CA2388383A1 - Agents for treating human diseases, especially for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth - Google Patents
Agents for treating human diseases, especially for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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Abstract
The invention relates to agents for treating human diseases which are based on substances that specifically influence the binding of .beta.-catenin on LEF-1/TCF- transcription factors, APC or conductin/axin. The invention particularly relates to the identification and use of hydrophobic pockets on the molecular surface in the proximity of the essential binding points for the binding partners of .szlig.-catenin with the aim of optimizing these substances. The invention also relates to the use of the substances, preferably for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth.
Description
AGENTS FOR TREATING HUMAN DISEASES; ESPECIALLY FOR TREATING
TUMORS SUCH AS COLONIC CANCERS AND MELANOMAS OR FOR
REGENERATING TISSUE AND PROMOTING HAIR GROWTH
Description The invention relates to agents for treating human diseases based on substances affecting the interaction between 13-catenin and LEF-1-/TCF-transcription factors, APC or conductin/axin.
Preferably these agents are suitable for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth. Accordingly, fields of application of the invention are pharmaceutical industry and medicine.
13-catenin is a cytoplasmic protein which fulfils various functions in the cell. In complex with the cell adhesion molecules of the cadherin family 13-catenin establishes the connection with the cytosceleton (Huelsken J. et al., E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J-Cell-Biol. 127: 2061-9, 1994). In addition, 13-catenin is a component of the Wnt signal transduction which plays an important role in embryonic development. The transcription factor LEF-1 was identified as interaction partner of 13-catenin in this signal cascade (Behrens, J. et al., Functional interaction of beta catenin with the transcription factor LEF-1. Nature, 382: 638-42, 1996). The mechanism of signal transduction by 13-catenin and LEF-1 has been clarified: It consists of the transport of 13-catenin into the cell nucleus mediated by LEF-1. And regulation of the gene expression in the cell nucleus by the LEF-1 induced DNA bending modified in the complex and by the carboxy-terminal transactivation domain of 13-catenin. In the mean time, there has been shown that also other members of the LEF-1/TCF family of transcription factors, e.g. TCF-4, are able to mediate this signal transduction (Korinek, V. et al., Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma. Science, 275: 1784-87, 1997).
Stabilizing the cytoplasmic pool of free 13-catenin not bound to cadherin is the prerequisite to this signal transduction depending on 13-catenin. This pool is negatively regulated by glycogen synthetase kinase 313, by the tumor suppressor gene product APC and conductin/axin.
There was shown for cancers and melanomas that mutations in the N-terminal area of 13-catenin or in the 13-catenin binding domain of APC stop this regulation (Morin, P.J. et al., Activation of beta-catenin-Tcf signaling in colonic cancer by mutations in beta-catenin or APC. Science, 275: 1787-90, 1997). Accordingly,the 13-catenin pool is stabilized. In melanomas this stabilization results in a LEF-1 mediated translocation of 13-catenin into the cell nucleus whereas in colonic cancers this function is primarily fulfilled by TCF-4. The transcriptional activity of the complex in cancer cell lines is detected by activating a reporter gene. In addition, it has been shown that this activity is inhibited in APC-deficient colon carcinoma cell lines after transfection of APC.
APC mutations were identified in the overwhelming majority of colonic cancers whereas not-APC-deficient tumors show mutations in the 13-catenin gene. The result of these mutations of APC or 13-catenin is an activation of signal transduction by the 13-catenin-LEF/TCF complex.
This underlines the key role played by 13-catenin in the development of tumors. As APC
mutations were identified as an early event in the development of colonic tumors the activation of the 13-catenin-LEF/TCF complex is certainly a central step in the development of tumors.
It was demonstrated in the mouse, that a deletion of the LEF-1 gene or the expression of 13-catenin-mutants, which are stabilized against degradation in the cell,among others lead to troubles of the development of hair follicles . Interestingly is, the expression of stabilised 13-catenin leads to increasing the quantity of hair follicle (Gat U. et., l 998.
De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta- catenin in skin. Cell 95:605-14) the inactivation of LEF-1 to trouble to the development of hair follicles,breast glands, and tooths (van Genderen et a1.,1994. Development of several organs that require inductive epithelial- mesenchymal interactions is impaired in LEF-ldeficient mice. Genes Dev. 8:2691-703).
These results point to, that the complex of LEF/TCF and 13-Catenin joins these development processes.
Attempts have been made to utilize the key role played by 13-catenin in the development of tumors for the development of therapeutic agents for treating tumors. Nearly at the same time, two patent applications were filed in the USA which, in the mean time, were published as WO
papers. In WO 98/41631 (John Hopkins University - B. Vogelstein) the influence on interactions of 13-catenin, TCF-4 and the tumor suppressor protein APC aimed at preventing the development of cancer is claimed. There was shown that products of mutated APC genes detected in colorectal tumors are no longer able to regulate the activation of the f~-catenin/TCF-4 transcription. Furthermore, colorectal tumors with intact APC
genes show activation mutations of 13-catenin in the N-terminal area which affects the functioning of the most important phosphorylation sites. Based on this data, the conclusion is drawn that the regulation of 13-catenin is critical for the tumor suppressor effect of APC
and this regulation may be evaded by mutations in APC or in 13-catenin. The main claim relates to the intron-free DNA molecule coding for TCF-4.
WO 98/42296 (Onyx Pharmaceuticals Inc. - Rubinfeld) relates to compositions and methods of diagnosing and treating illnesses caused by interactions between 13-catenin and transcription factors. The main claim relates to the isolated, stabilized 13-catenin and its fragments, yet such fragments were not indicated.
Furthermore it was proposed, to find peptids or derived structures, which are from the origin of 13-catenin or its interaction compounds which influence the interactions specifically (DE
198 07 390.9 of February 21, 98).
The aim of this invention is providing new agents for the treatment of tumors and aberrant the development of tissues and organs. The invention is based on the task to affect the interaction between 13-catenin and LEF/TCF transcription factors, APC and conductin/axin as a prerequisite to the translocation and the activity of the complex in the cell nucleus. This activity has to be specific, i.e. it must not interfere with other interactions of 13-catenin, at the same time.
The invention is realised accordingly the claims. An essential basis is - to our surprise - the detection of seperate essential binding sites of these interaction participants in the 13-catenin molecule.
The main idea is (1) to search for hydrophobic pockets near the essential binding sites of LEF-1/TCF, APC
or conductin based on crystal structures and surface calculations for 13-catenin. Such a hydrophobic pocket was found near the LEF/TCF binding site and characterized.
(2) Hydrophobic pockets near the essential binding sites are used for the computer-aided finding of low-molecular substances which may bind in these pockets.
TUMORS SUCH AS COLONIC CANCERS AND MELANOMAS OR FOR
REGENERATING TISSUE AND PROMOTING HAIR GROWTH
Description The invention relates to agents for treating human diseases based on substances affecting the interaction between 13-catenin and LEF-1-/TCF-transcription factors, APC or conductin/axin.
Preferably these agents are suitable for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth. Accordingly, fields of application of the invention are pharmaceutical industry and medicine.
13-catenin is a cytoplasmic protein which fulfils various functions in the cell. In complex with the cell adhesion molecules of the cadherin family 13-catenin establishes the connection with the cytosceleton (Huelsken J. et al., E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J-Cell-Biol. 127: 2061-9, 1994). In addition, 13-catenin is a component of the Wnt signal transduction which plays an important role in embryonic development. The transcription factor LEF-1 was identified as interaction partner of 13-catenin in this signal cascade (Behrens, J. et al., Functional interaction of beta catenin with the transcription factor LEF-1. Nature, 382: 638-42, 1996). The mechanism of signal transduction by 13-catenin and LEF-1 has been clarified: It consists of the transport of 13-catenin into the cell nucleus mediated by LEF-1. And regulation of the gene expression in the cell nucleus by the LEF-1 induced DNA bending modified in the complex and by the carboxy-terminal transactivation domain of 13-catenin. In the mean time, there has been shown that also other members of the LEF-1/TCF family of transcription factors, e.g. TCF-4, are able to mediate this signal transduction (Korinek, V. et al., Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma. Science, 275: 1784-87, 1997).
Stabilizing the cytoplasmic pool of free 13-catenin not bound to cadherin is the prerequisite to this signal transduction depending on 13-catenin. This pool is negatively regulated by glycogen synthetase kinase 313, by the tumor suppressor gene product APC and conductin/axin.
There was shown for cancers and melanomas that mutations in the N-terminal area of 13-catenin or in the 13-catenin binding domain of APC stop this regulation (Morin, P.J. et al., Activation of beta-catenin-Tcf signaling in colonic cancer by mutations in beta-catenin or APC. Science, 275: 1787-90, 1997). Accordingly,the 13-catenin pool is stabilized. In melanomas this stabilization results in a LEF-1 mediated translocation of 13-catenin into the cell nucleus whereas in colonic cancers this function is primarily fulfilled by TCF-4. The transcriptional activity of the complex in cancer cell lines is detected by activating a reporter gene. In addition, it has been shown that this activity is inhibited in APC-deficient colon carcinoma cell lines after transfection of APC.
APC mutations were identified in the overwhelming majority of colonic cancers whereas not-APC-deficient tumors show mutations in the 13-catenin gene. The result of these mutations of APC or 13-catenin is an activation of signal transduction by the 13-catenin-LEF/TCF complex.
This underlines the key role played by 13-catenin in the development of tumors. As APC
mutations were identified as an early event in the development of colonic tumors the activation of the 13-catenin-LEF/TCF complex is certainly a central step in the development of tumors.
It was demonstrated in the mouse, that a deletion of the LEF-1 gene or the expression of 13-catenin-mutants, which are stabilized against degradation in the cell,among others lead to troubles of the development of hair follicles . Interestingly is, the expression of stabilised 13-catenin leads to increasing the quantity of hair follicle (Gat U. et., l 998.
De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta- catenin in skin. Cell 95:605-14) the inactivation of LEF-1 to trouble to the development of hair follicles,breast glands, and tooths (van Genderen et a1.,1994. Development of several organs that require inductive epithelial- mesenchymal interactions is impaired in LEF-ldeficient mice. Genes Dev. 8:2691-703).
These results point to, that the complex of LEF/TCF and 13-Catenin joins these development processes.
Attempts have been made to utilize the key role played by 13-catenin in the development of tumors for the development of therapeutic agents for treating tumors. Nearly at the same time, two patent applications were filed in the USA which, in the mean time, were published as WO
papers. In WO 98/41631 (John Hopkins University - B. Vogelstein) the influence on interactions of 13-catenin, TCF-4 and the tumor suppressor protein APC aimed at preventing the development of cancer is claimed. There was shown that products of mutated APC genes detected in colorectal tumors are no longer able to regulate the activation of the f~-catenin/TCF-4 transcription. Furthermore, colorectal tumors with intact APC
genes show activation mutations of 13-catenin in the N-terminal area which affects the functioning of the most important phosphorylation sites. Based on this data, the conclusion is drawn that the regulation of 13-catenin is critical for the tumor suppressor effect of APC
and this regulation may be evaded by mutations in APC or in 13-catenin. The main claim relates to the intron-free DNA molecule coding for TCF-4.
WO 98/42296 (Onyx Pharmaceuticals Inc. - Rubinfeld) relates to compositions and methods of diagnosing and treating illnesses caused by interactions between 13-catenin and transcription factors. The main claim relates to the isolated, stabilized 13-catenin and its fragments, yet such fragments were not indicated.
Furthermore it was proposed, to find peptids or derived structures, which are from the origin of 13-catenin or its interaction compounds which influence the interactions specifically (DE
198 07 390.9 of February 21, 98).
The aim of this invention is providing new agents for the treatment of tumors and aberrant the development of tissues and organs. The invention is based on the task to affect the interaction between 13-catenin and LEF/TCF transcription factors, APC and conductin/axin as a prerequisite to the translocation and the activity of the complex in the cell nucleus. This activity has to be specific, i.e. it must not interfere with other interactions of 13-catenin, at the same time.
The invention is realised accordingly the claims. An essential basis is - to our surprise - the detection of seperate essential binding sites of these interaction participants in the 13-catenin molecule.
The main idea is (1) to search for hydrophobic pockets near the essential binding sites of LEF-1/TCF, APC
or conductin based on crystal structures and surface calculations for 13-catenin. Such a hydrophobic pocket was found near the LEF/TCF binding site and characterized.
(2) Hydrophobic pockets near the essential binding sites are used for the computer-aided finding of low-molecular substances which may bind in these pockets.
(3) These substances are tested in an ELISA with recombinant 13-catenin and individual interaction participants for the efficiency of inhibition.
(4) Based on crystallographic analytical data of 13-catenin the substances shall be optimized by introducing side groups which are additionally stabilized by bonds with the adjacent amino acid residues of the essential binding sites (e.g. the LEF/TCF
binding site: Lys 435, Arg 469, His 470, Lys 508 and Arg 515 or the APC
binding site for the 20 amino acid repeat domain: Lys 345, Trp 383 or the 15 amino acid repeat domain or the conductin binding site: Phe 253, His 260, Lys 292).
In following the invention is explained in detail:
In the first realisation of invention got 13-catenin- mutants were identified which mark the respective essentieal binding site for LEF-1/TCF-4, for the 20 amino acid repeats of APC or of conductin (Fig.l). The substitution of single basic amino acids by alanin-rest produced this pointmutants. Because of the essential binding sites of this factors are located in separated subregions of 13-catenin, the substances near to this binding sites influence always one interaction influence specifically.According to the invention, these substances able treat tumors or regenerate tissues.
In a second step, based on the X-ray crystallographic analytical data of the armadillo domain of 13-catenin the surfaces in this region are calculated. Using various computer programs such as e.g. Grasp or Ludi a hydrophobic pocket near the binding site for LEF-1/TCF-4 was identified (flanked by the amino acids Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438). This hydrophobic pocket forms an ideal molecular target for generating substances which bind in this pocket and establish contacts to the closely adjacent essential binding site. For energetic reasons this bond is favoured as a hydrate shell has not to be displaced to make a bond. It is possible that a phenyl alanine residue of LEF/TCF (Phe 24 of LEF-1 or Phe 21 of TCF-4) which is normally essential for binding to 13-catenin (DE 199 09 251 of 22/02/99) binds in this pocket. Thus, the substances might block this point of contact in 13-catenin solely by their binding in the pocket.
In a third step low-molecular substances from molecule data bases were computer-aided fitted into this pocket and selected owing to the number of the stabilizing intractions with 13-catenin.
These substances are mentioned hereinafter. The basic structure of these low-molecular compounds shall be modified after experimentally checking their inhibition function (e.g. in an ELISA) to optimize their function. This is guided by the idea that the modification of the substances, e.g. by adding acid groups, allows additional interactions with the basic residues of the amino acids Lys 435, Arg 469, His 470 or Lys 508 of !3-catenin. As these residues mark the essential binding site of the LEF/TCF factors these interactions should intensify the efficiency of the substances.
The adoption of this strategy to all essential binding sites of 13-catenin allows to generate new pharmaca, which by specifically affecting the respective interaction of 13-catenin, e.g. with the transcription factors LEF/TCF, the tumor suppressor gene product APC or conductin, inhibit the development of tumors or are used for regenerating skin and promoting growth of hair.
These possibilities of affecting arise from the findings published which relate to the function of the respective interaction between 13-catenin and its binding participants in these developmental processes.
In particular the following investigations were carried out:
1. Identification of separate, essential binding sites of 13-catenin for the interaction with LEF-1/TCF-4 with the 20 and 15 amino acid repeats of APC or conductin/axin.
Based on the X-ray crystallographic analysis of the armadillo domain of 13-catenin (Huber et al., 1997) selected basic and aromatic amino acid residues were substituted by alanine residues and the point mutants were analysed for their interaction with the binding participants of 13-catenin in a yeast-2-hybrid system (Fig. 1 ). The mutations blocking specific interactions form clusters in separate subregions of the domain. They mark the essential points of contact in the specific binding sites of 13-catenin for its interaction with LEF/TCF (Lys 435, Arg 469, His 470, Lys 508, Arg 515), with the 20 amino acid repeats of APC (Trp 383, Lys 345), with the 1 S amino acid repeats of APC
(Arg 386), or with conductin (Phe 253, His 260, Lys 292). The extended interaction sites are represented in Fig. 1.
2. Analyses of the molecule surface of 13-catenin in the area of the essential binding site for LEF/TCF
Proceeding from the X-ray crytallographic analytical data of 13-catenin the molecule surface was calculated by means of the programs Grasp and Ludi in the area of the binding site. Thus, ist was possible to identify a hydrophobic pocket (Fig. 2) flanked by the following amino acids: Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438. This pocket is localised in the closest vicinity of the essential binding site for LEF/TCF. It is of great importance for the computer-aided screening of substance libraries for selecting molecules binding in this pocket. The idea to select primarily hydrophobic interactions in the pocket as starting point provides the energetic advantage that the substances need not compete with the hydrate shell of the loaded amino acid residues of the surface. Thus, after their purposeful modification these substances are potent therapeutic agents for treating tumors as the interaction of LEF/TCF with !3-catenin is an early event in the development of tumors. They are experimentally tested for their ability to inhibit the oncogenic interaction (ELISA) and are modified. The molecular modelling of the substances in the pocket is based on the idea to stabilize the interactions proceeding in the proper pocket and to establish additional contacts to the essential amino acid residues of the LEF/TCF binding site. By means of this strategy the effect of these substances is to be optimized.
3. Identification of substances binding in the hydrophobic pocket Substance libraries were screened computer-aided and the molecules were selected according to their stabilizing interactions in the hydrophobic pocket. The selected substances were checked experimentally to inhibition of the complex formation of I3-catenin with LEF-1 in an ELISA. The basic structures of the molecules detected form various molecule classes which are represented in the following:
Molecule class I:
A: Cephalosporines of similiar structure as the substance "cefamandole" with an inhibition of complex formation for 13-catenin and LEF/TCF (ICSO = 25-100 pM).
The lead structure consists of an aromatic ring (in the case of cefamandole at carbon atom 17, compare "positive list"), an organic component resembling the structure of the ala-ala dipeptide and of a 13-lactam and thiazole ring. By means of the armatic ring the substance was fit into the hydrophobic pocket described. Related cephalosporines without this aromatic ring do not show an inhibitory effect. Additionally, stabilizing hydrogen bridges between a carbonyl group (carbon atom 17) of the substance with the Ser 389 of 13-catenin and a carboxyl group (carbon atom 8, at the thiazole ring) and the Lys 394 of 13-catenin were calculated. Further representatives of this molecule class with an experimentally confirmed inhibition of complex formation are the cephalosporines cefsulodine and cefadroxil (ICSo = 50-100 pM). Basically claim is raised for all cephalosporines with the same lead structure as that of cefamandole to provide the guiding structure for the development of a therapeutic agent or unmodified as a therapeutic agent for combating tumors and cancer.
B. As to its structure substance AC-(6-0-stearoyl) muramyl-ala-D-isoglutamine forms part of another subclass of inhibitors of complex formation (ICso = 100 pM).
C: As to its structure substance 3,6 dihydroxybenzonobornane forms part of another subclass of inhibitors of complex formation (ICSO= 100 pM).
Molecule class II:
Substances binding also in the hydrophobic pocket, in the vicinity of the essential binding site of 13-catenin for LEF/TCF, however not inhibiting complex formation as they are either too small to interfere with the complex formation or bind in another direction in the pocket than the substances of molecule class I. However, in the experiment a strong effect of the action of molecules of class I was detected for these substances by competing with binding in the pocket. Thus, these molecules serve also as guiding structures for modifying and developing potent inhibitors of complex formation of LEF/TCF in tumors.
Here in after, the invention shall be explained in greater detail by way of examples:
1. Preparation and testing of mutants of 13-catenin modulating the interaction with LEF-1, APC and conductin The mutagenesis of 13-catenin in the armadillo repeats 3-8 was carried out by means of the "mutagenesis kit" of the company Clontech according to the producer's record and the mutants were checked by sequencing. In all mutants the original amino acid was substituted by alanine. For analyzing the interactions the cDNA of human 13-catenin (armadillo repeat 3 up to the C-terminal end of the protein) coding for the amino acids Leu218-Leu781 or its mutants was cloned into the fusion vector for the activation domain of Gal-4 (pGAD424, Clontech). The cDNA for the binding domains of the interaction participants was cloned into the LexA fusion vector BTM116. To this end, the cDNA of LEF-I for the amino acids 1-99, conductin for the amino acids A1a342-ARG465; of human APC for the amino acids His1012-G1u1215 (APC 15 amino acid repeats) and for the amino acids Ser1259-Asp 1400 (APC 20 amino acid repeats) were amplified with the respective primers PCR. The interaction of the Lex-A hybrids with 13-catenin and its mutants was quantified by means of the f3-galactosidase reporter activity in the yeast 2-hybrid system (report:"Matchmaker", Clontech) (Fig. 1).
2. Identification of a hydrophobic pocket in 13-catenin in the vicinity of the essential binding site for LEF-1. To calculate the molecule surface of 13-catenin various computer programs such as GRASP, SPOCK and LUDI of MSI were used.
3. Identification of substances binding in the hydrophobic pocket at the essential binding site for LEF- I
Various substance libraries, e.g. the available chemicals data base, ACD of MSI and the programs LUDI, SPOCK and GRASP were used for searching substances binding in the hydrophobic pocket.
4. ELISA for testing substances for their inhibition of the complex formation of 13-catenin and LEF-1.
Selected substances were tested for their inhibition of the interaction between LEF-1 and 13-catenin in producing at first proteins in bacteria recombinant with N-terminate histidin-sequences and dry-cleaning with nickel-chromatography (Behrens et a1.1996).About 50 ng LEF-1 was adsorbed on the wells of ELISA-plates for 60 minutes at roomtemperature in PBS/BSA (0,1 mg BSA/ml).Following the wells were covered with 2,5% skimmed milk/
0,5%BSA for 2 hours at 8°C.AII the further steps followed at room temperature in PBS/BSA
(lOg.g BSA/ ml).
After washing the wells with PBS the substances dissolved in ad 10 % DMSO
final and the indicated final concentrations of the substances were added.
The incubation with 50-100 ng 13-catenin was realized for 15 minutes in PBS/BSA (O,Smg BSA/ml). The complex formation of LEF-1 and 13-catenin was detected with the antibody PA2 against carboxy-terminus of 13-catenin (Hulsken et al 1994). PA2 was added for 15 minutes in a titre dilution of 1:10000 in 1% BSA/PBS.
After washing the wells with PBS the quantification of the complex-formation followed by peroxidase conjugated antibodies (1:2000 in 1% BSA/PBS, Dianova) and by photometrically measuring of the turnover of o-phenylendiamin as substrate at 450 nm (Ultramark ELISA
reader, BioRad). The substances were used in concentrations between 1 gM and 10 mM. To check the specific inhibition of the complex formation of LEF-land 13-catenin, 13-catenin was absorbed in the wells and detected after incubation with the substances.
Substances binding in the hydrophobic pocket of 13-catenin without impeding the complex formation with LEF-1 were identified owing to their competition for this binding site with substances showing inhibitory effects. As example, cefamandole was used as a substance showing an inhibitory effect in its semimaximally efficient concentration (ICSO = 100 pM) together with another substances of the same concentration ( 100 ~M) in each binding reaction in an ELISA.
Substances, which bond at the same place as cefamandole, but used don't inhibit alone the complex formation, don't influence the cefamandole binding.
Legends for the Figures and Tables:
Fig. 1: Identification of the essential binding sites of 13-catenin for LEF-1, the 20 amino acid and 15 amino acid repeats of APC or conductin (A-D) Interaction of f3-catenin mutants with their binding partners in the yeast-2-hybrid system. The amino acid residues substituted by alanine in the 13-catenin mutants and their position in the arm repeats 3-8 are given hereinafter. The interaction was quantified by determining the 13-galactosidase reporter activity and is indicated compared with the interaction with wild type 13-catenin. (E)Description separately essential binding sites in the Armadillo- domain of 13-catenin (RasMol).
Fig. 2: Characterization of a hydrophobic pocket adjacent to the essential binding sites of 13-catenin for LEF-1 /TCF
(A) View of the hydrophobic pocket at the molecule surface of !3-catenin (RasMol). The pocket is flanked by amino acids marked in orange or yellow colours. The amino acid residues of the essential binding site fvr LEF/TCF are marked in blue colour.
The respective amino acids have been marked. (B) Side view of the hydrophobic pocket.
Fig. 3: Substances binding in the hydrophobic pocket of 13-catenin (A) Representation of the surface of the hydrophobic pocket region (Grasp).
The amino acid residues of the essential binding site for LEF/TCF are marked in blue colour (for mutations blocking the interaction between 13-catenin and LEF/TCF: Lys435, Arg 469 and His 470). (B) In the 13-catenin molecule one of the low-molecular substances binding in the pocket is represented.
Fig. 4: Cefamandole as a representative of molecule class I inhibits the complex formation of LEF-1 and 13-catenin in an ELISA.
Rising concentrations of cefamandole ( I S-250 ~M) result in a reduction of the complex formation of LEF-I and 13-catenin protein prepared recombinantly and purified in an ELISA
(IC50=25 pM).
Table 1: Substances potentially binding in the hydrophobic pocket ("drug list"), calculated by computer calculations ( for example LUDI/MSI) Table 2: "Positive list" of substances inhibiting the complex formation of 13-catenin and LEF-1 in ELISA or influence the effect from cefamandole.
to
binding site: Lys 435, Arg 469, His 470, Lys 508 and Arg 515 or the APC
binding site for the 20 amino acid repeat domain: Lys 345, Trp 383 or the 15 amino acid repeat domain or the conductin binding site: Phe 253, His 260, Lys 292).
In following the invention is explained in detail:
In the first realisation of invention got 13-catenin- mutants were identified which mark the respective essentieal binding site for LEF-1/TCF-4, for the 20 amino acid repeats of APC or of conductin (Fig.l). The substitution of single basic amino acids by alanin-rest produced this pointmutants. Because of the essential binding sites of this factors are located in separated subregions of 13-catenin, the substances near to this binding sites influence always one interaction influence specifically.According to the invention, these substances able treat tumors or regenerate tissues.
In a second step, based on the X-ray crystallographic analytical data of the armadillo domain of 13-catenin the surfaces in this region are calculated. Using various computer programs such as e.g. Grasp or Ludi a hydrophobic pocket near the binding site for LEF-1/TCF-4 was identified (flanked by the amino acids Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438). This hydrophobic pocket forms an ideal molecular target for generating substances which bind in this pocket and establish contacts to the closely adjacent essential binding site. For energetic reasons this bond is favoured as a hydrate shell has not to be displaced to make a bond. It is possible that a phenyl alanine residue of LEF/TCF (Phe 24 of LEF-1 or Phe 21 of TCF-4) which is normally essential for binding to 13-catenin (DE 199 09 251 of 22/02/99) binds in this pocket. Thus, the substances might block this point of contact in 13-catenin solely by their binding in the pocket.
In a third step low-molecular substances from molecule data bases were computer-aided fitted into this pocket and selected owing to the number of the stabilizing intractions with 13-catenin.
These substances are mentioned hereinafter. The basic structure of these low-molecular compounds shall be modified after experimentally checking their inhibition function (e.g. in an ELISA) to optimize their function. This is guided by the idea that the modification of the substances, e.g. by adding acid groups, allows additional interactions with the basic residues of the amino acids Lys 435, Arg 469, His 470 or Lys 508 of !3-catenin. As these residues mark the essential binding site of the LEF/TCF factors these interactions should intensify the efficiency of the substances.
The adoption of this strategy to all essential binding sites of 13-catenin allows to generate new pharmaca, which by specifically affecting the respective interaction of 13-catenin, e.g. with the transcription factors LEF/TCF, the tumor suppressor gene product APC or conductin, inhibit the development of tumors or are used for regenerating skin and promoting growth of hair.
These possibilities of affecting arise from the findings published which relate to the function of the respective interaction between 13-catenin and its binding participants in these developmental processes.
In particular the following investigations were carried out:
1. Identification of separate, essential binding sites of 13-catenin for the interaction with LEF-1/TCF-4 with the 20 and 15 amino acid repeats of APC or conductin/axin.
Based on the X-ray crystallographic analysis of the armadillo domain of 13-catenin (Huber et al., 1997) selected basic and aromatic amino acid residues were substituted by alanine residues and the point mutants were analysed for their interaction with the binding participants of 13-catenin in a yeast-2-hybrid system (Fig. 1 ). The mutations blocking specific interactions form clusters in separate subregions of the domain. They mark the essential points of contact in the specific binding sites of 13-catenin for its interaction with LEF/TCF (Lys 435, Arg 469, His 470, Lys 508, Arg 515), with the 20 amino acid repeats of APC (Trp 383, Lys 345), with the 1 S amino acid repeats of APC
(Arg 386), or with conductin (Phe 253, His 260, Lys 292). The extended interaction sites are represented in Fig. 1.
2. Analyses of the molecule surface of 13-catenin in the area of the essential binding site for LEF/TCF
Proceeding from the X-ray crytallographic analytical data of 13-catenin the molecule surface was calculated by means of the programs Grasp and Ludi in the area of the binding site. Thus, ist was possible to identify a hydrophobic pocket (Fig. 2) flanked by the following amino acids: Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438. This pocket is localised in the closest vicinity of the essential binding site for LEF/TCF. It is of great importance for the computer-aided screening of substance libraries for selecting molecules binding in this pocket. The idea to select primarily hydrophobic interactions in the pocket as starting point provides the energetic advantage that the substances need not compete with the hydrate shell of the loaded amino acid residues of the surface. Thus, after their purposeful modification these substances are potent therapeutic agents for treating tumors as the interaction of LEF/TCF with !3-catenin is an early event in the development of tumors. They are experimentally tested for their ability to inhibit the oncogenic interaction (ELISA) and are modified. The molecular modelling of the substances in the pocket is based on the idea to stabilize the interactions proceeding in the proper pocket and to establish additional contacts to the essential amino acid residues of the LEF/TCF binding site. By means of this strategy the effect of these substances is to be optimized.
3. Identification of substances binding in the hydrophobic pocket Substance libraries were screened computer-aided and the molecules were selected according to their stabilizing interactions in the hydrophobic pocket. The selected substances were checked experimentally to inhibition of the complex formation of I3-catenin with LEF-1 in an ELISA. The basic structures of the molecules detected form various molecule classes which are represented in the following:
Molecule class I:
A: Cephalosporines of similiar structure as the substance "cefamandole" with an inhibition of complex formation for 13-catenin and LEF/TCF (ICSO = 25-100 pM).
The lead structure consists of an aromatic ring (in the case of cefamandole at carbon atom 17, compare "positive list"), an organic component resembling the structure of the ala-ala dipeptide and of a 13-lactam and thiazole ring. By means of the armatic ring the substance was fit into the hydrophobic pocket described. Related cephalosporines without this aromatic ring do not show an inhibitory effect. Additionally, stabilizing hydrogen bridges between a carbonyl group (carbon atom 17) of the substance with the Ser 389 of 13-catenin and a carboxyl group (carbon atom 8, at the thiazole ring) and the Lys 394 of 13-catenin were calculated. Further representatives of this molecule class with an experimentally confirmed inhibition of complex formation are the cephalosporines cefsulodine and cefadroxil (ICSo = 50-100 pM). Basically claim is raised for all cephalosporines with the same lead structure as that of cefamandole to provide the guiding structure for the development of a therapeutic agent or unmodified as a therapeutic agent for combating tumors and cancer.
B. As to its structure substance AC-(6-0-stearoyl) muramyl-ala-D-isoglutamine forms part of another subclass of inhibitors of complex formation (ICso = 100 pM).
C: As to its structure substance 3,6 dihydroxybenzonobornane forms part of another subclass of inhibitors of complex formation (ICSO= 100 pM).
Molecule class II:
Substances binding also in the hydrophobic pocket, in the vicinity of the essential binding site of 13-catenin for LEF/TCF, however not inhibiting complex formation as they are either too small to interfere with the complex formation or bind in another direction in the pocket than the substances of molecule class I. However, in the experiment a strong effect of the action of molecules of class I was detected for these substances by competing with binding in the pocket. Thus, these molecules serve also as guiding structures for modifying and developing potent inhibitors of complex formation of LEF/TCF in tumors.
Here in after, the invention shall be explained in greater detail by way of examples:
1. Preparation and testing of mutants of 13-catenin modulating the interaction with LEF-1, APC and conductin The mutagenesis of 13-catenin in the armadillo repeats 3-8 was carried out by means of the "mutagenesis kit" of the company Clontech according to the producer's record and the mutants were checked by sequencing. In all mutants the original amino acid was substituted by alanine. For analyzing the interactions the cDNA of human 13-catenin (armadillo repeat 3 up to the C-terminal end of the protein) coding for the amino acids Leu218-Leu781 or its mutants was cloned into the fusion vector for the activation domain of Gal-4 (pGAD424, Clontech). The cDNA for the binding domains of the interaction participants was cloned into the LexA fusion vector BTM116. To this end, the cDNA of LEF-I for the amino acids 1-99, conductin for the amino acids A1a342-ARG465; of human APC for the amino acids His1012-G1u1215 (APC 15 amino acid repeats) and for the amino acids Ser1259-Asp 1400 (APC 20 amino acid repeats) were amplified with the respective primers PCR. The interaction of the Lex-A hybrids with 13-catenin and its mutants was quantified by means of the f3-galactosidase reporter activity in the yeast 2-hybrid system (report:"Matchmaker", Clontech) (Fig. 1).
2. Identification of a hydrophobic pocket in 13-catenin in the vicinity of the essential binding site for LEF-1. To calculate the molecule surface of 13-catenin various computer programs such as GRASP, SPOCK and LUDI of MSI were used.
3. Identification of substances binding in the hydrophobic pocket at the essential binding site for LEF- I
Various substance libraries, e.g. the available chemicals data base, ACD of MSI and the programs LUDI, SPOCK and GRASP were used for searching substances binding in the hydrophobic pocket.
4. ELISA for testing substances for their inhibition of the complex formation of 13-catenin and LEF-1.
Selected substances were tested for their inhibition of the interaction between LEF-1 and 13-catenin in producing at first proteins in bacteria recombinant with N-terminate histidin-sequences and dry-cleaning with nickel-chromatography (Behrens et a1.1996).About 50 ng LEF-1 was adsorbed on the wells of ELISA-plates for 60 minutes at roomtemperature in PBS/BSA (0,1 mg BSA/ml).Following the wells were covered with 2,5% skimmed milk/
0,5%BSA for 2 hours at 8°C.AII the further steps followed at room temperature in PBS/BSA
(lOg.g BSA/ ml).
After washing the wells with PBS the substances dissolved in ad 10 % DMSO
final and the indicated final concentrations of the substances were added.
The incubation with 50-100 ng 13-catenin was realized for 15 minutes in PBS/BSA (O,Smg BSA/ml). The complex formation of LEF-1 and 13-catenin was detected with the antibody PA2 against carboxy-terminus of 13-catenin (Hulsken et al 1994). PA2 was added for 15 minutes in a titre dilution of 1:10000 in 1% BSA/PBS.
After washing the wells with PBS the quantification of the complex-formation followed by peroxidase conjugated antibodies (1:2000 in 1% BSA/PBS, Dianova) and by photometrically measuring of the turnover of o-phenylendiamin as substrate at 450 nm (Ultramark ELISA
reader, BioRad). The substances were used in concentrations between 1 gM and 10 mM. To check the specific inhibition of the complex formation of LEF-land 13-catenin, 13-catenin was absorbed in the wells and detected after incubation with the substances.
Substances binding in the hydrophobic pocket of 13-catenin without impeding the complex formation with LEF-1 were identified owing to their competition for this binding site with substances showing inhibitory effects. As example, cefamandole was used as a substance showing an inhibitory effect in its semimaximally efficient concentration (ICSO = 100 pM) together with another substances of the same concentration ( 100 ~M) in each binding reaction in an ELISA.
Substances, which bond at the same place as cefamandole, but used don't inhibit alone the complex formation, don't influence the cefamandole binding.
Legends for the Figures and Tables:
Fig. 1: Identification of the essential binding sites of 13-catenin for LEF-1, the 20 amino acid and 15 amino acid repeats of APC or conductin (A-D) Interaction of f3-catenin mutants with their binding partners in the yeast-2-hybrid system. The amino acid residues substituted by alanine in the 13-catenin mutants and their position in the arm repeats 3-8 are given hereinafter. The interaction was quantified by determining the 13-galactosidase reporter activity and is indicated compared with the interaction with wild type 13-catenin. (E)Description separately essential binding sites in the Armadillo- domain of 13-catenin (RasMol).
Fig. 2: Characterization of a hydrophobic pocket adjacent to the essential binding sites of 13-catenin for LEF-1 /TCF
(A) View of the hydrophobic pocket at the molecule surface of !3-catenin (RasMol). The pocket is flanked by amino acids marked in orange or yellow colours. The amino acid residues of the essential binding site fvr LEF/TCF are marked in blue colour.
The respective amino acids have been marked. (B) Side view of the hydrophobic pocket.
Fig. 3: Substances binding in the hydrophobic pocket of 13-catenin (A) Representation of the surface of the hydrophobic pocket region (Grasp).
The amino acid residues of the essential binding site for LEF/TCF are marked in blue colour (for mutations blocking the interaction between 13-catenin and LEF/TCF: Lys435, Arg 469 and His 470). (B) In the 13-catenin molecule one of the low-molecular substances binding in the pocket is represented.
Fig. 4: Cefamandole as a representative of molecule class I inhibits the complex formation of LEF-1 and 13-catenin in an ELISA.
Rising concentrations of cefamandole ( I S-250 ~M) result in a reduction of the complex formation of LEF-I and 13-catenin protein prepared recombinantly and purified in an ELISA
(IC50=25 pM).
Table 1: Substances potentially binding in the hydrophobic pocket ("drug list"), calculated by computer calculations ( for example LUDI/MSI) Table 2: "Positive list" of substances inhibiting the complex formation of 13-catenin and LEF-1 in ELISA or influence the effect from cefamandole.
to
Claims (9)
1. Agents for treating human disease, contained substances, which prevent the binding of .beta.-catenin at LEF-1/TCF- transcriptionsfactors, APC or conductin/axin.
2. Method for detecting substances preventing binding of .beta.-catenin selectively with LEF-1/TCF transcription factors, APC or conductin/axin wherein in the .beta.-catenin molecule in the vicinity of essential binding sites hydrophobic pockets are identified and subsequently therapeutic substances fitting into this pocket are synthesised and tested.
3. Method according to claim 2 wherein .beta.-catenin mutants were identified which mark the respective essential binding site for LEF-1/TCF transcription factors, APC or conductin, - the surfaces in this region are calculated based on X-ray crystallographic analytical data of the armadillo region, thus identifying the existing hydrophobic pockets, - low-molecular compounds are fitted in this pocket and are selected in a biological assay owing to their stabilizing interactions with .beta.-catenin and their selective inhibition or promotion of complex formation with LEF/TCF, APC or conductin and - these compounds are further modified, if necessary by adding acid groups.
4. Method according to claims 2 and 3, wherein the .beta.-catenin-mutants Lys 435, Arg 469, His470, Lys 508 Arg 515, which mark the essential binding site for LEF-1 - the .beta.-catenin-mutants Phe 235, His 260, Lys 292 which mark the essential binding site for conductin, - the .beta.-catenin-mutants Lys 345, Trp 383, Arg 386, which mark the essential binding site for APC
were identified, - the molecule surface is calculated with the aid of the programs Grasp, Ludi and similar programs, - a hydrophobic pocket (flanked by the amino acids Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438) was identified in the vicinity of the LEF-1/TCF binding site, the compounds according to the annexed "drug list" and positive list" which thus become a subject of the claim are fitted into the identified hydrophobic pocket, subsequently experimentally checked with ELISA for their inhibition of the complex formation of .beta.-catenin and its binding partners and optimized by chemical modification.
were identified, - the molecule surface is calculated with the aid of the programs Grasp, Ludi and similar programs, - a hydrophobic pocket (flanked by the amino acids Val 358, Met 363, Ala 391, Ala 392, Thr 393, Lys 394, Gln 395, Met 398, Leu 401, Leu 402, Ile 423, Asn 426, Leu 427, Thr 428, Cys 429, Asn 430, Asn 431, Asn 434, Met 437, Val 438) was identified in the vicinity of the LEF-1/TCF binding site, the compounds according to the annexed "drug list" and positive list" which thus become a subject of the claim are fitted into the identified hydrophobic pocket, subsequently experimentally checked with ELISA for their inhibition of the complex formation of .beta.-catenin and its binding partners and optimized by chemical modification.
5. Substances with a lead structure essential for binding in the hydrophobic pocket and inhibition of the formation of complexes with .beta.-catenin marked by cephalosporines of the cefamandole type (molecule class IA), with the lead structure essential for binding in the hydrophobic pocket and inhibition of the formation of complexes with .beta.-catenin consisting of an aromatic ring, an organic component similar to the structure of the ala-ala dipeptide and a .beta.-lactam and thiazole ring, which are preferably cephalosporines of the cefamandole type such as e.g. cefsulodine, cefadroxil or cefamandole nafates.
6. Substances for the inhibition of the complex formation of .beta.-catenin with LEF/TCF
marked by a structure corresponding to AC-(6-0-stearoyl)-muramyl-ala-D-isoglutamine (molecule class IB).
marked by a structure corresponding to AC-(6-0-stearoyl)-muramyl-ala-D-isoglutamine (molecule class IB).
7. Substances for the inhibition of the complex formation of .beta.-catenin with LEF/TCF
marked by a structure corresponding to that of 3,6-dihydroxybenzonobornane (molecule class IC).
marked by a structure corresponding to that of 3,6-dihydroxybenzonobornane (molecule class IC).
8. Substances binding in the hydrophobic pocket without inhibiting the formation of complexes, however affecting binding of cefamandole to .beta.-catenin as guiding structures for developing potent inhibitors, preferably substances of the «positive list»
table which thus becomes an object of the claim.
table which thus becomes an object of the claim.
9. Agents according to claim 1 to 8 for treating tumors,such as colonic cancers and melanomas,or for regenerating and promoting hair growth.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19944404A DE19944404A1 (en) | 1999-09-16 | 1999-09-16 | Agents for the therapy of human diseases, in particular for the therapy of tumors such as colon carcinomas and melanomas or for tissue regeneration and promotion of hair growth |
| DE19944404.8 | 1999-09-16 | ||
| PCT/DE2000/003104 WO2001019353A2 (en) | 1999-09-16 | 2000-09-07 | Agents for treating human diseases, especially for treating tumors such as colonic cancers and melanomas or for regenerating tissue and promoting hair growth |
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| CA2388383A1 true CA2388383A1 (en) | 2001-03-22 |
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| EP (1) | EP1214345B1 (en) |
| JP (1) | JP2003509364A (en) |
| AT (1) | ATE286071T1 (en) |
| AU (1) | AU1128801A (en) |
| CA (1) | CA2388383A1 (en) |
| DE (3) | DE19944404A1 (en) |
| WO (1) | WO2001019353A2 (en) |
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| CA2453175A1 (en) * | 2001-07-09 | 2003-01-23 | Pharmacia Italia Spa | Interaction inhibitors of tcf-4 with beta-catenin |
| WO2004013308A2 (en) * | 2002-08-06 | 2004-02-12 | Exelixis, Inc. | Maxs as modifiers of the axin pathway and methods of use |
| EP1977747A1 (en) * | 2007-04-05 | 2008-10-08 | Max-Delbrück-Centrum | Adaptive cardiac remodeling by beta-catenin downregulation |
| EP2144606A2 (en) | 2007-04-05 | 2010-01-20 | Max-Delbrück-Centrum Für Molekulare Medizin | Remedy for the treatment of cardio-vascular diseases or disorders |
| CN108066345A (en) * | 2016-11-14 | 2018-05-25 | 武汉华杰世纪生物医药有限公司 | A kind of compound with antitumor action |
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| CA2283932A1 (en) * | 1997-03-24 | 1998-10-01 | Onyx Pharmaceuticals, Inc. | Compositions and methods for diagnosing/treating disease based on beta-catenin/transcription factor interactions |
| EP1054899A2 (en) * | 1998-02-21 | 2000-11-29 | Max-Delbrück-Centrum Für Molekulare Medizin | AGENTS FOR TREATING HUMAN ILLNESSES BASED ON $g(b)-CATENIN, AND THE PRODUCTION AND USE THEREOF |
-
1999
- 1999-09-16 DE DE19944404A patent/DE19944404A1/en not_active Withdrawn
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2000
- 2000-09-07 AU AU11288/01A patent/AU1128801A/en not_active Abandoned
- 2000-09-07 AT AT00972578T patent/ATE286071T1/en not_active IP Right Cessation
- 2000-09-07 JP JP2001522988A patent/JP2003509364A/en active Pending
- 2000-09-07 CA CA002388383A patent/CA2388383A1/en not_active Abandoned
- 2000-09-07 DE DE10082729T patent/DE10082729D2/en not_active Ceased
- 2000-09-07 EP EP00972578A patent/EP1214345B1/en not_active Expired - Lifetime
- 2000-09-07 DE DE50009133T patent/DE50009133D1/en not_active Expired - Fee Related
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| EP1214345B1 (en) | 2004-12-29 |
| WO2001019353A2 (en) | 2001-03-22 |
| EP1214345A2 (en) | 2002-06-19 |
| DE10082729D2 (en) | 2002-01-24 |
| WO2001019353A3 (en) | 2002-03-28 |
| AU1128801A (en) | 2001-04-17 |
| DE50009133D1 (en) | 2005-02-03 |
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