vol. 257, Academic Press, Inc., San Diego, Chapter 8, 1995).

The polypeptide according to this invention can also be obtained by chemical synthesis, e.g. solid phase method of peptide synthesis (Merrifield et al., J. Am. Chem. Soc. 85:2149, 1964). Standard methods in the solution, well known in this field can be the ü is also used for the synthesis of the polypeptide, containing SEQ ID NO:2 or 4, or its homologue (Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, Berlin, 1984; Bodanszky, Peptide Chemistry, Springer-Verlag, Berlin, 1993). Newly synthesized polypeptide can be isolated, for example, high performance liquid chromatography and can be characterized using mass spectrometry or analysis of amino acid sequence.

The polypeptide in accordance with this invention can be also obtained by means of recombinant DNA. Nucleic acid encoding SEQ ID NO:2 or 4 or their homologues, provided by this invention can be cloned into a suitable vector for expression. This vector is commercially available or can be constructed by specialists with expertise in this field and contains the elements of expression, necessary for transcription and translation. The selected vector may also be used in the system of prokaryotic or eukaryotic host, as it is convenient, provided that the elements of expression and regulatory elements have compatible origin. Recombinant polypeptide produced in the cell-the owner or secreted from the cell, can be selected using, for example, affinity chromatography with an antibody against SEQ ID NO:2 or 4 or a fragment, ion exchange chromatography, HPLC, gel filtration chromatogr is the philosophy, crystallization with the use of ammonium sulfate, electrofocusing or preparative gel electrophoresis (see, generally Ausubel et al., supra; Sambrook et al., supra). Selected purified protein usually found in the form of a single strip on LTO-page, colored Kumasi blue.

This invention also provides fused proteins containing SEQ ID NO:2 or 4, or a homologue, and a heterologous polypeptide. Such fused proteins can be obtained by covalent binding of two protein segments or standard procedures used in the field of molecular biology. For example, the methods of recombinant DNA can be used to obtain a fused proteins by the formation of DNA-structure, which contains the coding sequences selected from SEQ ID NO:2 or 4, in proper reading frame with nucleotides encoding the second protein segment and the expression of this DNA constructs in the cell host, as it is known in this field. Many kits for constructing fused proteins are available from companies that supply research laboratory instruments for experiments, including, for example, Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), Clontech (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Applications

This invention provides excellent the provides compounds of formula (I), which inhibit transcription induced by a subpopulation of β-catenin/TCF. For example, as described in detail in the example, COMPOUND 1 selectively blocks the interaction of β-catenin with CBP without inhibiting the interaction of β-catenin with R, which is closely related in relation to SVR. Treatment with COMPOUND 1 causes a redistribution of β-catenin from the nucleus to the cytoplasm, selectively inhibits the Association of CBP with some promoters of target genes (e.g. c-myc and cyclin D1) and, therefore, inhibits the expression of these genes. In addition, COMPOUND 1 selectively activates apoptotic caspase in transformed but not in normal cells of the colon, causing the stop of the G1/S-phase of cancer cells and reduces the proliferation of transformed colorectal cells. Thus, the compounds of this invention can have a variety of applications such as cancer treatment, reducing tumor growth, increased apoptosis, modulation induced β-catenin gene expression, etc.

In one aspect, the invention provides a method of selective inhibition of the interaction of β-catenin/CBP regarding the interaction of β-catenin/R, providing for the introduction of compounds in the composition, where the composition comprises β-catenin, CBP and R, and this compound selectively inhibits the interaction of β-who atenean/CBP regarding the interaction of β-catenin/R.

In another aspect, the invention provides a method of selective inhibition of the interaction of β-catenin/R regarding the interaction of β-catenin/CBP, providing for the introduction of compounds in the composition, where the composition comprises β-catenin, CBP and R, and this compound selectively inhibits the interaction of β-catenin/R regarding the interaction of β-catenin/CBP. Some analogs of COMPOUND 1 are selective in relation to the protein complex of β-catenin/R.

Protein-protein interaction (for example, the interaction between β-catenin and R and the interaction between β-catenin and CBP), and the agent action on protein-protein interactions can be described and/or measured by any appropriate methods known in this field. Such methods may include analysis of binding in vitro using affinity purified recombinant proteins, β-catenin, CBP and R or their fragments. In some embodiments, the implementation of one protein component may first be immobilized on a solid carrier (e.g., tablet ELISA) to facilitate detection and measurement of protein-protein interactions. Protein-protein interactions can also be described using analyses of binding in vivo, such as immunoprecipitation and Western blot analysis, described Primero.

In another aspect, the invention provides a method for enhancing the movement of β-catenin from the nucleus to the cytosol, providing for the introduction of compounds into the cell, where the cell contains a nucleus and the cytosol and this kernel contains β-catenin, and this connection causes the movement of β-catenin from the nucleus to the cytosol. The movement of β-catenin from the nucleus to the cytosol can be detected by analysis of immunofluorescence assay, as described in the examples.

In another aspect, the invention provides a method of selective inhibition of the expression of target genes of the pathway Wnt/β-catenin, providing for the introduction of compounds into the composition, and this composition contains the target genes of the pathway Wnt/β-catenin, and this compound causes a change in expression of target genes of the pathway Wnt/β-catenin.

As indicated above, this invention provides methods of influence on SVR-stimulated gene expression and in the preferred embodiment, provides preferred methods of influence on SVR-stimulated gene expression in comparison to the impact on R-stimulated gene expression. The invention also provides preferred methods of influence on R-stimulated gene expression in comparison to the impact on SVR-stimulated gene expression. The invention is related persons of the NGOs remarkable because of the structural similarities between SVR and R and the fact that many specialists with expertise in this area consider the SVR and R as having essentially equivalent biological function. This efficiency can be applied, for example, to the effects on the expression of survivin (blocking apoptosis protein in tumor cells).

In one aspect, the invention provides a method of modulation induced β-catenin gene expression, involving contacting the composition with an agent, where the composition comprises β-catenin, CBP and R, where β-catenin is the probability of associating with SVR in comparison with R, and this agent is in contact with this composition in an amount effective to modify the probability of binding of β-catenin with CBP in comparison with R. In exemplary aspects of this modulation may take the form of increasing the binding of CBP with β-catenin, optional with decreasing binding R with β-catenin. Or this modulation may take the form of increasing the binding R with β-catenin, not necessarily with a decrease in the binding of CBP with β-catenin. This composition can be a cell.

Expression of genes of interest and the actions of the agent on the expression of these genes can be performed by any suitable methods known in this field. Such methods include the use of chips cDNA, RT-PCR with primers for amplifica the AI genes of interest and the measurement of reporter activity, launched by the promoters of genes of interest, and ChIP assays.

In another aspect, the invention provides a method of modulating the activity of the Wnt pathway, including:

(a) contacting (i) a composition containing components of the Wnt pathway, (ii) a compound that activates the Wnt path, to ensure that activated the Wnt pathway; and

(b) modulation of the activity of the Wnt pathway with a chemical agent, which is fully or substantially inhibits the binding between R and β-catenin, but causes a small suppression or no suppression of the binding between SVR and β-catenin.

In another aspect, the invention provides a method of enhancing cell proliferation, including:

(a) providing a population of cells under conditions in which a portion of this population will proliferate and the part of this population will be differentiated; and

(b) adding a chemical agent to this population, where the agent causes an increase in the portion of cells that proliferate concerning the part of cells that are differentiated.

Cell proliferation and cell differentiation can be characterized by any appropriate methods known in this field. Such methods include flowing cytometrics analysis and assays in soft agar as described in the examples.

In another aspect, the invention provides the characteristic way of maintaining stem cells in an undifferentiated state, providing contacts of this stem cell with an agent that inhibits differentiation of cells or stimulates the proliferation of cells, in an amount effective for maintaining the stem cells in the undifferentiated state. In some embodiments, the implementation, the agent capable of reducing the interaction between β-catenin and R without suppressing the interaction between β-catenin and CBP.

Therapy using stem cells offers the possibility of treating many degenerative diseases caused by premature death or dysfunction of specific types of cells and the inability of the body to replace or restore them. Possible therapeutic applications of stem cells include immunological conditioning of patients for organ transplantation, treatment of autoimmune diseases, such as muscular dystrophy, multiple sclerosis and rheumatoid arthritis, recovery of damaged tissues, for example, in stroke, spinal cord injuries and burns, treatment of neurodegenerative diseases, such as disease Lou Gehrig and neurological conditions such as Parkinson's disease, Huntington's disease and Alzheimer's disease, treatment of leukemia, sickle cell anemia, heart disease and diabetes. For most methods using stem cells em is reonline stem cells or adult stem cells may be cultured in vitro, coil for differentiation into the desired cell type and to come to the patient. For the successful cultivation of stem cells these stem cells must be maintained in the undifferentiated state.

To maintain stem cells in an undifferentiated state, the compounds of this invention, for example, compounds that stimulate cell proliferation or inhibit differentiation of cells, can be used at different stages of the cultivation of stem cells. For example, this connection can be used in the selection of stem cells from their tissue source. Alternatively, it can be added to the culture medium after a certain period of cultivation. It can continuously present in the culture medium for maintaining stem cells in an undifferentiated state. The concentration of this compound can be optimized correction amount of this compound to a level at which stem cells are maintained in the undifferentiated state, or differentiation of stem cells is reduced in comparison with stem cells, cultured in the absence of these compounds, and other aspects of this culture cells (e.g., the ratio of cell viability and the rate of cell proliferation) do not experience harmful the impact.

These and other methods of the present invention can be practiced using a chemical agent such as a chemical agent, identified here as COMPOUND 1 and its analogs.

Screening tests

The compounds of formula (I), as well as other agents may be subjected to screening for activity, as described herein, and in accordance with the following methods.

For example, in one aspect this invention provides a way of identifying with a small molecule inhibitor of the interaction of β-catenin:CBP, providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with molecules containing CBP 1-111; (b) contacting the mixture of stage (a) part of the molecule containing the β-catenin; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule that contains β-catenin, stage (b)with a portion of the molecule containing CBP 1-111, stage (a); and (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the CBP 1-111, with part of the molecule that contains β-catenin, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:CBP. Optionally, this method may further comprise a stage (e) contactyou the ia identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (d) with the mixture, contains (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing R 1-111 with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

In another aspect, the invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:CBP, providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with part of the molecule containing the β-catenin; (b) contacting the mixture of stage (a) part of the molecule containing CBP 1-111; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule containing the CBP 1-111, stage (b)with a portion of the molecule containing the β-catenin, stage (a); and (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the β-catenin with a portion of the molecule containing the CBP 1-111, this small molecule stage (a) as an inhibitor of usaimage the effects of β-catenin:CBP. Optionally, this method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (d) with a mixture containing (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing R 1-111, with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

In another aspect, the invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:CBP, providing stage (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:CBP with part of the molecule that contains (1) β-catenin associated with CBP 1-111; (b) determine, through analysis, to dissociate whether the specified molecule stage (a) CBP 1-111 from β-catenin; and (C) identifying, after determining that the specified small molecule stages (a) to dissociate the binding of β-catenin from SVR 1-110, this small molecule stage (a) as an inhibitor inter is Astia β-catenin:CBP. Optionally, this method may further comprise a stage (d) contacting identified with a small molecule inhibitor of the interaction of β-catenin:CBP stage (C) with a mixture containing (1) the portion of the molecule containing R 1-111, and (2) β-catenin; (e) determine, through analysis, not inhibits whether the specified molecule stage (d) linking the specified portion of the molecule containing R 1-111, with β-catenin; and (f) confirm, after determining that the specified small molecule stage (d) did not inhibit the binding of the specified portion of the molecule containing R 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:CBP.

In one aspect, the invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:R providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R with molecules containing R 1-111; (b) contacting the mixture of stage (a) part of the molecule containing the β-catenin; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the molecule that contains β-catenin, stage (b)with a portion of the molecule containing R 1-111, stage (a); and (d) identifying, after determining that the specified minor who alcula stage (a) inhibits the binding of the specified portion of the molecule, containing R 1-111, with part of the molecule that contains β-catenin, this small molecule stage (a) as an inhibitor of the interaction of β-catenin:R. Optionally, this method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (d) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing the CBP 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:R.

In another aspect, the invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:R providing stages: (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R part of the molecule containing the β-catenin; (b) contacting the mixture of stage (a) part of the molecule containing R 1-111; (C) determine, through analysis, inhibits whether the specified molecule stage (a) binding part of the mole is uly, containing R 1-111, stage (b), with part of the molecule that contains β-catenin, stage (a); (d) identifying, after determining that the specified small molecule stage (a) inhibits the binding of the specified portion of the molecule containing the β-catenin with a portion of the molecule containing R 1-111, small molecule stage (a) as an inhibitor of the interaction of β-catenin:R. Optionally, this method may further comprise a stage (e) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (d) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (f) determine, through analysis, not inhibits whether the specified molecule stage (e) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (g) confirmation, after determining that the specified small molecule stage (e) did not inhibit the binding of the specified portion of the molecule containing the CBP 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:R.

In another aspect, the invention also provides a method of identifying with a small molecule inhibitor of the interaction of β-catenin:R providing stage (a) contacting estimated with a small molecule inhibitor of the interaction of β-catenin:R with a part of the molecules is, contains (1) β-catenin associated with R 1-111; (b) determine, through analysis, to dissociate whether the specified molecule stage (a) R 1-111 from β-catenin; and (C) identifying, after determining that the specified small molecule stages (a) to dissociate the binding of β-catenin from R 1-110, small molecule stage (a) as an inhibitor of the interaction of β-catenin:R. Optionally, the above method may further comprise a stage (d) contacting identified with a small molecule inhibitor of the interaction of β-catenin:R stage (C) with a mixture containing (1) the portion of the molecule containing CBP 1-111, and (2) β-catenin; (e) determine, through analysis, not inhibits whether the specified molecule stage (d) linking the specified portion of the molecule containing the CBP 1-111, with β-catenin; and (f) confirm, after determining that the specified small molecule stage (d) did not inhibit the binding of the specified portion of the molecule containing the CBP 1-111, with the specified β-catenin that this small molecule is a selective inhibitor of the interaction of β-catenin:R.

Protein-protein interaction (for example, the interaction between β-catenin and R and the interaction between β-catenin and CBP), and the agent action on protein-protein interactions can be described and/or measured by any appropriate methods known in D. the authorized area. For example, a suitable method of analysis for the methods of this invention is the isothermal tetrazona calorimetry (ITC). ITC experiments can be performed using isothermal titrating calorimeter (MicroCal MCS (MicroCal, Northampton, MA), essentially as recommended by the manufacturer. Briefly, a fragment of CBP C1 intensively cialiswhat against buffer for dialysis containing 50 mm PIPES (pH 7.5) and 0.1 mm EDTA. The sample protein add DMSO to a final concentration of 0.05% to match the diluted sample medicines. The protein concentration determined using the Bradford protein analysis (Bio-Rad laboratories, Hercules, CA) using gamma-globulin bovine plasma as a standard (Bio-Rad). Due to the limitations of the solubility of the ITC experiments performed by the introduction of 5-15 µl SVR C1 [223 μm] in the cell sample, filled with 23.3 microns estimated with a small molecule inhibitor. The heat of dilution appreciate after saturation of this estimated with a small molecule inhibitor in the cell sample and thermodynamic parameters calculated using the software package Origin 5.0 (MicroCal). Higher heat of dilution indicates a stronger binding between the estimates with a small molecule inhibitor, and this fragment of CBP. Stronger binding between the at estimated with a small molecule inhibitor, and this fragment of CBP identifies a small molecule, which may be more effective in the destruction of the binding of CBP, for example, binding of CBP with β-catenin.

Pharmaceutical composition and introduction

Molecules of nucleic acids, peptides and compounds in accordance with this invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically contain a molecule of nucleic acid, peptide or compound and a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” is called the solvent, the distribution environment, coatings, antibacterial and antifungal agents, isotonic and delaying absorption agents and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in this field. In these compositions may also include additional active compounds.

The pharmaceutical composition of this invention can be administered parenterally, topically, orally or locally for therapeutic treatment. Can be used in different aqueous media, for example, water, buffered water, and 0.4% saline, 0.3% glycine and the like, and they may include other proteins to enhance stability, such as albumin, lipoprotein, globulin, etc. of the Obtained composition can be presterilized the Ana common well-known sterilization methods. These solutions can be packaged for use or filtered under aseptic conditions and lyophilized, and the dried product combined with a sterile solution prior to administration.

Oral compositions generally include an inert diluent or suitable for food media. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic injection of the active compound may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Pharmaceutically compatible binding agents, and/or adjuvant substances may be included as part of this composition. Tablets, pills, capsules, lozenges and the like can contain any of the following ingredients, or compounds of a similar nature: a binder (for example, microcrystalline cellulose, tragakant or gelatin); excipient (for example, starch or lactose), disintegrity agent (for example, alginic acid, primogel or corn starch), lubricants (e.g. magnesium stearate or stearates); glidant (for example, colloidal silicon dioxide); sweetening agent (such as sucrose or saccharin) or a flavouring agent such as peppermint, methyl salicylate or orange aromati the ATOR).

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be provided transmucosal or transdermal means. For transmucosal or transdermal administration in the preparation of the composition used wetting reagents (penetrants). Such penetrants are usually known in this field and include, for example, for transmucosal administration, detergents, bile acids and derivatives of fuseboy acid. Transmucosal introduction can be performed with the use of nasal sprays or suppositories. For TRANS-dermal active compounds are prepared in the form of ointments, liniments, gels, or creams as generally known in this field.

In one embodiment, the active compounds are prepared with carriers that will protect this compound against rapid elimination from the body, for example, in the form of a composition of controlled release, including implants and microencapsulating delivery systems. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, on intoevery and polylactic acid. Methods of preparing such forms will be obvious to specialists with expertise in this area. These substances can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

Especially, it is preferable to prepare oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The unit dosage form is called, in this context, physically discrete units suitable to individual doses for the subject being treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations that exist in the field of production of dosage forms such an active compound for the treatment of individuals, and directly depends on these parameters.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and ED5 (dose, therapeutically effective in 50% of the population). The ratio of doses between toxic and therapeutic effects is therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, measures should be taken to create a delivery system that directs such compounds to the site of affected tissue to minimize potential damage in relation to uninfected cells and, thereby, reduce side effects.

Data obtained from tests on cell cultures and animal studies can be used in the preparation of the dose range for use in humans. The dosage of such compounds lies in the range of circulating concentrations that include the ED50with low toxicity or no toxicity. This dose may vary within this range depending on the shape of the dose and used as a way of introduction. For any compound used in the method of the present invention, therapeutically effective dose can be approximately determined first from tests on cell cultures. The dose can be cooked in animal models to achieve the range is on circulating concentrations in plasma, which includes IC50(i.e. the concentration of test compound which gives premaxillae inhibition of symptoms)as defined in cell culture. Such information can be used for more accurate determination of the applicable human dose. Levels in plasma may be measured, for example, high performance liquid chromatography (HPLC).

The following examples are provided to illustrate this invention and should not be construed as a limitation.

EXAMPLES RETRIEVE

EXAMPLE OBTAIN 1

Obtaining (N-Fmoc-N'-R3-hydrazino)acetic acid

(1) preparation of N-Fmoc-N'-methylhydrazino

Dvuhgolosy round bottom flask 2 l supplied glass tube and the tube with calcium chloride. Solution was added sulfate methylhydrazine (20 g, 139 mmol, where R3is stands) in THF (300 ml) and the solution DiBoc (33 g, 153 mmol) in THF. Saturated aqueous sodium bicarbonate solution (500 ml) was added dropwise via addition funnel over 2 hours with intensive stirring. After 6 hours was slowly added a solution of Fmoc-Cl (39 g, 153 mmol) in THF. The resulting suspension was stirred for 6 hours at 0aboutC. the Mixture was extracted with ethyl acetate (EA, 500 ml) and the organic layer was retained. The solution was dried with sodium sulfate and evaporated in vacuum. The following stud is Yu performed without purification.

Dvuhgolosy a round bottom flask of 1 l provided with a glass tube and the tube with calcium chloride. A solution of the product from the previous stage in the Meon (300 ml) was added and slowly added concentrated HCl (30 ml, 12 B.C.) through an addition funnel with stirring on a magnetic stirrer in a bath with a mixture of ice water and stirred over night. This mixture was extracted with EA (1000 ml) and the organic layer was retained. The solution was dried using sodium sulfate and evaporated in vacuum. The residue was purified by recrystallization with n-hexane and EA with obtaining N-Fmoc-N'-methylhydrazino (32,2 g, 83%).1H NMR (DMSO-d6) δ of 7.90-7,88 (d, J=6 Hz, 2H), 7,73-of 7.70 (d, J=9 Hz, 2H), 7,44-7,31 (m, 4H), to 4.52-4,50 (d, J=6 Hz, 2H), or 4.31-4.26 deaths (t, J=6 Hz, 1H), 2,69 (s, 1H).

(2) Obtain tert-butyl ether (N-Fmoc-N'-methylhydrazino)acetic acid

Dvuhgolosy a round bottom flask of 1 l provided with a glass stopper and reflux condenser connected to a tube with calcium chloride. Solution was added N-Fmoc-N'-methylhydrazino (20 g, 75 mmol) in toluene (300 ml). Was slowly added a solution of tert-butylbromide (22 g, 111 mmol) in toluene (50 ml). Was slowly added Cs2CO3(49 g, 149 mmol). Was slowly added NaI (11 g, 74 mmol) with vigorous stirring. The reaction mixture was stirred at the temperature of reflux distilled for 1 day. The mixture of products of the filter is and was extracted with EA (500 ml). The solution was dried over sodium sulfate and evaporated in vacuum. The product was purified by chromatography with a solution of hexane:EA = 2:1 to obtain tert-butyl ether (N-Fmoc-N'-methylhydrazino)acetic acid (19,8 g, 70%).

1H NMR (CDCl3-d) δ 7,78 to 7.75 (d, J=9 Hz, 2H), to 7.61-to 7.59 (d, J=6 Hz, 2H), 7,43-7,26 (m, 4H), 4,42-and 4.40 (d, J=6 Hz, 2H), 4,23 (W, 1H), only 3.57 (s, 2H), 2,78 (s, 3H), 1.50 in (C, N).

(3) Receiving (N-Fmoc-N'-methylhydrazino)acetic acid

Dvuhgolosy a round bottom flask of 1 l provided with a glass stopper and reflux condenser connected to a tube with calcium chloride. Solution was added tert-butyl ether (N-Fmoc-N'-methylhydrazino)acetic acid (20 g, 52 mmol). Was slowly added a solution of HCl (150 ml, 4 M solution in dioxane) with vigorous stirring in a bath with a mixture of water with ice. The reaction mixture was stirred at room temperature for 1 day. The solution was concentrated completely under reduced pressure at 40aboutC. was Added a saturated aqueous solution of NaHCO3(100 ml) and the aqueous layer washed with diethyl ether (100 ml). Was added dropwise slowly concentrated HCl at 0about(PH 2-3). This mixture was extracted and the organic layer was retained (500 ml, MS). The solution was dried with sodium sulfate and evaporated in vacuum. The residue was purified by recrystallization with n-hexane and ethyl acetate to obtain (N-Fmoc-N'-mating is draino)acetic acid (12 g, 72%).1H NMR (DMSO-d6) δ 12,38 (s, 1H), 8,56 (W, 1H), 7,89-7,86 (d, J=9 Hz, 2H), 7,70-to 7.67 (d, J=9 Hz, 2H), 7,43-7,29 (m, 4H), 4,29-4,27 (d, J=6 Hz, 2H), 4,25-4,20 (t, J=6 Hz, 1H), 3,47 (s, 2H), has 2.56 (s, 3H).

EXAMPLE of GETTING 2

Obtaining (N-Mos-N'-R7-hydrazino)acetic acid

(1) obtaining the ethyl ester of (N'-methoxycarbonylamino)acetic acid

MOS-NH-NH2(50 g, 0.55 mol) was dissolved in DMF (300 ml) and then the reaction vessel was added ethylbromoacetate (68 ml, 0,555 mol) and potassium carbonate (77 g, 0,555 mol). This mixture was heated to 50aboutC for 5 hours. After completion of the reaction the mixture was filtered and diluted with EtOAc and washed with brine (3 times). The crude product was purified column chromatography (eluent: hexane/EtOAc = 4/1) to obtain 72 g of a colorless oil.

(2) Ethyl ether [N-R7-N'-methoxycarbonylamino]acetic acid

Ethyl ester (10 g, 0.05 mol), potassium carbonate (6.9 g, 0.05 mol) and R7-bromide (14.1 g, 0.06 mol) was dissolved in DMF (200 ml) and the mixture was heated to 50aboutC for 5 hours. After completion of the reaction the mixture was filtered and diluted with EtOAc and washed with brine (3 times). The crude product was purified by chromatography (eluent: hexane/EtOAc = 4/1).

(3) [N-R7-N'-methoxycarbonylamino]acetic acid

Alquileres the config ethyl ester (9.5 g, 0.03 mol) was dissolved in a mixture of THF/water (1/1, ml) was added 2 n NaOH solution (28,3 ml) at 0aboutC. the Mixture was stirred at room temperature for 2 hours. After the original ether not detected in the UV, this solution was diluted with EA, then shared. The aqueous layer was acidified to pH 3~4 1 N. HCl and the compound was extracted with DHM (3 times). The combined organic layer was dried over MgSO4and was evaporated to obtain a yellow solid.

EXAMPLE for the preparation of 3

(1) preparation of Nβ-Mos-Nα-benzylideneaniline

This compound was obtained as described in the literature procedure. (Chequillaume et al., Synlett 2000, 3, 331).

(2) Obtaining 1-methoxycarbonyl-2,8-dibenzyl-6-methyl-4,7-dioxotetrahydrofuran[2,1-c][1,2,4]triazine

Bromatology resin (60 mg, 0.98 mmol/g) and a solution of benzylamine in DMSO (2.5 ml, 2 M) were placed in a vial with a screw cap. The reaction mixture was shaken at 60aboutWith the use of rotary kilns [Robbins Scientific] within 12 hours. The resin was collected by filtration and washed with DMF, then DHM with receipt of the first component.

A solution of Fmoc-alanine (4 EQ, commercially available, the second component), HATU (PerSeptive Biosystems, 4 EQ) and DIEA (4 EQ) in NMP (Advanced ChemTech) was added to the resin. After shaking the PE klonoa mixture for 4 hours at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

To this resin was added 20% piperidine in DMF. After shaking this reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

A solution of Nβ-Mos-Nα-benzylideneaniline (4 EQ, compound (3) in preparative example 2, where R7denotes a benzyl, a third component component), HOBT [Advanced ChemTech] (4 EQ) and DIC (4 equiv) in DMF was added to a prepared as described above, the resin. After shaking the reaction mixture for 3 hours at room temperature, the resin was collected by filtration and washed with DMF, DHM and then Meon. The resin was dried in vacuum at room temperature.

This resin was treated with formic acid (2.5 ml) for 18 hours at room temperature. After removing the resin by filtration, the filtrate are condensed under reduced pressure to obtain the product in the form of butter.1H NMR (400 MHz, CDCl3) δ ppm; is 1.51 (d, 3H), 2,99 (m, 1H), 3,39 (d, 1H), 3,69 (d, 1H), 3,75 (m, 1H), 3,82 (s, 3H), was 4.02 (d, 1H), 4,24 (d, 1H), 4,39 (d, 1H), 4.75 in (d, 1H), 5,14 (kV, 1H), 5,58 (DD, 1H), 7,10-7,38 (m, 10H).

EXAMPLE 4

(1) preparation of N'-Fmoc-N-methylhydrosiloxane

Chilled on ice two-phase mixture N-fluoren-9-Eletropaulo ether N-methylhydrocinnamic acid (107 mg, 0.4 mmol) in 15 m is CH 2Cl2and 15 ml of a saturated aqueous solution of NaHCO3quickly mixed with the addition of 1.93 M phosgene in toluene (of 1.03 ml, 2 mmol) in one portion. The reaction mixture was stirred for 30 minutes, the organic phase was collected and the aqueous phase was extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuum to obtain 128 mg (97%) of carbamoylated in the form of a foamy solid. [Caution: vapors are very toxic phosgene. Use phosgene in the hood]. This product is used for the next solid-phase synthesis without additional purification.

(2) Obtaining benzylamine 2,5-dimethyl-7-benzyl-3,6-dioxohexane[1,2,4]triazolo[4,5-a]pyrazin-1-carboxylic acid

Bromatology resin (30 mg, 0.98 mmol/g) and a solution of benzylamine in DMSO (1.5 ml, 2 M) were placed in a vial with a screw cap. The reaction mixture was shaken at 60aboutWith the use of rotary kilns [Robbins Scientific] within 12 hours. The resin was collected by filtration and washed with DMF, then DHM with receipt of the first component.

A solution of Fmoc-alanine (3 EQ, the second component, commercially available), HATU (PerSeptive Biosystems, 3 EQ) and DIEA (3 EQ) in NMP (Advanced ChemTech) was added to the resin. After shaking the reaction mixture for 4 hours p is at room temperature, the resin was collected by filtration and washed with DMF, DHM and then DMF for joining thus the second component to the first component to the component.

To this resin was added 20% piperidine in DMF. After shaking this reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

A solution of N'-Fmoc-N-methylhydrosiloxane (combined third and fourth components, 5 EQ)obtained in the above stage (1), DIEA (5 EQ) in DHM added to prepared as described above, the resin. After shaking the reaction mixture for 4 hours at room temperature the resin was collected by filtration and washed with DMF, DHM and DMF.

To this resin was added 20% piperidine in DMF (10 ml per 1 g of resin). After shaking the reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

This resin was treated with a mixture benzylisothiocyanate (4 EQ) and DIEA (4 EQ) in DHM for 4 hours at room temperature. Then the resin was collected by filtration and washed with DMF, DHM and then Meon. The resin was dried in vacuum at room temperature.

This resin was treated with formic acid for 14 hours at room temperature. After removing the resin by filtration, fil the rat are condensed under reduced pressure to obtain the product in the form of oil.

1H NMR (400 MHz, CDCl3) δ ppm; to 1.48 (d, 3H), 2,98 (s, 3H), 3,18 (m, 1H), 3.46 in (m, 1H), 4,37-4,74 (m, 5H), to 5.66 (DD, 1H), 6,18 (m, 1H), 7,10-7,40 (m, 10H).

EXAMPLE of GETTING 5

Getting benzylamine 2,5,7-trimethyl-3,6-dioxohexane[1,2,4]triazolo[4,5-a]pyrazin-1-carboxylic acid

Specified in the header connection receive according to the procedure described in preparative example 4, but with the reaction bromatology resin with a solution of methylamine instead of benzylamine.1H NMR (400 MHz, CDCl3) δ ppm; to 1.48 (d, 3H), 2,99 (s, 3H), 3,03 (s, 3H), 3,38 (m, 1H), 3,53 (DD, 1H), 4,36 (DD, 1H), to 4.52 (q, 1H), 4,59 (DD, 1H), 5,72 (DD, 1H), to 6.19 (W t, 1H), 7,10-7,38 (m, 5H).

An EXAMPLE of OBTAINING 6

Getting benzylamine 2-methyl-5-(p-hydroxyphenylethyl)-7-naphthylmethyl-3,6-dioxohexane[1,2,4]triazolo[4,5-a]pyrazin-1-carboxylic acid

Bromatology resin (30 mg, 0.98 mmol/g) and a solution of naphthylethylene in DMSO (1.5 ml, 2 M) were placed in a vial with a screw cap. The reaction mixture was shaken at 60aboutWith the use of rotary kilns [Robbins Scientific] within 12 hours. The resin was collected by filtration and washed with DMF, then DHM with receipt of the first component.

A solution of Fmoc-Tyr(OBut) - OH (3 EQ), HATU (PerSeptive Biosystems, 3 EQ) and DIEA (3 EQ) in NMP (Advanced ChemTech) was added to the resin. After shaking the reaction mixture for 4 hours at room temperature the resin was collected by filtration and washed with DMF, DHM and Sitemap for joining thus the second component to the first component to the component.

To this resin was added 20% piperidine in DMF. After shaking this reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

A solution of N'-Fmoc-N-methylhydrosiloxane (5 EQ), DIEA (5 EQ) in DHM was added to the resin obtained as described above. After shaking the reaction mixture for 4 hours at room temperature the resin was collected by filtration and washed with DMF, DHM and DMF.

To this resin was added 20% piperidine in DMF (10 ml per 1 g of resin). After shaking the reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

This resin was treated with a mixture benzylisothiocyanate (4 EQ) and DIEA (4 EQ) in DHM for 4 hours at room temperature. Then the resin was collected by filtration and washed with DMF, DHM and then Meon. The resin was dried in vacuum at room temperature.

This resin was treated with formic acid for 14 hours at room temperature. After removing the resin by filtration, the filtrate are condensed under reduced pressure to obtain the product in the form of oil.

1H NMR (400 MHz, CDCl3) δ ppm; 2,80 are 2.98 (m, 5H), 3,21-3,37 (m, 2H), 4,22-to 4.52 (m, 2H), 4,59 (t, 1H), 4,71 (d, 1H), 5,02 (DD, 1H), 5,35 (d, 1H), 5,51 (d, 1H), 6,66 (t, 2H), 6,94 (DD, 2H), 7,21-8,21 (m, N).

EXAMPLE of GETTING 7

Getting benzylated is 2-methyl-6-(p-hydroxyphenylethyl)-8-naphthyl-4,7-dioxotetrahydrofuran[2,1-c][1,2,4]triazine-1-carboxylic acid

Bromatology resin (60 mg, 0.98 mmol/g) and a solution of naphtylamine in DMSO (2.5 ml, 2 M) were placed in a vial with a screw cap. The reaction mixture was shaken at 60aboutWith the use of rotary kilns [Robbins Scientific] within 12 hours. The resin was collected by filtration and washed with DMF, then THM.

A solution of Fmoc-Tyr(OBut) - OH (4 EQ), HATU (PerSeptive Biosystems, 4 EQ) and DIEA (4 EQ) in NMP (Advanced ChemTech) was added to the resin. After shaking the reaction mixture for 4 hours at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

To this resin was added 20% piperidine in DMF. After shaking this reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

A solution of Nβ-Fmoc-Nα-benzylideneaniline (4 equiv), HOBT [Advanced ChemTech] (4 EQ) and DIC (4 equiv) in DMF was added to a prepared as described above, the resin. After shaking the reaction mixture for 3 hours at room temperature the resin was collected by filtration and washed with DMF and then DHM. To this resin was added 20% piperidine in DMF (10 ml for 1 g of resin). After shaking the reaction mixture for 8 minutes at room temperature the resin was collected by filtration and washed with DMF, DHM and then DMF).

This resin was treated with a mixture benzylisothiocyanate (4 EQ) and DIEA (4 EQ) in DHM within 4 hours is at room temperature. Then the resin was collected by filtration and washed with DMF, DHM and then Meon. After drying the resin under vacuum at room temperature the resin was treated with formic acid (2.5 ml) for 18 hours at room temperature. The resin was removed by filtration and the filtrate are condensed under reduced pressure to obtain the product in the form of oil.

1H NMR (400 MHz, CDCl3) δ ppm; 2,73 (s, 3H), 3,13 (d, 1H), 3,21-to 3.38 (m, 3H), 3,55 (d, 1H), 3,75 (t, 1H), 4,22 (DD, 1H), 4,36 (DD, 1H), 4,79 (d, 1H), 5,22 (t, 1H), vs. 5.47 (m, 2H), of 6.68 (d, 2H), 6,99 (d, 2H), 7,21-8,21 (m, N);

MS (m/z, ESI) 564,1 (MN+) 586,3 (MNa+).

EXAMPLES

Plasmids

Deletion constructs SVR and R expressed in the commercially available vector pTriEx-3 (Novagen, Madison, WI). The number of deletions of mouse CBP generated using PCR from a plasmid with a full-sized mouse CBP, kind gift of Dr. Richard Goodman of the Vollum Institute, Portland, OR. Amplificatoare areas have a BamHI site at the 5'end and a NotI site at the 3'-end to create the possibility of cloning in reading frame with the ATG expressing vector pTriEx-3. For the recognition and treatment of these plasmids were cloned in frame readout with 6x-his-tag label and the label of herpes simplex virus (HSV) at the COOH-end. Direct primer used for cloning shorter-end designs SVR, was 5'-GATATCTGAGCTCGTGGATCCGATGGCCGAGAACTTGCTG-3' (SEQ ID NO:7). Reverse primers used for the SVR-the 1 (1-334), C2 (1-634), -C3 (1-1594), -C4 (1-2062), -C5 (1-2623), C6 (1-3094), C7 (1-3694)were: S1: 5'-CGTGTATACAGCTGTGCGGCC-GCGTTTGTACTGTTCGGCTG-3' (SEQ ID NO:8), S2: 5'-CGTGTATACAGCTGTGCGGCCGCTCC-ATTCATGACTTGAGC-3' (SEQ ID NO:9), C3: 5'-GCTGTATACAGCTGTGCGGCCGCGCGTTTTT-CAGGGTCTGC-3' (SEQ ID NO:10), C4: 5'-CGTGTATACAGCTGTGCGGCCGC AGCTGGTAAAGC-TGGCTG-3' (SEQ ID NO:11), C5: 5'-CGTGTATACAGCTGTG CGGCCGCATGTTGGAGAGAGGGC-AT-3' (SEQ ID NO:12), C6: 5'-CGTGTATACAGCTGTGCGGCCGCAGAACCTTGTAAA TCCTC-3' (SEQ ID NO:13), C7: 5'-CGTGTATACAGCTGTGCGGCCGCGCTGTAGTAGGCTGCATC-3' (SEQ ID NO:14). Shortened at the N-end design SVR was obtained with the following reverse primer: 5'-GTATACAGCTGTGCGGCCGCCAAACCCTCCACAA ACTTTTC-3' (SEQ ID NO:15). Direct primers for SVR-C8 (4081-7324), -C9 (4534-7324), -C10 (5074-7324), -C11 (5674-7324), -C12 (6286-7324), -R13 (6754-7324) were: C8: 5'-GATATCTGAGCTCG-TGGATCCGGAAGCTGGGGAGGTTT TT-3' (SEQ ID NO:16), C9: 5'-GATATCTGAGCTCGTGGAT-CCGAAGAAGATGC TGGACAAG-3' (SEQ ID NO:17), C10: 5'-GATATCTGAGCTCGTGGATCCGTCC AAATGGTCCACTCTG-3' (SEQ ID NO:18), C11: 5'-GATATCTGAGCTCGTGGAT CCGTCTCCTACCTCAGCACCA-3' (SEQ ID NO:19), C12: 5'-GATATCTGAGCTC GTGGATCCGAACATCCTTAA-ATCAAAC-3' (SEQ ID NO:20), C13: 5'-GATATCT GAGCCGTGGATCCGCAGCAGCAACGCATG-CAA-3' (SEQ ID NO:21). Using the direct primer shorter-end designs and reverse primer shortened at the N-end designs was amplified full-SVR mouse, and it was cloned in the vector pTriEx-3. SVR (Δ1-111+NLS) were generated using PCR full-SVR using the following direct, purified by electrophoresis in SDS page, primer containing a BamHI site to the left of the NLS sequence (underlined) SVR: 5'-ATCTGAGCTCGTGGATCCGGGACCGCCCAACCCCAAACGAGCCAAACTCCAGCCGAACAGTACAAACATGGCCAGCTTA-3' (SEQ ID NO:22)and the reverse primer was primer used for Generalov is of shorter N-end designs SVR, mentioned above. Insert cloned in sites BamHI-NotI plasmid pTriEx-3.

Deletion constructs R generated using PCR plasmids R person (CMVβ-R-SLEEP), kind gift of Dr. David Livingstone (Harvard, MA). The PCR products were cloned into the Hindlll site-NotI vector pTriEx-3. Direct primer for shorter-end designs R was: 5'-GACGGTACCGGTTCGAAGCTTA-TGGCCGAGAATGTGGT-3' (SEQ ID NO:23). The reverse primers for: R-P1 (1-334), R-P2 (1-634) and R-P3 (1-1054) are as follows: P1: 5'-CGTGTATACAGCTGTGCGGCCGC-CAAACCTAATC CAGGACT-3' (SEQ ID NO:24), P2: 5'-CGTG-TATACAGCTGTGCGGCCGCGTTGC CAGCACTTCCCAT-3' (SEQ ID NO:25), P3: 5'-CGTGTATACA-GCTGTGCGGCCG CGGCCTGTTCCCGGCGCTG-3' (SEQ ID NO:26).

Plasmid β-catenin/TCF-reporter was generated by insertion of 4 tandem binding sites TCF4 (CCAACCTTTGATCTTACCCCCTTTGATCTTACCCCCTTTGATCAG-GAATTCGGTTGGAAACTAGAATGGGGGAAACTAGAATGGGGGAAACTAGT CCTTAAG) (SEQ ID NO:27) in sites XhoI-kpni restriction sites of the plasmid pGL3 (Promega) to the left of the SV40 promoter, triggering the expression on the right luciferase gene.

All primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Restriction enzymes used for cloning are underlined and were purchased from New England Biolabs, Beverly, MA.

Cell culture

Cell lines colon human SW480 and NST and normal colonocytes CCD18Co (ATCC, Manassas, VA) were grown in DMEM (Invitrogen Gibco-BRL, Baltimore, MD), supplemented with 10% fetal calf serum in an atmosphere of 5% CO2at 37aboutC.

Transfection

Exponentially growing cells SW480 and NST (105)were cultured in 24-hole tablets or cups 100 mm and transfusional 0.5 μg of β-catenin/TCF-reporter and increasing concentrations of the effector plasmid or 10 µg expressing vectors, respectively. The cells were transfusional using FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN) or Superfect (Qiagen, Valencia, CA)as indicated. Nuclear extracts were obtained according to the procedure described in the set of NE-PER (Pierce Biotechnology, Rockford, IL).

Luciferase assays

Luciferase assays for different groups were carried out in 20 μl of cell lysate using dual analysis of luciferase (Promega), after 16-24 h after transfection as indicated. For the normalization used increasing concentrations of expressing empty vector pTriEx-3.

Analyses using soft agar

Analysis of the formation of colonies in soft agar was performed with SW480 cells by means of some modifications of the procedure described previously (Moody et al., “A vasoactive intestinal peptide antagonist inhibits non-small cell lung cancer growth,” Proc. Natl. Acad. Sci. USA. 90:4345-49 (1993)).

Each well (35 mm) 6-hole Cup (Nalge Nunc International, Roskide, Denmark) were coated with 1 ml of 0.8% bottom agar in DMEM containing 10% fetal calf serum. After curing 1 ml of DMEM medium containing 0.4% top agar, 10% fetal calf serum, connection, concentrated in 2 times and each well was added 5000 viable cells. These cultures were incubated at 37aboutWith humid thermostat with 5% CO2. Colonies in soft agar were subjected to monitoring once a day and photographed item is after incubation for 8 days. Colonies with a diameter of >60 μm, believed.

Immunoprecipitate

Cells were literally Lisinym buffer containing 20 mm HEPES pH of 7.9, 100 mm NaCl, 0.5 mm EDTA, 0.5% of Nonidet P-40, 6 mm MgCl2, 5 mm 2-mercaptoethanol and 1 tablet a mixture of protease inhibitors Complete™ (Roche Molecular Biochemicals)for 30 minutes on ice and osvetleni by centrifugation. Lysates of whole cells were incubated with the indicated antibodies for CBP-C1 antibody a-22 from Santa Cruz Biotechnology, Inc., for R-P1, antibody (N-15 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) and for full-length endogenous β-catenin monoclonal antibody (Transduction Laboratories, Lexington, KY), pre-linked to Protein a-agarose pellets (Pierce, Rockford, IL)for 1 hour at room temperature. Immune complexes were washed several times in HBS (100-150 mm NaCl, as indicated, 10 mm HEPES pH of 7.9, 5 mm 2-mercaptoethanol and 1 tablet a mixture of protease inhibitors Complete™) and subjected to Western-blotting, see below. Then, these pellets were washed seven times with buffer containing 20 mm HEPES pH of 7.9, 500 mm NaCl and 5 mm 2-mercaptoethanol containing 1 tablet a mixture of protease inhibitors Complete™).

“Drop-down” analysis

25-100 μm biotinylated COMPOUND 2 was bound over night at room temperature with 100 μl of a 50% suspension of streptavidin-agarose pellet (Amersham Pharmacia Biotech, Arlington Heights, IL) in buffer containing 50% DMSO and 50% binding protein buffer, RVV (20 mm Hepes R is 7,9, 20% glycerol, 0.05 mm EDTA, 60 mm NaCl, 6 mm MgCl2, 0,1% Nonidet P-40, 5 mm 2-mercaptoethanol and 1 tablet a mixture of protease inhibitors Complete™). These pellets were washed 3 RVV to remove unbound COMPOUNDS 2. 100-200 μl of lysates of whole cells containing sverkhekspressiya plasmids (see thus), incubated with these pellets for 3-4 hours at room temperature or overnight at 4aboutC. Then, these pellets were washed 3x in RVV-buffer and then erwerbende proteins were subjected to electrophoresis in LTO-page and Western-blotting (see below). In competitive assays, the excess of COMPOUND 1 as described for each experiment, was preincubate with these lysates for 1 hour at room temperature.

ChIP analysis

Analyses with crosslinking with formaldehyde and immunoprecipitated chromatin SW480 cells was performed as described previously (Barlev et al., “Acetylation of p53 activates reduced through recruitment of coactivators/histone acetyltransferases,” Mol. Cell 8:1243-54 (2001); El-Osta et al., “Analysis of Chromatin-immunopurified MeCP2-associated fragments,” Biochem. Biophys. Res. Commun. 289:733-37 (2001); Shang et al., “Formation of the androgen receptor reduced Complex,” Mol. Cell 9:601-10 (2002)). The primers used for PCR of the promoters of c-myc and cyclin D1, are as follows: forward primer c-myc: 5'-TGGTAGGCGCGCGTAGTTA-3' (SEQ ID NO:28) and reverse primer: 5'-GGGCGGAGATTAGCGAGAG-3' (SEQ ID NO:29). Direct primer cyclin D1: 5'-TGCTTAACAACA-GTAACGT-3' (SEQ ID NO:30) and reverse primer: 5'-GGGGCTCTTCCTGGGCAGC-3' (SEQ ID NO:31). These PR is emery ChIP were designed at about 20-30 base pairs to the right of the domain of TCF4 binding to the promoter region near the site of transcription initiation. The PCR products have a size of approximately 200 base pairs. Anti-SVR-antibody, al-26, was a kind gift of Dr. David Livingstone.

Western blot analysis

Immune complexes obtained as described above were separated by electrophoresis in LTO-SDS page followed by transfer to membrane immobilon-P (PVDF) (Millipore, Bedford, MA). These membranes were blocked by 5% non-fat dry milk in TBST (15 mm Tris-HCl, pH 7.4, 0.9% NaCl and 0.05% tween-20) followed by blotting with indicated antibodies. Anti-His-antibody from Qiagen Inc. used for detection of proteins produced in pTriEx-3. Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.), used for detection. Immunoblot were analyzed using set to ECL detection (Amersham Pharmacia Biotech).

Immunofluorescence

The immunofluorescence was used to determine the location SVR and β-catenin in SW480 cells and NSC treated with COMPOUND 1 (25 μm) or control (0.5% DMSO). Cells in logarithmic phase were sown and after 24 hours, these cells were treated with COMPOUND 1 or control. 24 hours after treatment the cells were fixed. Cover glasses were incubated with antibodies induced against CBP (a-22) (Santa Cruz Biotechnology) and β-catenin (Transduction Laboratories), respectively. Slides were examined using the confocal microsc the PA laser scanning Nikon PCM 2000 after application of the secondary antibody, conjugated with either FITC or TRITC (Jackson ImmunoResearch, Westgrove, PA).

Running cytometrics analysis (FACS)

For FACS analysis of approximately 5 x 106cells from PNRI-processed or processed by the media, cells were fixed with 70% cold ethanol and kept at -20aboutC for at least 30 minutes. These cells were washed once with 1 x SFR and incubated with a solution of iodide of propecia (fluorescent dye) (85 μg/ml of iodide of propecia, 0,1% Nonidet P-40, 10 mg/ml RNase for 30 minutes at room temperature, 10000 stained cells for each sample were obtained using flow cytometry Beckman Coulter EPICS XL-MCL and the percentage of cells in different cell cycle phases was determined using Expo32 ADC (Coulter Corporation, Miami, Florida, 33196).

Purification of proteins

CBP (1-111) and R (1-111) expressed in the form of a fused protein with a 6X-His tag and was affinity purified from bacterial lysates using their 6X-His tags. Transformed bacterial precipitation (culture 1 l) resuspendable in 5-10 ml lisanova buffer (20 mm Hepes pH of 7.9, 150 mm NaCl, 0.1% Nonidet P-40, 5 mm 2-mercaptoethanol and 1 tablet a mixture of protease inhibitors Complete™). Cells were literally sonification. Clarified lysates were incubated for 1 hour at 4aboutWith 500 ál of Ni-NTA-agarose pellet (Qiagen). Associated proteins were suirable 500 ál of buffer for elution (20 mm Hepe pH of 7.9, 150 mm NaCl, 1 tablet a mixture of protease inhibitors Complete™, 5 mm 2-mercaptoethanol and 250 mm imidazole). Erwerbende proteins were frozen in the form of a small aliquot.

PCR analysis reverse transcription real-time

Set RNeasy Midi (Qiagen) was used for extraction of RNA and real-time PCR was performed according to the Protocol provided with the kit SYBR Green PCR Master Mix (Perkin Elmer Biosystems, Shelton, CT). Primers used for amplification of cyclin D1 and axin2, hnkd, c-myc, c-jun and fra-1 were: forward primer cyclin D1: 5'-AGATCGAAGC-CCTGCTG-3' (SEQ ID NO:32) and reverse primer: 5'-AGGGGGAAAGAGCAAAGG-3' (SEQ ID NO:33), leading to a product size of approximately 300 BP; direct primer axin2: 5'-GTGTGAGGTCCAC GGAAA-CT-3' (SEQ ID NO:34) and reverse primer: 5'-CTCGGGAAATGAGGTA-GAGA-3' (SEQ ID NO:35); direct primer hnkd: 5'-CTGGCTGCTGCTACCACCA TTGCGT-3' (SEQ ID NO:36) and reverse primer: 5'-CCAGGCCCAAATTGGG ACGT-3' (SEQ ID NO:37); direct primer c-myc: 5'-GAA-GAAATTCGAGCTG CTGC-3' (SEQ ID NO:38) and reverse primer: 5'-CACATACAGTCCTGGATGAT-G-3' (SEQ ID NO:39); direct primer c-jun: 5'-AGATGCCCGGCGAGACACCG-3' (SEQ ID NO:40) and reverse primer: 5'-AGCCCCCGACGGTCTCTTT-3' (SEQ ID NO:41); direct primer fra-1: 5'-ACC-CCGGCCAGGAG-TCATCCGGGCCC-3' (SEQ ID NO:42) and reverse primer: 5'-AGGCGCCTCACAAAGCGAGGAGGG-TT-3' (SEQ ID NO:43). β-actin was used for normalization. Primers of β-actin were: forward primer: 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' (SEQ ID NO:44) and reverse primer: 5'-CGTCATACTCCTCCTTGCYGATCCACA TCTGC-3'(SEQ ID NO:45). Each set of primers amplified at 95aboutC for 10 minutes and 40 cycles of 95aboutC for 15 seconds and 60 aboutC for 1 minute.

Analysis of the activity of caspase-3

Cells SW480, NST and CCD18Co were sown in 105cells per well (96 well plates) for 24 hours before treatment. 25 μm COMPOUND 1 or control (0.5% DMSO) was added to each well. 24 hours after treatment cells were literally and caspase activity was measured using a kit for determining caspase-3/7 (Apo-One Homogeneous caspase-3/7 assay, #G77905, Promega). Relative fluorescence units (RFU) was obtained by subtraction of the values of units of the blind experience (control, no cells) from the experimentally measured values.

Table 1 shows the results of quantitative analysis of PCR with reverse transcription (RT-PCR) real-time SW480 cells treated for 4, 8 or 24 hours or COMPOUND 1 (25 mg)or control (0.5% DMSO). 1 µg of mRNA for each time point were subjected to RT-PCR real-time. The level of endogenous expression of cyclin D1, c-myc, fibronectin, hnkd, axin2, c-jun and BMP-4 were measured relative to β-actin. Quantitative determination Δ thresholds (CT) were performed by subtracting the mean values of each set of corresponding mean values obtained for β-actin. All experiments were performed in two replications.

CONNECTION 1 prevents β-catenin/TCF transcription effect on SVR

Due to mutations in the APC cell carcinoma of the colon SW480 have the given constitutive movement of β-catenin in the nucleus and, therefore, high basic transcription of β-catenin/TCF, as assessed reporter system TOPFLASH (Korinek et al., “Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma,” Science 275:1784-87 (1997)). Related reporter used for screening libraries of small molecules with template secondary structure (Ogbu et al., “Highly efficient and versatile synthesis of libraries of constrained b-strand mimetics.” Bioorg. Med. Chem. Lett. 8:2321-26 (1998); Eguchi et al., “Solid-phase synthesis and structural analysis of Bicyclic β-turn mimetics incorporating functionality at the i to i+3 positions,” Amer. Chem. Soci. 102:22031-32 (1999)inhibitors mediated by a complex of β-catenin/TCF transcription. From the initial screening, the authors have chosen CONNECTION 1 (figure 1A), which had IC505 μm (figure 1B) and a very good selectivity in comparison with a number of other CBP-dependent reporters, including NFAT (figure 1C), CRE and AP-1 (data not shown). COMPOUND 1 exhibits a similar activity in cells NST, which are defective in phosphorylation sites of β-catenin, but Express ARS wild type (data not shown).

To identify molecular targets (targets) CONNECTION 1, the authors have derivateservlet it for use as an affinity reagent to obtain the COMPOUND 2 (figure 1A). Nuclear extracts were obtained from SW480 cells after pretreatment with COMPOUND 1 or the media, and then incubated with COMPOUND 2. Then these complexes were separated on a streptavidin-Groznyj granules and subjected to gel electrophoresis. Big band held from nuclear extracts of SW480 cells by affinity column with COMPOUND 2 had an average molecular weight of 225 KDa was identified by Western blot turns as CREB-binding protein (CBP) (figure 1D). COMPOUND 1 was specifically buyrevia SVR associated with COMPOUND 2 (figure 1D, compare lane 7 with 8) and the incubation of nuclear extracts with COMPOUND 1 (20 μm) before affinity chromatography blocked the binding of CBP (figure 1D, lane 9). Used antibody was specific in relation to SVR and did not cross-react with R. Thus, these results indicate that COMPOUND 1 binds CBP.

A number of additional studies were performed to further validate that the CONNECTION 1 connects SVR.14C-labeled version of COMPOUND 1 was synthesized by the inclusion of14C-labeled tyrosine in this synthesis. Nuclear lysates of cells SW480, nitrostilbene or transfected expressing vectors or β-catenin, or CBP, was treated14C-labeled COMPOUND 1 with either DMSO or with a “cold” CONNECTION 1 during the night. Then cell lysates were absoluely using columns G-25 to remove unbound14C-labeled COMPOUNDS 1 and was measured by incorporation of radioactivity. As seen in figure 1E, nuclear lysates, transfetsirovannyh SVR, kaliprasanna 4-6 times increased inclusion of 14C-labeled COMPOUND 1 in comparison with control (figure 1E, compare lane 2 with 6), which was removed competitive “cold” CONNECTION 1 dependent dose-dependent manner (figure 1E, compare lane 6 with 7 and 8).

On the basis of these data it is concluded that COMPOUND 1 binds CBP. Thus, one aspect of the present invention provides a method comprising combining the composition containing the SVR, with the agent, where the agent binds CBP. The binding of CBP prevents any other binding assays, which otherwise would be subject to the SVR. Thus, this invention provides a method of treatment of the subject by binding to CBP, introducing an effective amount of an agent to a subject in need of it.

COMPOUND 1 specifically interacts with the first 111 amino acids of CBP

Biotinylated analogue, COMPOUND 2 (figure 1A)was used to determine the minimum area SVR required for binding of COMPOUND 1. Cell lysates that contained sverkhekspressiya fragments of CBP (figure 2B, top panel), incubated with streptavidin-agarose beads of pre-associated with a CONNECTION 2 for several hours. Then associated proteins were suirable of these granules and subjected to gel electrophoresis and Western blot turns with COI is whether the anti-His antibodies for detection of the bound fragment (corresponding fragments) SVR. As shown (figure 2B, bottom panel), the minimum area, which is specifically associated COMPOUND 2, was the area of amino acids 1-111 on NH2-the end of the SVR (compare lanes 2, 3, and 4 others). As is clear from the experiments coimmunoprecipitation, the binding was not detected with any of the fragments R, sverkhekspressiya in the SW480 cell (data not shown, see below). When CBP (1-111), SVR (1-211) and SVR (1-351) together with R (1-111), R (1-211) and R (1-351) was sverkhekspressiya in SW480 cells (figure 2C, bottom panel), an excess of COMPOUND 1 significantly competitive deleted binding fragments of CBP (figure 2C, top panel, compare lanes 4-6 7-9), but has not had any effect on the binding of fragments R (figure 2C, compare lanes 10-12 13-15). Thus, it is seen that COMPOUND 1 binds to the first 111 amino acids of CBP, but not related protein, R.

To exclude the possibility of indirect Association between SVR and COMPOUND 1, mediated by other cellular component, and to test for direct binding of the authors affinity purified CBP (1-111) and R (1-111) using the 6x-His-tagged expressed E. coli proteins (figure 2D, right). Using CONNECTION 2 associated with streptavidin-agarose bead, the authors demonstrated specific binding of CBP (1-111), but not R (1-111) with COMPOUND 2, which show jumping is NTO removed the excess COMPOUND 1 (figure 2D, on the right, compare lanes 3-5 6-8). These results confirm a direct Association between CBP (1-111) and COMPOUND 1 in vitro. Because the CONNECTION 1 associated with SVR, expressed in E. coli, this reduces the likelihood that the SVR must be phosphorylated by kinases of the mammal to associate with the CONNECTION 1.

Additional confirmation of the crucial role of the amino-end of the SVR was obtained with the use of design SVR (Δ1-111 + NLS). While the expression of full-SVR rescued the inhibition of COMPOUND 1 (25 μm) activity of β-catenin/TCF promoter-dependent dose-dependent manner, the expression of CBP (Δ1-111 + NLS) were not affected by this action (file).

Thus, in one aspect, the invention provides a method of modulating β-catenin-induced gene expression, involving contacting the composition with an agent, where the composition comprises β-catenin, CBP and R, and this agent are contacted with the composition in an amount effective to reduce the binding of β-catenin with CBP with a small effect or no effect on the binding of β-catenin with R.

CONNECTION 1 competes with β-catenin for SVR

The SVR is the limiting speed factor in many transcriptional events. As can be seen in figure 3A, transfection of increasing concentration of CBP, but not β-catenin, increased IC50 CONNECTION 1 (12.5 μm) dependent on dose. The analyses thus performed in SW480 cells to determine that the CONNECTION 1 destroys the binding of β-catenin with CBP. Immunoprecipitated β-catenin SVR inhibited by COMPOUND 1 dependent on concentration (figure 3B, compare lanes 2, 3 and 4, lane 1 is the control without addition of antibodies). The binding of COMPOUND 1 with SVR is very specific, because this compound does not prevent the binding of β-catenin with R (FIGU, bottom panel, compare lanes 2-4), despite the fact that SVR and R are vysokomolochnye.

For additional confirmation of the above-mentioned interaction between β-catenin/CBP SW480 cells were transfusional the above deletion constructs SVR and R (figa). After subsequent washing of the pellets CBP (1-111), SVR (1-211) and SVR (1-351)and R (1-111), R (1-211) and R (1-351) was specifically associated with immunoprecipitated β-catenin (figs, compare lanes 2-4 with 5-15). These studies linking clearly show that the first 111 amino acids NH2all as SVR and R associated with β-catenin. To confirm that this area overlaps the binding site SVR CONNECTION 1, the authors evaluated the binding of CBP (1-111), SVR (1-211) and SVR (1-351) and R (1-111), R (1-211) and R (1-351) with β-catenin in the presence of an excess of the COMPOUNDS IS OF 1. Fig.3D, bottom panel, shows comparable levels of expression of these fragments in SW480 cells. Exhibiting an excess of COMPOUNDS 1 strikingly removes competitive binding fragments SVR with β-catenin, but not affect the binding of fragments R with β-catenin (fig.3D, top panel, compare lanes 4-9 10-15). From the presented data shows that COMPOUND 1 specifically binds to the amino end of CBP (amino acids 1-111), distinguishing between vysokomolochnye coactivators, SVR and R, and competitive removes the interaction of β-catenin with CBP.

These studies linking clearly show that the first 111 amino acids as SVR and R are minimal region of interaction with β-catenin. Comparison of the sequences of these regions shows a striking similarity with previously published motifs bind β-catenin detected in TCF, APC and E-cadherin (fige and 3F). Data mapping sequences strongly suggest that the SVR, like other interacting with β-catenin proteins, captures conservative stretch of negatively charged amino acids required for binding of β-catenin.

COMPOUND 1 reduces the level of nuclear β-catenin

Investigated the subcellular distribution of β-catenin and CBP to determine whether the localization of each of the CONNECTION 1. A large part of the endogenous β-catenin in SW480 cells detected in the nucleus, as well as SVR (figa). Treatment with COMPOUND 1 caused the transport of β-catenin in the cytoplasm of SW480 cells in which expression of endogenous E-cadherin is limited (figa, compare the control, top panel, with the processed cells, lower panel) (de Vries et al., “In vivo and in vitro invasion in relation to phenotypic characterisnics of human colorectal carcinoma cells,” Br. J. Cancer 71:271-77 (1995)). Treatment with an inhibitor of nuclear transport leptomycin To eliminate the induced CONNECTION 1 cytoplasmic transport of β-catenin, suggesting that nuclear export of β-catenin observed in figa, was caused by the destruction of the COMPOUND 1 complex of β-catenin/CBP (data not shown). Thus, the authors conclude that the destruction of the binding of β-catenin with CBP leads to reduced nuclear levels of β-catenin. Induced CONNECTION 1 moving of β-catenin from the nucleus to the cytoplasm of SW480 cells was observed using Western blot analysis (pigv).

Differential regulation and use of coactivator genes to target β-catenin

Gene cyclin D1 is expressed inappropriately in many different tumor types, and it is known that he is a direct target path Wnt/β-catenin (Shtutman et al., The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway,” Proc. Natl. Acad. Sci. USA 96:5522-27 (1999); Tetsu et al. “Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells,” Nature 398:422-26 (1999)). To determine the effect of COMPOUND 1 on the expression of the straight line, the target path β-catenin/TCF, PCR with reverse transcription (RT-PCR) real time was performed on mRNA extracted from cells treated with COMPOUND 1 (25 μm) or control at time points 4, 8 and 24 hours after treatment (table 1). Table 1 shows the results of quantitative analysis of PCR with reverse transcription (RT-PCR) real-time SW480 cells treated for 4, 8 or 24 hours or COMPOUND 1 (25 μm)or control (0.5% DMSO). 1 µg of mRNA for each time point were subjected to RT-PCR real-time. The levels of expression of endogenous cyclin D1, C-myc, fibronectin, hnkd, axin2, c-jun and BMP-4 were measured relative to β-actin. Quantitative determination Δ thresholds (CT) were performed by subtracting the mean values of each set of corresponding mean values obtained for β-actin. All experiments were performed in two replications.

TABLE 1
RT-PCR real-time
GeneThe incubation period
(watch)

CONNECTION 1
(25 µm)
mRNA
cyclin D14
24
0,6
3,1
axin24
8
0,3
0,8
hnkd4
8
0,3
1,0
c-jun4
24
0
-2,9
fra-14
8
of-1.0
of-1.4
c-myc4
8
-0,9
-2,4

Gene β-actin was used to normalize the data. Δ thresholds (CT) were performed by subtracting the mean values of each set of corresponding mean values obtained for gene β-actin. The lower the amount of mRNA in the cells, the higher will be the amount Δ. As summarized in table 1, observed an increase in Δ-values for the matrix cyclin D1 in cells treated with COMPOUND 1, dependent on time in comparison with control cells. Evaluated the protein levels cycline D1 cells. Cell lysates treated whole cells SW480 were subjected to gel electrophoresis and Western blot analysis. As shown nfigure 5A, there was a distinct reduction in the level cycline D1 after treatment with COMPOUND 1 (25 μm), starting with 4 hours and increased at 24 hours after treatment (compare lanes 1, 2, 3, 4 and 5 with 6).

In addition, it was chosen subpopulation of genes, which were reported previously in the literature, are direct targets of transcription of the β-catenin/TCF, for analysis of RT-PCR real-time. Among this set of levels of matrix genes axin2 and human naked cuticle (hnkd) (Yan et al., “Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta-catenin signaling is activated in human colon tumors,” Proc. Natl. Acad. Sci. USA 98:14973-78 (2001)) was negatively regulated, as it was clear (table 1). However, for several regulated β-catenin/TCF gene levels matrices was significantly increased, for example, c-myc (He et al., “Identification of c-MYC and as a target of the APC pathway,” Science 291:1509-12 (1998)), C-jun and fra-1 (table 1) (Mann et al., Target genes of beta-catenin-T-cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas,” Proc. Natl. Acad. Sci. USA 96:1603-08 (1999)). Thus, COMPOUND 1 inhibits the expression of only a subpopulation of target genes of β-catenin.

The discovery that COMPOUND 1 inhibited the expression of cyclin D1, but not c-myc, two known gene transfer path of the signal, β-catenin, together with the specificity of COMPOUND 1 with respect to CBP, but not in relation to R, suggests that selective use of R promoter of C-myc may allow you to avoid repression CONNECTION 1. To assess the Prim is namesti of coactivator in endogenous promoter of C-myc was performed analyses thus chromatin (ChIP) in SW480 cells, processed or COMPOUND 1 (25 μm)or control (0.5% DMSO). As shown in figure 5B, the promoter of c-myc busy both coactivators SVR and R in the treated control cells, and most of the busy SVR. Treatment with COMPOUND 1 completely and selectively blocks the Association of CBP with the promoter of c-myc and simultaneously increases the level of associate R. As in the case with the promoter of c-myc, treatment with COMPOUND 1 completely and selectively blocks the Association of CBP with the promoter of cyclin D1 (figure 5B, bottom panel). In sharp contradiction, R cannot replace SVR in relation to associate with the promoter of the gene cyclin D1. This correlates well with data obtained FROM real-time PCR and Western blot analysis (table 1 and figure 5A). Thus, COMPOUND 1 selectively reduces the Association of CBP, but not R with subpopulation regulated β-catenin promoters.

For further study of the selectivity of COMPOUND 1 was performed analysis of cDNA chips using the Clontech Atlas Human Cancer 1.2 Array (#7851-1). These data demonstrated that COMPOUND 1 had a very selective effect on global gene transcription (Table 2-5). After 8 hours treatment of SW480 cells 25 μm COMPOUND 1, ~2% of the analyzed genes positively regulated more than 2-fold, whereas ~0,3% of these genes negatively regulated by more than h the m 50% (table 2-3).

TABLE 2
Genes positively regulated by 8 hours of processing 25 μm COMPOUND 1 in SW480 cells
Code geneRatioProtein/gene
A12d4,20the receptor for epidermal growth factor (EGFR)
As2,48fos-related antigen (FRA1)
A14d1,36ERBB-3 receptor protein, the precursor tyrosine kinase
A14n1,94the glucose Transporter brain (GTR3)
B08h2,56PTPCAAX1 nuclear tyrosinosis (PRL-1)
Sa1,84protein WSL+TRAMP+Apo-3+receptor 3 domain of death (DDR3)
C05d6,92induced by stopping the growth and DNA damage protein 153 (GADD153)
C09i536 induced by stopping the growth and DNA damage protein
(GADD45)
D03b1,47DNA-binding protein SRVR
D03e2,82integrin alpha (ITGA3); galactopoiesis B3 (GAPB3)
D03k1,77the receptor for nerve growth factor with low affinity
(NGF-receptor; NGFR)
D06e1,84integrin beta 4 (ITGB4); CD104 antigen
D08e1,69the precursor integrin alpha 7B (IGA7B)
D08f2,14paxillin
D09b2,11nuclear protein
E03dwith 3.27nerve growth factor-induced homolog RS
E07f1,77the precursor of interleukin-1 beta (IL-1; IL1B); catabolic
E09e,94 inhibitory cytokine 1 macrophages (MIC1)
F04i5,85associated with gelatinase neutrophil precursor
lipocalin (NGAL)
F05e1,82ornithindecarboxilase
F06n3,15protein alpha of early growth response (EGR alpha)
F08f1,59keratin 18 cytoskeleton type I; cytokeratin 18 (K18)
F09g6,40the Gravin
F13k2,23glycyl-tRNA synthetase

TABLE 3
Genes negatively regulated by 8 hours of processing 25 μm COMPOUND 1 in SW480 cells
Code geneRatioProtein/gene
A05i0,57G2/mitotic-specific cyclin B1 (CCNB1)
A0f 0,77metalloproteinase 11 matrix (MMR); stromelysin 3
B01m0,74linker for activation of T cells (LAT)
B07l0,85cellisvisible protein placenta; canvascolor
B12j0,62ras-related substrate 2 toxin botulinum C3; P21-rac2
B14n0,74receptor beta retinoic acid (RXR-beta; RXRB)
C06f0,38the factor that allows the DNA replication MSM; CDC21 homolog
C13e0,38cyclic nuclear antigen of proliferating cells (PCNA);
cyclin
D08b0,73the histone H4
D11c0,60the precursor of subunit Epsilon glycoprotein CD3
surface T-cell
E01g0,88preds the factory worker interleukin-13 (IL-13); NC30
F07e0,53subunit M2 ribonucleopeptide

td align="center"> 2,50
TABLE 4
Genes positively regulated 24-hour processing of 25 μm COMPOUND 1 in SW480 cells
Code geneRatioProtein/gene
Asof 1.34protooncogen C-jun; transcription factor AP-1
A12dof 2.51epidermal growth factor (EGFR)
As1,99fos-related antigen (FRA1)
C01j1,71protein elk-3 domain ets; NET; SRF-accessory protein 2
(SAP2)
C05d3,66induced by stopping the growth and DNA damage protein 153
(GADD153)
D03e2,99integrin alpha 3 (ITGA3); galactopoiesis B3 (GAPB3)
D03kthe receptor for nerve growth factor with low affinity
(NGF-receptor; NGFR)
D06e2,68integrin beta 4 (ITGB4); CD104 antigen
D08haccounted for 10.39N-sam; predecessor receptor 1 growth factor
fibroblasts (FGFR1)
E02m1,36shortened lymphocyte antigen HLA-G MHC class I
E02n2,35predecessor regulated glucose protein 78 kDa
(GRP 78)
E07f1,98the precursor of interleukin-1 beta (IL-1; IL1B); catabolic
E09e2,79inhibitory cytokine 1 macrophages (MIC1)
F04gof 1.34vimentin (VIM)
F04i23,85associated with gelatinase neutrophil precursor
lipocalin (NGAL)
F06n1,79 protein alpha of early growth response (EGR alpha)
F09g8,46the Gravin
F09h3,25protein TRAM
F12l2,07BENE
F13k2,02glycyl-tRNA synthetase
G311,29alpha-subunit of C-4 antigen HLA
class I (HLAC)

TABLE 5
Genes negatively regulated 24-hour processing of 25 μm COMPOUND 1 in SW480 cells
Code geneRatioprotein/gene
A02b0,52protein EW
A03g0,43c-myc-binding protein MM-1
A06i0,47G1/S-specific cyclin D1 (CCND1), cyclin PRAD1; bcl-1 oncogene
A10k0,28regulatory subunit 1 cyclin-dependent kinase (CKS1)
A11k0,40regulatory subunit of the cyclin-dependent kinase (CKS2)
B02a0,21the carrier protein ADP/ATP
B03m0,45protein 14-3-3 Sigma; stratified; marker protein 1
epithelial cells
B07l0,50cellisvisible protein placenta; canvascolor
C04b0,73protein associated with the receptor necrosis factor
tumor type 1 (TRAP1)
C04h0,36HHR23A; protein RAD23A reparations cut nucleotides UV
C05f0,23the factor that allows the DNA replication MSM; nuclear protein WM
C13e0,33cyclic nuclear antigen of proliferating cells (PCNA), cyclin
D06m0,45cytosolic superoxide dismutase 1 (SOD-1)
D07b0,35protein HMG2 high mobility
D07m0,34glutathionylated (GSH synthetase; GSH-S)
D08b0,27the histone H4
D09b0,49nuclear protein
D12a0,70subunit R48 factor chromatin Assembly (subunit R48
CAF1)
E04i0,45PDGF-associated protein
E11l0,66the precursor glycoprotein CD59
F03e0,34the fatty acid synthase
F06d0,40subunit N L-lactate dehydrogenase (LDHB)
F10c0,68inosine-5'-mono is ofaccelerated 2
F13j0,55the elongation factor 2 (EF2)
G290,72subunit 1-specific brain tubulin
(TUBA1)
G450,65highly basic protein of 23 kDa; 60S-ribosomal protein L13A

CONNECTION 1 stop G1/S-phase and activates caspase activity.

It was shown that inhibition of gene expression cycline D1 causes the stop G1/S phase of the cell cycle (Shintani et al., “Infrequent alternations of RB pathway (Rb-p16INK4A-cyclin D1) in adenoid cystic carcinoma of salivary glands,” Anticancer Res. 20:2169-75 (2000)). Cells NST (figure 6A, top panel) and SW480 (figure 6A, bottom panel) were treated with COMPOUND 1 (25 μm) (figure 6A, right) or control (0.5% DMSO) (figure 6A, left) within 24 hours. Then these cells were stained with iodide of propecia (PI) and analyzed the DNA content using a FACS-cytofluorometry. As expected, control cells (figure 6A, left) had a normal cell cycle, while treated with COMPOUND 1 cells (figure 6A, right) found increased accumulation of G1/S-phase of the cell cycle. Thus, it can be seen that COMPOUND 1 causes a delay of cells in the G1phase.

Caspase are the cysteine proteases that are normally activated in a population of cells that run apoptotic stimuli. To assess the induction of apoptosis in SW480 cells, NST and colonocytes wild-type (CCD18Co cells) these cells were treated with either COMPOUND 1 (25 μm)or control (0.5% DMSO) for 24 hours followed by analysis on the activity of caspase 3/7. As shown in figure 6B, COMPOUND 1 specifically and significantly activated the path of caspase-3/7 in SW480 cells and NST in comparison with CCD18Co cells.

COMPOUND 1 reduces the proliferation of transformed colorectal cells

Analyses of the formation of colonies in soft agar was performed using SW480 cells treated with COMPOUND 1 (0.25 to 5 μm) and 5-fluorouracil (5FU) (0.5 to 32 μm). As shown in figure 7A, COMPOUND 1 shows a dose-dependent decrease in the number of formed colonies. The value of the IC50COMPOUNDS 1 and 5-FU was 0,87±0,11 μm and up to 1.98±0.17 microns, respectively. Thus, COMPOUND 1 increased the activity of caspase and reduced growth in vitro colorectal cells, which are transformed by mutations that activate signaling β-catenin.

COMPOUND 4 and COMPOUND 5 reduce tumor growth in mice Min.

COMPOUND 4, COMPOUND 5 or the media was injected into wild-type mice and mice Min. COMPOUND 4 is the e analogue COMPOUND 1 (figure 1A). We measured the number of polyps formed in the small intestine and colon of these mice after different treatments (table 6). These data showed that COMPOUND 4 and COMPOUND 5 with the introduction at approximately 300 mg/kg reduced the number of polyps in Min mice in comparison with the number of polyps in the control mice treated only with the media.

TABLE 6
Activity of a COMPOUND 4 and COMPOUND 5 on the number of polyps in mice Min
GroupThe number of polyps (mean ± S.D.)P (total) vs. VH% inhibition vs. VH
The small intestineThe colonThe total number of
Wild type0,0±0,00,0±0,00,0±0,0--
Media65,8±15,91,8±1,567,7±15,3--
Connected the s 5 -100 mg/kg 69,2±20,81,7±1,571,4±23,0--
Connection 5 -300 mg/kg46,1±17,11,1±1,247,0±16,9<0,0131
Connection 4 -300 mg/kg45,2±22,11,4±0,946,8±17,0<0,0131
Sulindac -160 ppm48,0±20,70,5±0,548,5±20,9<0,0528

Cytotoxicity of COMPOUND 3

Cytotoxicity of COMPOUND 3 (analog COMPOUND 1, figure 1A) and other anti-cancer therapeutic agents was measured using cancer cells of different origin. These results show that COMPOUND 3 at concentrations lower than the concentrations of other anti-cancer therapeutic agents, or similar concentrations of other anti-cancer funds (i.e., cisplatin, 5-FU, ADR (adriamycin)), cause the death of cancer cells (table 7).

TABLE 7
Cytotoxicity
OriginCellConnection 3Cisplatin5-FUADR
LeukemiaHL601,243>107,0100,086
ProstateRS1,207>10>100,267
EasyA1,386>101,0070,117
Kidney2930,7316,6412,015<0,03
MelanomaRPM179510,9365,0100,9200,171
Mammary gland MCF77,355>101,7511,424

The values in table 7 are presented in µg/ml.

Metabolism of COMPOUND 3 in the rat and man

Metabolism of COMPOUND 3 was analyzed by incubation of compounds with microsomal rat liver or human for 5 minutes - 1 hour, fractionation of extracts treated microsome assay using HPLC and exposure of these fractions mass spectrometric analysis. The results are shown in figures 10A and 10B. Several metabolites (e.g., M1, M2, M3) were observed in both systems.

A study of the bioavailability of the COMPOUND 3, COMPOUND 4 and COMPOUND 5

Bioavailability of COMPOUND 3, COMPOUND 4 and COMPOUND 5 was investigated in mouse and rat. The structures of these compounds are shown in figure 1A. All these compounds were injected (i.v. and p.o., 10 mg/kg) using the same media (namely, 20% Tween 80). Bioavailability of COMPOUNDS 3, 4 and 5 in the mouse equivalent to less than 2%, less than 2% and almost 0%, respectively. Bioavailability of COMPOUND 3 in the rat is approximately 24%.

Discussion

There are increasing and draws attention to the data that improper regulation of the pathway β-catenin participates in the development and progression of cancer (Morin P.J., “Beta-catenin signaling and cancer,” Bioessays 21:1021-30 (1999); Moon et al., “The promise andperils of Wnt signaling through beta-catenin,” Science 296:1644-46 (2002); Oving et al., “Molecular causes of colon cancer,” Eur. J. Clin. Invest. 32:448-57 (2002)). Here, the inventors describe the discovery that the compounds of formula (I) inhibit transcription subcomplex of β-catenin/TCF. The biological activity of these low molecular weight inhibitors characterized by screening of reporter genes using a chemical library of template secondary structures in the cells of carcinoma of the colon SW480, which have a mutation in a gene called APC, leading to increased constitutive transcription complex β-catenin/TCF. Affinity chromatography using biotinylated analogue (COMPOUND 2) has made possible the identification coactivator protein, CBP, as the molecular targets of COMPOUNDS 1.

It was shown that transfection of CBP, but not β-catenin significantly increases the binding14C-labeled COMPOUND 1 with nuclear lysates SW480. For validation SVR as molecular targets of COMPOUNDS 1, it was shown that transfection of expressing CBP vector could outweigh the inhibition of COMPOUND 1 gene construct β-catenin/TCF-reporter. In addition, COMPOUND 1 selectively blocked the interaction of β-catenin and CBP, without inhibiting the interaction of β-catenin with the closely related coactivator R. In addition, COMPOUND 1 caused a redistribution of β-catenin from the nucleus to titop is the AZM in SW480 cells. Finally, mediated by COMPOUND 1 inhibits the expression of cycline D1 stops in the G1/S cell cycle, and prolonged treatment causes activation of caspase in SW480 cells (or NST), but not in normal colonocytes, leading to apoptosis in transformed cell lines and carcinoma of the colon. Thus, the CONNECTION 1 is an inhibitor of the pathway β-catenin and CBP is its cellular target.

For further study of the selectivity of COMPOUND 1 was performed analysis of cDNA chips using the Clontech Atlas Human Cancer 1.2 Array (#7851-1). These data demonstrated that COMPOUND 1 had a very selective effect on global gene transcription (Table 2-5). After 8 hours treatment of SW480 cells 25 μm COMPOUND 1, ~2% of the analyzed genes positively regulated more than 2-fold, whereas ~0,3% of these genes negatively regulated by more than 50% (table 2-3).

Promoter-dependent selectivity of coactivator contributes to the complexity of the path β-catenin/TCF. As expected, COMPOUND 1 inhibits the expression of genes cyclin D1, hnkd and axin 2 (Yan et al., “Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta-catenin signaling is activated in human colon tumors,” Proc. Natl. Acad. Sci. USA 98:14973-78 (2001)). In contrast, expression of c-myc (He et al., “Identification of c-MYC and as a target of the APC pathway,” Science 281:1509-12 (1998)) and C-jun (Mann et al., Target genes of beta-catenin-T-cell-factor/lymphoid-enhancer-factorsignaling in human colorectal carcinomas,” Proc. Natl. Acad. Sci. USA 96:1603-08 (1999)) increases due to differential use of coactivation in the path of β-catenin/TCF (table 1 and figure 7B). Using ChIP analyses demonstrated that COMPOUND 1 selectively inhibits the Association of CBP with the endogenous promoters of c-myc and cyclin D1, and that in the treated cells increases the activity R promoter of C-myc, but not of the promoter of cyclin D1. This is a very good correlation with data obtained in experiments co-IP with β-catenin, and data FROM real-time PCR for cyclin D1. CONNECTION 1, which is selective in relation to the inhibition of the interaction of β-catenin/CBP, and related analogs that are selective against β-catenin/R (Kahn et al., unpublished data), provide new Gimignano tools to uncover the mechanisms by which the complex of β-catenin/TCF activates the transcription of genes in a promoter-dependent and coactivator-specific manner (figure 7B).

In the literature, there is considerable controversy concerning the specific contact sites of interaction between β-catenin and coactivator proteins CBP and R. This is presumably associated with random and usually low-moderate binding affinity of both CBP and R with β-catenin relative to many target proteins. The inventors expected that they would COI is lesofat the binding specificity of COMPOUND 1 against CBP to clarify this situation. Research linking COMPOUND 1 with fragments of CBP resulted in the disclosure of the minimum area of interaction for NH2-end of CBP (amino acids 1-111). In addition, the CONNECTION 1 is not associated with the homologous sequence in R. COMPOUND 1 selectively blocked the interaction between CBP (1-111) and β-catenin in cells without inhibiting the interaction R (1-111)/β-catenin. Comparison of the sequences of this region shows a striking similarity with previously published motifs bind β-catenin detected in TCF, APC and E-cadherin (figure 5A) Huber et al., “The structure of The beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin,” Cell 105:391-402 (2001)). As CBP (1-111)and R (1-111) contain key negatively charged buttons (DELIXXXXE) for interactions with β-catenin (Graham et al., “Tcf4 can specifically recognize beta-catenin using alternative conformations,” Nat. Struct. Biol. 8:1048-52 (2001)). The motive SXSSXS, where X denotes an amino acid with a nonpolar aliphatic group of R found in ARS and E-cadherine, is also present in the RAF, SASSP (amino acids 89-93), but not in R. Although the differential binding of COMPOUND 1 with CBP (1-111) in comparison with R (1-111) in SW480 cells could, in principle, be due to differential phosphorylation, application of known inhibitors of GSK-3β (Nikoulina et al., “Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle,” Diabtes 51:2190-98 (2002)) or PKC (Bollag et al., “Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes,” J. Invest. Dermatol. 100:240-46 (1993)) had no apparent effect on the binding of COMPOUND 1 (data not shown). In addition, purified recombinant CBP (1-111), expressed in E. coli, was able to connect with CONNECTION 1, which further testifies against dependent coactivator phosphorylation as a discriminating factor. Thus, using COMPOUND 1 as the tool authors specifically mapped the minimal region of interaction of CBP with β-catenin in the first 111 amino acids of the RAF.

Additional research support authors mapping comes from the existence of a binding site on SVR receptors for retinoic acid (RA), RXR/RAR, in close proximity to the binding motif of β-catenin on SVR (figure 3F). Previously it was shown that treatment of RA inhibits signaling β-catenin/TCF (Earswaran et al., “Cross-regulation of beta-catenin-LEF/TCF and retinoid signaling pathways,” Curr. Biol. 9:1415-18 (1999)). Consensus (LXXLL) (SEQ ID NO:46) binding site RXR/RAR (LSELL) is located at amino acid residues 70-74 in the alleged authors of the binding site of β-catenin as SVR and R (Minucci et al., “Retinoid receptors in health and disease: co-regulators and the chromatin connection,” Semin. Cell Dev. Biol. 10:215-25 (1999)).

CONNECTION 1 allows authors to solve the problem of promoter-dependent activeconnections transmission signal of β-catenin. Treatment with COMPOUND 1 did not inhibit the interaction R with β-catenin (figure 3D), and in fact it actually increases the formation of complexes of β-catenin/l in the treated cells (figure 3B). As shown in the comparison of the sequences, although there are similarities in the mapped motifs bind β-catenin, there are differences between the two coactivators, which may be responsible for the observed specificity of COMPOUND 1 with respect to CBP, but not in relation to R. Based on these studies it seems that the interaction between N-terminal 111 amino acids of CBP/R and β-catenin is required for activation of transcription of the β-catenin/TCF.

Despite great interest in the detection with selective small molecule inhibitors of transcription of the β-catenin/TCF, to the authors ' knowledge of the invention, COMPOUND 1 represents the first example of direct with a small molecule inhibitor of this pathway. Despite the elegant struture studies on the interaction between β-catenin and TCF (Graham et al., “Crystal structure of beta-catenin/TCF complex,” Cell 103:885-96 (2000); Graham et al., “TCF4 can specifically recognize beta-catenin using alternative conformations,” Nat. Struct. Biol. 8:1048-52 (2001); Poy et al., “Structure of human Tcf4-beta-catenin complex,” Nat. Struct. Biol. 8:1053-57 (2001)), a priori attractive method for the inhibition of this pathway, there are problems related to the development of special the specific inhibitors, due to different partners, along with TCF (for example, APC and E-cadherin), which is also associated with the Central repeats Arm (armadillo repeats) β-catenin (Huber et al., “The structure of beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin,” Cell 105:391-402 (2001)). This elegant selectivity of COMPOUND 1 by specific inhibition of the interaction of β-catenin/CBP, in contrast vysokogomogennogo coactivator R (which has up to 96% identity at the amino acid level with SVR) has provided a unique tool chemogenomic for the study of β-catenin/TCF-mediated transcription. The specificity of the COMPOUND 1, its ability to selectively activate caspase apoptosis in transformed but not in normal colonocytes, and its effectiveness in the analysis of the formation of colonies in soft agar, are all encouraging signs regarding its potential therapeutic applicability in the case of colon cancer. In addition, analogs of COMPOUND 1 was found efficacy in vivo, with limited toxicity in murine cancer models (models naked mice injected cells SW480 and mice Min; Moser et al., “ApcMin: a mouse model for intestinal and mammary tumorigenesis,” Eur. J. Cancer A:1061-64 (1995); Kahn et al., unpublished data), in addition validityof use of selective inhibitors of transcription of β-catenin/TCF/SVR for p the potential use in cancer chemotherapy, and other hyperproliferative disorders.

All of the above U.S. Patents, published patent applications U.S. patent application U.S., foreign patents, foreign patent applications and non-patent publications cited in this description.

From the previous description it will be understood that, although specific embodiments of this invention have been described herein for purposes of illustration, various modifications may be made without deviating from the idea and scope of this invention. Thus, this invention is not limited by anything except the attached claims.

1. A method of modulating expression of the target genes induced β-catenin, using an agent that increases the binding R with β-catenin and reduces the binding of CBP with β-catenin, including
the casting composition containing β-catenin, CBP and R, where β-catenin is the probability of associating with SVR in comparison with R in contact with the agent in amounts effective to change the probability of binding of β-catenin with CBP in comparison with R,
moreover, the specified agent is a compound having a structure selected from formula (I)or its stereoisomers:

where a denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G denotes XR 7)n-, W denotes -(C=O)NH-, X is nitrogen or CH, Z represents CH, n=0 or 1; R1denotes optionally substituted C6-12arylalkyl or optionally substituted C6-12heteroaromatic containing one or two nitrogen atom or oxygen; R2denotes optionally substituted C6-14arylalkyl or optionally substituted C6-14heteroaromatic containing one or two nitrogen atom or oxygen or sulfur; R4denotes benzyl or benzyl substituted by hydroxyl, R6denotes hydrogen, R7denotes hydrogen or a saturated or unsaturated With1-4alkyl.

2. The method according to claim 1, where the composition is ex vivo.

3. The method according to claim 1, where the composition is a cell of a mammal.

4. The method according to claim 3, where the mammal is suffering from cancer and the amount is effective to treat cancer.

5. The method according to claim 4, where the cancer is cancer of the colon.

6. The method according to claim 1, where the agent is a compound 1 having the formula
.

7. Composition for modulation of expression of the target genes induced β-catenin containing
an agent that increases the binding R with β-catenin and reduces the binding of CBP with β-catenin,
β-catenin, CBP and R,
where β-catenin is the probability of associating with SVR in comparison with R, and the agent is present in the HDMI is tion in the quantity effective to change the probability of binding of β-catenin with CBP in comparison with R,
moreover, the specified agent is a compound having a structure selected from formula (I)or its stereoisomers:

where a denotes -(C=O)-, means -(CHR4)-, D is -(C=O)-, E denotes -(ZR6)-, G represents -(XR7)n-, W denotes -(C=O)NH-, X is nitrogen or CH, Z represents CH, n=0 or 1; R1denotes optionally substituted C6-12arylalkyl or optionally substituted C6-12heteroaromatic containing one or two nitrogen atom or oxygen; R2denotes optionally substituted
With6-14arylalkyl or optionally substituted C6-14heteroaromatic containing one or two nitrogen atom or oxygen or sulfur; R4denotes benzyl or benzyl substituted by hydroxyl, R6denotes hydrogen, R7denotes hydrogen or a saturated or unsaturated With1-4alkyl.

8. The composition according to claim 7 in state ex vivo.

9. The composition according to claim 7, where the agent is a compound 1 having the formula



 

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2 dwg, 1 tbl, 3 ex

FIELD: veterinary science.

SUBSTANCE: alleged invention concerns veterinary protozoology. There is disclosed method for preparing anaplama antigen for serm diagnostics in animals, including infection with cattle anaplasmosis agent, centrifugation anaplasma recovery from blood, evaluation of activity in "РДСК" differing is plasma prepared in centrifugation and recovered from blood of infected animals, is cleared from deposited anaplasmas, processed with 20-25% polyethylene glycol 6000 taken in the same ratio as blood plasma; further the prepared mixture is kept at room temperature for 13-20 min, centrifugated again at 6000 rpm for 16-25 min; then supernatant fluid is removed, and the produced sediment is used as anaplasma exoantigen in serum tests with its activity to be 95-97%.

EFFECT: invention provides preparing high active anaplasma antigen.

2 tbl

FIELD: medicine.

SUBSTANCE: invention can be applied in assessment of human disadaptive processes at various pathological states. Method involves determination of met-hemoglobin, membrane-linked hemoglobin and oxidised nucleotide content and total optic erythrocyte density before and after functional sampling in the form of short-term local ischemia. Coefficient of erythrocyte resistance to ischemia K is defined on the basis of obtained data. If K is below 0 then erythrocyte resistance to ischemia is high, K values within 0 to 0.1 indicate standard resistance, and K over 0.1 indicates low resistance. Invention allows for assessment of erythrocyte resistance to functional stress, such as tissue hypoxia.

EFFECT: fast, reliable and objective detection of different types of cell adaptation to ischemia.

3 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I) and their pharmaceutically acceptable salts which have PDE9A inhibition properties. In formula (I) R1 represents alkyl with 1-8 carbon atoms or cycloalkyl with 5-6 carbon atoms which, if necessary, can have up to three substitutes independently selected from: alkyl with 1-6 carbon atoms, hydroxy, halogen and trifluoromethyl, where the alkyl with 1-6 carbon atoms, if necessary, can be substituted with 1-3 substitutes independently selected from halogen and trifluoromethyl, R2 represents phenyl or aromatic mono- or bicyclic heteroaryl with 5-10 atoms in the ring and up to 5 heteroatoms selected from: sulphur, oxygen and/or nitrogen, where phenyl is substituted with 1-3 substitutes, and the heteroaryl, if necessary, can be substituted with 1-3 substitutes in each case independently selected from: alkyl with 1-6 carbon atoms, alkoxy with 1-6 carbon atoms, trifluoromethyl, trifluoromethoxy, amino, hydroxyl and halogen.

EFFECT: compounds can be used for preparing medicinal agents for enhancing perception, ability to concentrate, learning capability and memory enhancement.

9 cl, 1 dwg, 2 tbl, 78 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyrimidine-condensed derivatives of formula , where n is selected from 0, 1, 2, 3 and 4, Z1 is selected from N, C(O) and CR3, where R3 represents hydrogen, Z2 is selected from N and CR4, where R4 is selected from hydrogen and halogen, where the bond between Z1 and Z2 is selected from a single bond and a double bond, R1 is selected from C1-C4alkyl and C1-C4alkoxy, R2 is selected from NR5C(O)R6, C(O)NR5R6 and NR5R6, where R5 represents hydrogen, and R6 is selected from hydrogen, C1-C4alkyl and phenyl, where phenyl as R6 is optionally substituted with 1-2 radicals independently selected from a group comprising halogen(C1-C4)alkyl, heteroaryl(C0-C4)alkyl and heterocycloalkyl(C0-C4)alkyl, where any heteroaryl or heterocycloalkyl substitute R6 can be optionally substituted with a substitute independently selected from C1-C4alkyl and heterocycloalkyl, where the said heteroaryl and heterocyclyl represent a saturated or unsaturated 5-6-member ring containing 1 or 2 N atoms as a heteroatom, and to their pharmaceutically acceptable salts, hydrates, solvates and isomers. The invention also relates to a pharmaceutical composition base on a formula I compound and to use of formula I compound for preparing a medicinal agent which can be used for treating diseases or disorders associated with anomalous or disrupted kinase activity, primarily diseases or disorders related to anomalous activation of kinase Ab1, Bcr-Ab1, BMX, BTK, CHK2, c-RAF, CSK, c-SRC, Fes, FGFR3, Flt3, IKKα, IKKβ, JNK2α2, Lck, Met, MKK4, MKK6, MCST2, NEK2, p70S6K, PDGFRβ, PKA, PKBα, PKD2, Rsk1, SAPK2α, SAPK2β, SAPK3, SGK, Tie2 and TrkB.

EFFECT: novel compounds have useful biological properties.

7 cl, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula : in which R1 represents a hydrogen atom or alkyl optionally substituted with (1) aralkyloxy group, (2) aroyl, (3) isoquinolinyl or (4) aryl, optionally substituted with an alkoxy group; the solid line and the dashed line between A1 and A2 represent a double bond (A1=A2) or a single bond (A1-A2); A1 is a group of formula C(R4), and A2 is a nitrogen atom when the solid line and the dashed line between A1 and A2 represents a double bond (A1=A2); A1 is a group of formula C=O, and A2 is a group of formula N(R5) when the solid line or the dashed line between A1 and A2 represent a single bond (A1-A2); R2 represents alkyl optionally substituted with a cyano group, aryl optionally substituted with an alkoxy group, aralkyl optionally substituted with a halogen atom, a cyano group, an alkoxy group, an alkyl or carbamoyl or alkynyl; R3 represents a hydrogen atom, a halogen atom, cyano, formyl, carboxyl, alkyl optionally substituted with (1) amino group optionally substituted with alkyl, or (2) alkoxy group, aryl optionally substituted with an alkoxy group, tetrazolyl, alkylcarbonyl, cycloalkylcarbonyl, heteroarylcarbonyl, where heteroaryl is a 4-6-member monocyclic radical containing 1-2 heteroatoms selected from a nitrogen atom or oxygen atom, alkoxycarbonyl, carbamoyl optionally substituted with alkyl, cycloalkyl or cycloalkylalkyl, hydroxyl, alkoxy group or a group of formula: -Rd-C(O)O-Re, where Rd represents a single bond, and Re represents a group of formula: -CH(R4a)OC(O)R4b, where R4a represents alkyl or R4b represents cycloalkyloxy or aryloxy; R represents a hydrogen atom, hydroxyl, cyano, alkyl, carbamoyl, carboxyl, aryloxy optionally substituted with an alkoxy group or carbamoyl, alkylsulfonyl, alkylcarbonyl or alkoxycarbonyl; R5 represents a hydrogen atom or alkyl; -Y represents a group of formula (A) given below: in which m1 equals 2, and R6 is absent, or to pharmaceutically acceptable salts of the said compounds. The invention also relates to compounds of formula (VI), to pharmaceutical compositions, to a dipeptidyl peptidase IV inhibitor, as well as to use of the said compounds.

EFFECT: obtaining novel biologically active compounds with dipeptidyl peptidase IV inhibition properties.

20 cl, 76 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: described are novel compounds with general formula , their stereoisomers and pharmaceutically acceptable salts or solvates, where the dashed line can represent a double bond (together with the present single bond); R represents phenyl or benzodioxolyl, each of which can be substituted; R1, R3 and R4 independently represent hydrogen or C1-C6alkyl; R5 represents C1-C6alkyl; R7 represents hydrogen; R12 represents R3 or -C(O)R2, where R2 represents C1-C4 alkyl; D and G represent -CH2 - or -CH- when they are bonded to each other by a double bond; m equals 1; a pharmaceutical composition containing said compounds, and use of the novel compounds in treating conditions mediated by corticotropin-releasing factor (CRF).

EFFECT: increased effectiveness of compounds.

11 cl, 13 ex, 11 tbl

FIELD: medicine.

SUBSTANCE: there are described new compounds of general formula

where Xa represents 2 to 4 condensed cycloalkyl, aryl, heterocyclic rings containing 1 to 2 heteroatoms, chosen of N and O, and heteroaryl rings containing 1 to 4 heteroatoms, chosen of N, O or S where said rings can be additionally substituted. (Radical values R1-R4, R1, Y and n are specified in the patent claim), specific representatives of said compounds and a pharmaceutical composition containing them.

EFFECT: new compounds are effective in stimulation of endogenous development or release of growth hormone and can be used in treating obesity, osteoporosis and for increasing muscle bulk and muscle strength.

17 cl, 339 ex, 10 tbl

FIELD: chemistry.

SUBSTANCE: described are novel derivatives of pyrazolo[1,5-a]pyrimidine with general formula 1 (values of radicals are given in the formula of invention), a pharmaceutical composition containing said derivatives and use of the novel compounds for preparing a medicinal agent for treating one or more diseases associated with cyclin-dependant kinalse CDK2.

EFFECT: novel compounds have useful biological properties.

36 cl, 87 tbl, 607 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel derivatives of benzene sulphonamide of formula (I), tautomeric and stereoisomeric forms and physiologically acceptable salts thereof: where X is O, S; R1 is H, halogen; R2 is H, halogen; halogen; R3 is NO2, CN; R4 is: ,

where R71 is H; R72 is H; Z1 is -[CH2]P-, where p = 2.

EFFECT: compounds have antagonistic activity towards CCR3, which enables for their use in making medicinal agents.

13 cl, 1 tbl, 3 ex

FIELD: pharmacology.

SUBSTANCE: present invention relates to antagonists of serotonin 5-HT5 receptors with general formula 1 and their pharmaceutically acceptable salts and/or hydrates, particularly to substituted 3-sulphonyl-[1,2,3]triazolo[1,5-a]quinazolines and 3-sulphonyl-thieno[2,3-e][1,2,3]triazolo [1,5-a]pyrimidines, as active compounds for pharmaceutical compositions and medicinal agents, and methods of producing said compounds. In general formula 1 , Ar is a phenyl which is unsubstituted or substituted with halogen or at least one lower alkyl; R1 is a hydrogen atom or optionally substituted amine group, or optionally substituted 5-6 member azaheterocyclyl, bonded by a nitrogen atom to a carbon atom of a triazolopyrimidine ring with 1-2 heteroatoms selected from nitrogen, oxygen or sulphur, and optionally annulated with a benzene ring; where the substitutes are selected from hydrogen, optionally substituted C1-C5alkyl, optionally substituted C3-C8cycloalkyl, alkoxy group, acyl, saturated or unsaturated optionally annulated 5-7 member heterocyclyl, where heteroatoms are selected from nitrogen, oxygen or sulphur, optionally substituted phenyl; R2 and R3 together with carbon atoms to which they are bonded form an optionally substituted benzene or thiophene ring, where substitutes are selected from C1-C5alkyl or halogen atom.

EFFECT: invention also relates to pharmaceutical compositions and medicinal agents, a method of treating or preventing development of CNS diseases mediated by action of serotonin 5-HT5 receptors, for example Alzheimer's disease.

20 cl, 6 dwg, 4 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyrazolpyrimidine derivatives of formula (I) where p is 0 or 1; R1 and R2 can independently represent H, halogen, lower alkyl, lower alkoxy, possibly substituted with one or more halogens or CF3; R3 is lower alkyl, hydroxy-lower alkyl or NRaRb; Ra and Rb are independently selected from a group consisting of H; cycloalkyl containing 3-6 carbon atoms; phenyl; lower alkyl possibly substituted with one or more hydroxy, fluorine, C3-6cycloalkyl, phenyl, pyridyl or NRcRd, where Rc and Rd are independently selected from H or lower alkyl; or where Ra and Rb together with the nitrogen atoms to which they are bonded can form a 5- or 6-member hetero-ring, possibly additionally containing 1 or 2 heteroatoms selected from O or N, and possibly substituted with lower alkyl or hydroxy-lower alkyl; R4 is H, Cl, lower alkoxy, cycloalkyl, containing 3-6 carbon atoms, or straight lower alkyl which is possibly substituted with one or more F; R5 is H; halogen or lower alkyl; as well as to their pharmaceutically acceptable salts.

EFFECT: invention also relates to pharmaceutical compositions based on these compounds and their use in preparing medicine for treating or preventing acute and/or chronic neurological disorders in which activation of mGluR2 is involved.

19 cl, 179 ex

FIELD: pharmacology.

SUBSTANCE: invention refers to new 2-alkylamino-3-arylsulphonylcycloalkano[e]pyrazolo[1,5-a]pyrimidines of general formula 1 and 2-alkylamino-3-arylsulphonylcycloalkano[d]pyrazolo[1,5-a]pyrimidines of general formula 2 with properties of serotonin 5-NT6 receptor antagonists, to pharmaceutical compositions containing specified compounds as a principle, medical products and method of treatment and the prevention of CNS diseases. In general formulae 1 and 2, R1 represents hydrogen atom or C1-C3 alkyl; R2 represent C1-C3 alkyl; R3 represent hydrogen atom, one or two optionally substituted identical halogen atoms, C1-C3 alkyl or hydroxyl optionally substituted with C1-C3alkyl; n represents an integer 1, 2 or 3. The invention also relates to the method for making the compounds of general formula 1 or 2 by interaction of 3-amino-4-arylsulphonyl-2H-pyrazoles of general formula 3 with relevant β-dicarbonyl compounds of general formula 4 or their derivatives of general formula 5. 3, 4, 5, where: R1, R2, R3 and n have said values.

EFFECT: new 2-alkylamino-3-arylsulphonyl-cycloalkano[e or c1]pyrazolo[1,5]pyrimidines - serotonin 5-NT6 receptor antagonists, methods of making and applying thereof.

12 cl, 1 dwg, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: described are compounds of formula (I)

Values of radicals R1-R6 are given in the formula of invention. The compounds inhibit protein kinase MEK1/2. Also described is a pharmaceutical composition for administration in diseases mediated by MEK1/2.

EFFECT: compounds are highly efficient.

16 cl, 27 ex

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