MXPA01005557A - METHODS AND COMPOSITIONS FOR RESTORING CONFORMATIONAL STABILITY OF A PROTEIN OF THE p53 FAMILY - Google Patents

Abstract

The invention is in the field of cancer treatment. In particular, the present invention provides pharmaceutical compounds capable of interacting with mutant and non-mutant forms of cancer-related regulatory proteins such that the mutant protein regains the capacity to properly interact with other macromolecules, thereby restoring or stabilizing all or a portion of its wild type activity. Regulatory proteins include members of the p53 protein family such as, for example, p53, p63 and p73. The compounds of the invention are useful for cancer treatment. Methods for screening for such pharmacological compounds are also provided.

Description

METHODS AND COMPOSITIONS TO RESTORE THE CONFORMATIONAL STABILITY OF A FAMILY PROTEIN FROM p53 I. FIELD OF THE INVENTION The invention is situated in the field of cancer treatment. The present invention provides non-peptide organic compounds capable of interacting with a tumor suppressor protein of the p53 family, and stabilizing a functional conformation thereof. The invention is particularly applicable to the stabilization of mutant forms of tumor suppressor proteins in patients in whom correction of the functional capacity of such proteins can facilitate the treatment of cancer. Methods for conducting discriminant assays for such compounds are also provided.

II. BACKGROUND OF THE INVENTION The primary structure of a protein consists of the particular sequence of structural blocks of amino acids that are linked together to form the chain or polypeptide chains of the protein. In turn, these polypeptide chains are folded into a three-dimensional structure. It is currently believed that various diseases have their origin in a conformational perturbation of the three-dimensional structure of a cellular protein (see reviews by Thomas et al., 1995, TIBS 20: 456-459, Carrell et al., 1997, Lancet 350: 134- 138). For example, Alzheimer's disease is caused by poor folding and subsequent aggregation of beta-amyloid protein, which leads to deterioration of cell function. Similarly, it is believed that the etiological agents of Creutzfeld-Ja ob disease, the prions, cause the disease by initiating a chain reaction that converts normal prion proteins into abnormally folded prion proteins. Proteins that adopt abnormal conformations can do so because they are inherently susceptible to abnormal folding, or because they undergo mutations that thermodynamically destabilize the mutant protein relative to the wild-type protein. A palpable example of missense mutations that lead to disease is the p53 tumor suppressor protein. Wild-type p53 functions as a transcriptional regulator to coordinately control multiple pathways in the cell cycle, apoptosis and angiogenesis. Cellular pathways that control cell stresses, such as DNA damage, mismatch of the mitotic spindle, and hypoxia, all appear to converge on p53. The loss of p53 activity can lead to an uncontrolled proliferation of the affected cells, and to tumor growth. Although in itself the loss of p53 activity may or may not be the trigger for the transformation of a cell into a cancer cell, detectable cancers are more common, and are more likely to grow, in people with p53 mutations. In fact, p53 mutants are the most common genetic aberration in cancer. Recently, two additional proteins, p73 and p63, have been identified with homology to p53 (for a review see Kaelin, 1999, J. Natl. Cancer Inst. 91: 594-598, see also Yang et al., 1998, Molecular Cell 2 (3): 305-16, and Yoshika and others, 1999, Oncogene 18 (22): 3415-21). The p51 has also been designated p40, p51, KET or p73L. These proteins not only share homology of the amino acid sequence with p53, but can also activate promoters of the p53 response and induce apoptosis. In addition, the genes that encode these proteins seem to be ancestrally related to p53. Thus, there is a family of proteins related to p53, recognized in the state of the art, which have similar functions and related amino acid sequences. P53 is a complex macromolecule with three independent functional domains: an N-terminal end that includes a transcriptional activation domain (approximately amino acids 1-43); a central portion encoding a DNA binding domain (DBD) approximately 100-300 amino acids); and a C-terminal portion serving as an oligomerization domain (approximately amino acids 319-360). The crystal structure of the DBD of p53 shows an approximately spherical globular domain with a high content of beta sheets.

The activity of p53 is highly dependent on the ability of the protein to maintain its functional conformation. The analysis of tumors derived from many different cancers reveals that DBD is frequently mutated. Friedlander et al., 1996, J. Biol Chem. 271: 25468-25478. Although there is a wide variety of point mutations that occur within the DNA binding domain of p53 in major cancers (Pavletich et al., Genes &Development 7, 2556-2564), specific positions of residues within the DBP of p53, known as hot spots, they mutate frequently unusually high. The hot spot mutations commonly found in human tumors are scattered approximately randomly along the DBD. When exposed to urea, the p53 DBDs of all frequently mutated forms of p53 are less stable than the wild type DBD (Bullock et al., See above). In addition, p53 mutants are often associated in cells with heat shock proteins, which leads to speculation that they are less able to retain the native conformation (Finlay et al., 1998, Molecular and Cellular Biology 8: 531- 39). It has been found that interactions with the C-terminal domain of p53 activate the p53 cell cycle stop properties. Specifically, in-cycle cell injection of antibody to p53 specific for the C-terminus could arrest them (Mercer et al., 1982, Proc. Nat. Acad. Sci. USA 79, 6309-6312). More detailed studies have "shown that the C-terminal domain regulates the DNA-binding activity of the DBD domain." For example, Hupp and others have found that the monoclonal antibody Pab 421, which interacts with residues 373-381 of the C domain p53 terminal, is capable of enhancing the DNA-binding activity of some mutant forms of p53 (Hupp et al., 1993, Nucleic Acids Research 21: 3167-3174). Thus, Hupp et al. have focused on antibodies and peptides that neutralize an independent negative regulatory domain at the C-terminal end, in an attempt to restore p53 function (Selivanova et al., 1997, Nature Med. 3, 632-638) .However, mutants in position 273 that are restored by this approach they differ from other common mutants in that they retain a high basal DNA-binding activity, and exhibit thermodynamic stability characteristics similar to the wild-type protein (Bullock et al., 1997, Proc. Nat. Acad. Sci. USA 94, 14338-143421). Other researchers in this field have argued that the development of a compound that fixes the N-terminal domain of a mutant of p53 is the most efficient way to rescue the activity of wild-type p53. For example, Friedlander et al. Have tested different monoclonal antibodies that bind epitopes defined on p53, in terms of their ability to promote the DNA binding activity of temperature sensitive p53 mutants. Friedlander et al., 1996, J. Biol. Chem. 271: 25468-25478. Although the Pab 421 antibody, specific for the C-terminal end, restored the function of DNA binding in mutant p53 at low temperatures, antibodies to specific p53 N-terminal end, and in particular the monoclonal antibody Pab1801, were more effective in the promotion of the DNA binding activity of p53 mutants sensitive to temperature, at elevated temperatures. Based on these findings, Friedlander and others ventured the hypothesis that the development of a small molecule that simulated the recognition region of the 1801 epitope, attaching to the N-terminus, would facilitate mutant p53 binding activity of wild-type DNA. Notably, Friedlander and others have shown that an antibody specific for an epitope within the central portion (DBD domain) of the p53 protein, had no effect on DNA binding activity. As a possible explanation for your results, Friedlander and others suggested the hypothesis that the conformation of a domain within a protein had been stabilized by the use of a remote domain. Bullock and others have shown that the change in thermodynamic stability in mutants binding domain p53 DNA, commonly occurring, is quite small, and speculated that development of a therapy with small molecule p53 would be feasible, such as it had been suggested by Friedlander and others (ie, molecules that are fixed to the N-terminus). Bullock et al., 1997 (see above). More global to identifying anticancer compounds have focused on approaches determine direct anti-tumor activities of small molecules in cell-based (eg tumor cell lines) or animal testing trials. Several small molecules with possible antitumor activity have been described. Mazerska et al., 1990, Anti-Cancer Drug Design 5, 169-187; Su et al., 1995. J. Med. Chem. 38, 3226-3235; Nagy et al., 1996, Anticancer Research 16, 1915-1918; Wuonola et al., 1997, Anticancer Research 17, 3409-23. Mazerska et al. Describe a series of nitro-9-aminoacridines with a nitro group attached to the acridine group, whose antitumor properties have been attributed to its ability to bind DNA and produce covalent intracatenary crosslinks. Su et al. Describe a series of 9-anilinoacridine derivatives with various positions of the substituted aniline and acridine ring systems, which have been developed as topoisomerase II inhibitors. Nagy et al. Describe a series of compounds related to phenothiazine, linked by means of a short carbon linker to a group based on urea or a phthalimido. Nagy and others have postulated that the antitumor activity of this class of compounds derives from its ability to react with calcium channels and with calmodulin. Wuonola et al., In the work cited above, describe phenothiazine compounds which are similar to the compounds described by Nagy et al., In the work cited above. To date, no small non-peptide organic molecule has been reported to interact with a protein of the p53 family to restore or stabilize wild type activities, such as tumor suppression activity. In addition, the discovery of such compounds has been hampered by the lack of a large capacity discriminant assay.

BRIEF DESCRIPTION OF THE INVENTION Recognizing the importance of the identification of compounds that can conformationally stabilize thermodynamically unstable preteins or abnormally folded proteins associated with diseases in humans, and knowing the lack of a high capacity assay system in which such compounds could be quickly identified, The authors of the present invention have investigated the use of mutant mutant p53 DNA binding domain (DBD) isolated in in vitro and in vivo assays as a model system in which to rapidly identify agents that conformationally stabilize mutant p53. The invention provides a rapid, reliable and accurate method for objectively identifying compounds, including pharmaceutical agents for humans, that favor wild-type activity in a protein of the p53 family. Accordingly, the present invention provides the first demonstration that non-peptidic organic compounds can interact with a protein of the p53 family and promote its wild-type activity. At physiological temperature or at temperatures close to it, these active compounds have favored a wild type activity of p53 not only in various p53 mutant proteins, but also in wild-type p53 proteins. These compounds have an important use as anticancer pharmaceutical agents. Thus, the invention provides a new approach and useful compounds for antitumor therapy in cancers with mutant or wild-type activity, of a protein of the p53 family. In one aspect, the invention provides a method for promoting a wild type activity in a mutant form of a human protein of the p53 family, in which one or more functional activities of the protein are at least partially diminished by the inability of the protein to maintain a functional conformation under physiological conditions, the method comprising the steps of contacting the mutant protein with a non-peptide organic compound that is capable of binding to one or more domains of the mutant protein under physiological conditions and stabilizing a conformation. functional thereof, and allow the stabilized protein to interact with one or more macromolecules that participate in the wild-type activity. The human protein of the p53 family can be, for example, p53, p63 or p73. In preferred embodiments, the non-peptide organic compound interacts with p53, and even more preferably, with the DNA binding domain of p53. The invention also provides, in another embodiment, a method for treating a human subject in relation to a morbid condition associated with the expression of a mutant protein of the p53 family having one or more wild-type activities decreased, comprising the of administering to the subject a non-peptidic organic compound that is capable of binding to one or more domains of the mutant protein under physiological conditions, and stabilizing a functional conformation thereof; and allowing the stabilized protein in the patient to interact with one or more macromolecules that participate in wild-type activity. In yet another embodiment, the invention provides a method for treating a human subject suffering from cancer, comprising the steps of: administering to the subject a non-peptidic organic compound that is capable of binding to one or more domains of a human protein of the p53 family under physiological conditions, and stabilize a functional conformation thereof; and allowing the stabilized protein to interact with one or more macromolecules that participate in a wild type activity of the protein. In one aspect, the non-peptidic organic compounds for use in the invention can be a compound that contains both a hydrophobic group (for example a planar polycyclic) and a cationic group (preferably an amine) linked by a linker with a length specific. In a preferred aspect, the non-peptidic organic compounds for use in the invention are selected from the group consisting of: ((H) (III) where, for group I, R 5 is -N-R 18 R 19, wherein R 8 is H, C 1 -C 6 alkyl, or phenyl, and R 19 is H, C C β alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) P- (CHR22) m- (CH2) nNR20R21, or (CH2) p- (CHR22) m- (CH2) n-NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or CI-CT alkyl, and R20 and R21 are each independently selected from: (a) H, C1- alkyl C12, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, Ce-C14 aryl, Cs-Cg heteroaryl, (C? -Ce alkyl) C6-C12 alkyl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Cß alkoxy, (Ci-Cß alkyl) -3-C10 -heterocycloalkyl, or (Ci-CβJ-Cd-Cι-aryl alkyl; or (b) NR20R21, taken together, represents hydrogen, morphoiin, or 4- (C 1 -C 6 alkyl) -piperizine; R 6 is (a) Ci-Ce alkyl or C 2 -C 8 alkenyl, each being or optionally substituted with one or more phenyl groups, or (b) phenyl substituted with halo, C-β alkoxy; and R7 and R8 are identical or different, and are selected from H, nitro, Ci-C alco alkoxy, or halogen selected from fluoro, chloro, and bromo; where, for group II, R is Ci-Ce alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22 ) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-C alquilo alkyl, and R 20 and R 21 are each independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 10 heterocycloalkyl, Ce-Cι aryl, C 5 -C 9 heteroaryl, C 1 -C 6 alkyl -CβJ-C6-C12 aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, CrC6 alkyl, C-C alco alkoxy, C (-C6 alkyl (C?-C6) alkylcyclohexyl, alkyl (CrCβJ -heteroaryl C5-C9, or (C? -C6) alkyl-C13-C10aryl, wherein, for group III, R 10 is -N-R 18 R 19, wherein R 18 is H, Ci-Cß alkyl, or phenyl, and R 19 is H, Ci-Cß alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, cycloheteroalkyl C3-C8, -CONR18 (CH2) pNR20R21. - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5 , n is 0-5, R22 is hydroxy or C? -C6 alkyl, and R20 and R21 are each independently selected from: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, C6-C aryl, C5-C9 heteroaryl, (C6-C6 alkyl) -C6-C12 -aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C-Cß alkyl, C alco-alkoxy, Cß, (CrC-β alkyl) -3-C10 -heterocycloalkyl, (Ci-CeJ-C5-C9-heteroaryl alkyl, or (Ci-C-alkyl) -aryl-C-aryl, or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C? -C6 alkyl) -piperizine; A and B are the same or different, and each represents carbon or nitrogen, and R11 and R12 are the same or different, and are selected from H, nitro, Ci-Ce alkoxy, or halogen selected from fluoro, chloro and bromo, where, for group IV, R 13 is -N-R 18 R 19, wherein R 18 is H, C 1 -C 6 alkyl, or phenyl, and R 19 is H, C C β alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, cycloheteroalkyl C3-C8, -CONR18 (CH2) PNR20R21. - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5 , n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R 20 and R 21 are each independently selected from: (a) H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 10 heterocycloalkyl, ( C6-C6 alkyl) -C5-C9 heteroaryl, C5-C9 heteroaryl, C6-C6 aryl, and (Ci-CβJ-C6-C10 alkyl), wherein said groups are optionally substituted with one or more hydroxy, halo , amino, trifluoromethyl, Ci-Cß alkyl, CrC-6 alkoxy, Ci-CeJ alkyl-C3-C10 heterocycloalkyl, (C alquilo -Ce) alkyl-C5-C9 heteroaryl, and (Ci-CβJ-aryl C3 alkyl) -C10; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (Ci-CβJ-piperizine alkyl, A and B are the same or different, and each represents carbon or nitrogen, and R14 and R15 are the same or different, and are selected from H, nitro, Ci-Ce alkoxy, or halogen selected from fluoro, chloro and bromo, and wherein, for group V, A is carbon or nitrogen; R 16 is -N-R 18 R 19, wherein R 18 is H, C C β alkyl, or phenyl, and R 19 is H, C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n- NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R20 and R21 are each independently selected from: (a) H, alkyl C? -C? 2, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, C6-C10 aryl, C5-C9 heteroaryl, (C? -Ce alkyl) C6-C? Alkyl, and (d-Ce alkyl) - C5-C9 heteroaryl, or wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Ce alkyl, C1-C6 alkoxy, (C 1 -Ce) alkyl -heterocycloalkyl C 3 -C 10, (alkyl) Ci-Cβ) -heteroaryl Cs-Cg, or (C 1 -C 4 alkyl) -aryl Ce-Cι alkyl; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (aikyl Ci-CβJ-piperizine, and R17 is selected from H, nitro, C?-C6 alkoxy, or halogen selected from fluoro, chloro and In addition, many of the compounds useful in the practice of the invention are new in themselves, and the description herein of such compounds defines a further aspect of the invention. The invention also provides, in another aspect, a method to design additional compounds that favor a wild-type activity of a protein of the p53 family The method involves employing one of the active compounds of the invention to generate a hypothesis, identify a candidate compound that satisfies the hypothesis, and determine whether the The candidate compound favors a wild-type activity of a protein of the p53 family Another aspect of the invention is a composition comprising a complex of a family protein of p53 and a non-peptide compound that interacts with the protein and promotes a wild type activity of the protein. In yet another aspect, the invention provides a method for performing discriminant assays for compounds that favor a wild-type activity of a protein of the p53 family. In a preferred aspect, the method comprises performing assays for compounds that interact with the DNA binding domain (DBD) of p53, and measuring the conformation of the DBP of p53 in the presence of the compound. However, the invention also contemplates the use of full-length and part-length proteins of the p53 family in these methods to perform discriminant assays. In a particular embodiment, the operations of testing and measuring are performed simultaneously. Compounds that have been found to favor wild-type activity in a mutant form of a p53 family protein are optionally subjected to another in vivo discriminant assay for their ability to stop or repress tumor growth. Another aspect of the invention is a method for discovering drugs by the discriminant assay of non-peptidic organic compounds in search of a specific interaction with the DBP of p53. The success of the present invention in the identification of compounds that favor wild-type activity in a mutant or wild-type protein of the p53 family, demonstrates that the methods of the invention are broadly applicable to the discovery of drugs for a class of diseases that are induced by conformationally defective or unstable proteins. Examples of such target proteins include pp60src, ubiquitin activating E1 enzyme, regulator of transmembrane conductance in cystic fibrosis, hemoglobin, prion proteins, serpins, and beta-amyloid protein.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Modulation of epitopes dependent on the conformation in DBD of p53. DBD of p53 was immobilized in microtiter wells, and incubated at elevated temperatures. An ELISA assay determined the percentage of epitope for mAb1620 that remained in the heated wells, compared to control wells that had been kept on ice. Figure 1A: 0.5 ng DBD of wild-type p53 were incubated, and the epitope for mAb162 remaining as a percentage of the unheated control is shown. The standard deviations were <10%. Figure 1B: 1.25 ng of pDB DBD marked with FLAG were immobilized, heated to 45 ° C, and the remaining epitopes are shown for anti-FLAG, mAb1620, and mAb240, as a percentage of the unheated control. Figure 1C: 1.0 ng of DBD of wild-type p53 and mutant at position 143, which had approximately equal levels of the epitope for mAb1620, were heated at 37 ° C, and the stability of the epitope was determined as a percentage of unheated controls . The error bars indicate the standard deviation for 4 replicates. Figure 2. Stabilization of the 1620 epitope in DBD of mutant p53. Figure 2A: Representative compounds, designated Compound X, Compound Y and Compound Z, which have favored the conformational stability of p53. Figure 2B: 1 ng of wild-type p53 DBD was immobilized and heated at 45 ° C for 30 minutes in the presence of compounds or an equivalent concentration of DMSO vehicle. The epitope for mAb1620 is indicated as a percentage of the unheated control. Figure 2C: DBD preparations of wild-type and mutant p53 were immobilized, with approximately equal levels of epitope for mAb1620 (within a 10% range), and heated at 37 ° C for 30 minutes in the presence of the compound or vehicle. The epitope for mAb1620 is shown, remaining as a percentage with respect to the unheated controls. The error bars indicate the standard deviation for 4 replicates. Figure 3. Modulation of p53 conformation and transcription activity in cells with mutant p53. Figure 3A: H1299 transfectants expressing mutant p53 at position 173 were treated, in culture, with 16.5 μg / ml Compound X. Cell lysates were normalized for minor variations of the total p53 protein using Western blots with pan antibody p53, mAbDO-1, and in an ELISA assay the amount of p53 exhibiting the epitope for mAb1620 was determined. The increase in the positive p53 fraction for 1620 was corrected with respect to the positive p53 fraction for 1620 in the untreated cells. Figure 3B: H1299 transfectants paired with a luciferase reporter gene (H1299 / reporter) or reporter gene and mutant p53 at position 173 (H1299 / reporter + mutant p53) were treated in microtiter wells for 16 hours. The induced expression of the luciferase reporter gene, which is indicative of the wild-type p53 function, was corrected with respect to the basal level of expression in the absence of the compound. The values represent the mean 4 replicates. Figure 4. Introduction of WAF1 expression in cells with mutant p53. Saos-2 cells expressing transfected mutant p53 proteins (at position 173 or position 249) were treated, in culture, with 16.5 μg / ml Compound x for 16 hours. The cell lysates were normalized with respect to the total protein, and analyzed in Western stains. The upper portion of the stain was probed with mAbDO-1 for total p53, and the lower portion of the stain was probed with an antibody directed toward WAF1.

Figure 5. Promotion of conformational stability and p53 function in tumors. Mice harboring subcutaneous tumors derived from mutant H1299 / reporter + p53 cells were administered a single intraperitoneal injection of Compound X at a rate of 100 mg / kg, and duplicate tumor lysates were normalized to the total p53 content based on in densitometric scans of Western stains with mAbDO-1. In an ELISA assay, the amount of p53 exhibited by the epitope for mAb1620 was determined, and the increase in positive p53 fraction was corrected for 1620 with respect to the positive p53 fraction for 1620 in lysates from untreated tumors. The tumor lysates were also analyzed for luciferase expression to verify the intensification of transcription activity of p53. Luciferase expression was normalized with respect to protein concentration, and compared with lysates from untreated tumors. Figure 6. Suppression of tumor xenografts expressing mutated p53. Tumor cells were inoculated to mice, and treated by peritoneal injections of Compound X or vehicle, as indicated. The compound was administered for seven days, once a day (abbreviated, q. D.), Or at 12 hour intervals (abbreviated b.i. d). The vehicle treated mice received injections at 12 hour intervals. The volume of the tumor was determined by measuring the diameter of said tumor in two dimensions, and it was averaged between 5-7 mice within each group. The dotted lines represent the initial volume of the tumor when the treatment was started.

V. DETAILED DESCRIPTION OF THE INVENTION Loss of function in the p53 tumor suppressor gene product can lead to the uncontrolled proliferation and / or loss of apoptosis observed in many different types of cancers. Even if p53 has not mutated into a cancer cell, the promotion of wild-type p53 activity in one of these cells can inhibit the cancerous phenotype. The invention demonstrates, for the first time, that non-peptidic organic compounds can interact with a protein of the p53 family to stabilize the functional conformation thereof. Accordingly, such compounds have an important use as pharmaceutical agents for the treatment of all types of cancers. Thus, in one aspect, the invention provides a method for promoting a wild-type activity in a mutant form of a human protein of the p53 family, in which one or more functional activities of the protein are at least partially diminished by the inability of the protein to maintain a functional conformation under physiological conditions, the method comprising the steps of contacting the mutant protein with a non-peptide organic compound that is capable of binding to one or more domains of the mutant protein under physiological conditions and stabilizing a functional conformation thereof, and allowing the stabilized protein to interact with one or more macromolecules that participate in wild-type activity. The mutant human protein of the p53 family can be a mutant p53, p63 or p73 protein. In preferred embodiments, the non-peptide organic compound interacts with p53, and even more preferably, with the DNA binding domain of p53. The invention also provides, in another embodiment, a method for treating a human subject in relation to a morbid state associated with the expression of a mutant protein of the p53 family having one or more wild-type activities decreased, comprising the operations of administering to the subject a non-peptidic organic compound that is capable of binding to one or more domains of the mutant protein under physiological conditions, and stabilizing a functional conformation thereof, and allowing the stabilized protein in the patient to interact with one or more macromolecules that participate in wild-type activity. Still in another modality, the invention provides a method for treating a human subject suffering from cancer, comprising the steps of: administering to the subject a non-peptidic organic compound that is capable of binding to one or more domains of a human protein of the p53 family under physiological conditions, and to stabilize a functional conformation thereof, and allow the stabilized protein to interact with one or more macromolecules that participate in wild-type activity. The human protein of the p53 family that is stabilized in the methods of the invention can be a wild type protein or a mutant protein, for example p53, p63 or p73. Although the proteins of the p53 family are mutants in various cancers, in some cancers or cancer cell types, the structure or function of a protein of the p53 family (p53 itself has received most of the studies) is altered although the cells involved retain an allele that codes for the wild type. For example, see Kaelin, 1999, supra, for a discussion of viral-associated cancers in which a viral protein degrades the p53 protein, or p53 is inactivated or degraded by, for example, oncogene expression products. . Given the importance of p53 family proteins in cellular regulatory processes, it will be apparent that the compounds of the invention are also useful for stabilizing functional conformations of members of the non-mutant p53 family, under physiological conditions and in cells where the survival time and / or the structure and / or the activity of said proteins is normal. Thus, the compounds of the invention are useful in the treatment of cancers in which the function of the p53 protein, and the like, is not substantially affected by the presence of the cancerous state, and also in the treatment of tissues expressing precancerous cells whose anomalies do not yet extend detectably to the function, survival time and / or abnormal structure of p53 (or members of the p53 family). In addition, further stabilizing proteins of the p53 family (for example by causing an increased life span thereof), in healthy cells that are adjacent to sites of malignancy, or that otherwise come into contact with malignant cells in the body, The extent of the cancer can be controlled. The compounds of the invention are also useful in this regard. In accordance with the practice of the invention, a protein of the p53 family is defined as a mammalian p53, p63 or p73.; and / or a protein having a domain, all having at least 50, and more preferably 80%, amino acid sequence homology to one or more of (1) the N-terminal domain required for transcriptional activation, (2) ) the DNA binding domain, or (3) the oligomerization domain of a mammalian p53, p63, or p73, wherein said homology is measured by any of the recognized BLASTP v. 2.0 (www.ncbi.nlm.nih.gov) (Altschul et al., 1990, J. of Molec. Biol., 215: 403-410, "The BLAST Algorithm"; Altschul et al., 1997, Nuc. Acids. .25: 3389-3402), and WU BLAST-2.0 (available from Washington University, St. Louis, MO, USA), and wherein said protein demonstrates at least one function that is recognized in the art as well as characteristic of p53, p63, or p73 (for example, the ability to activate the responsive promoters gives p53 and induce apoptosis, for a review of properties recognized in the art see Kaelin, 1999, Yang et al., 1998, and Yoshikawa et al. , 1999, cited above). For a general discussion of the procedure and benefits of the BLAST, Smith-Waterman, and FASTA algorithms, see Nicholas et al., 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www.psc.edu), and references cited in them. Compounds that stabilize the wild-type conformation of a protein of the p53 family are compounds that, when in contact with a protein of the p53 family, favor or restore a wild-type activity of the protein, such as affinity of DNA fixation or the ability to interact with any macromolecule for a normal function of the p53 family protein. Other wild-type activities of p53 include, but are not limited to, transcriptional activation activity (e.g., induction of WAF1), cell cycle arrest, and apoptosis elicitation. In another aspect, the invention includes the use of the compounds of the invention, to inhibit tumor growth and / or treat cancer. A particular advantage of the invention is that it has been shown that the compounds thus identified by using these methods stabilize the active conformation not only of wild type p53 DBD and mutant p53 DBD used in the discriminant assays, but also of other DBPs of p53 and mutant p53s. Therefore, the compounds thus identified have a wide applicability in the treatment of various cancers. The present invention also provides a novel way of conducting discriminant assays for compounds that favor the wild type conformation of a p53 family protein, and can restore wild type activity to mutant proteins of the p53 family. The compounds identified by employing the methods of the invention are useful for treating diseases, such as cancer, which are associated with defects in the activity of proteins of the p53 family. The methods of the invention involve carrying out discriminant tests on compounds, in search of those that interact directly with a protein of the p53 family. Such methods may employ for the purposes of discriminant assays a full length protein of the p53 family (mutant or wild type) or a deletion derivative containing at least the DBD and optionally the N-terminal and / or domains. C-terminal However, in a preferred aspect of the invention, discriminant assays utilize a polypeptide fragment of a protein of the p53 family that contains only the DBD, without the intact N- or C-terminal domains. this, for the purposes of this application, it is understood that the expressions "the DNA binding domain" or "the DBD" include just the DBD of a protein of the p53 family, without any N-terminal or C-terminus intact (unless otherwise indicated)., these DBD domains can be fused to heterologous polypeptides depending on the format of the assay (eg, a FLAG epitope or a glutathione-S-transferase protein). Furthermore, instead of merely eliminating a negative regulatory effect on DNA binding, the methods and compounds of the invention favor the increased conformational stability of p53 family proteins of both wild type and mutants. Accordingly, in an aspect illustrated below by means of a non-limiting working example, the invention provides a method for conducting discriminant assays for compounds that interact specifically with the DBP of p53, and measuring the conformation of the DBD of p53 in the presence of the test compound. Optionally, the DBD of p53 is a DBD of mutant p53. However, it is easier to produce wild-type p53 DBD in large quantities. Although the discriminant assay can be performed in a cell-based format, for high-throughput scans specific to the search for compounds that target the p53 DBD, an in vitro-based assay is more direct and desired. Compounds identified in a preliminary screening against DBP of p53 can be further tested for their effects on intact p53 function (including missense p53 mutants). The compounds identified by the use of these methods are also within the scope of the invention. For the purposes of the present invention, assays are designed for compounds that interact with the DNA binding domain of a protein of the p53 family, so that the compounds discovered are those that specifically target the DBD and not other domains of the protein. For example, a compound that specifically "interacts with" or "acts on" the DBD does not necessarily need to be stably fixed to the DBD (although it can do so); it is sufficient that the compound has some effect on the conformation of a protein of the p53 family in the presence of the compound. Accordingly, it is possible that the compounds are first subjected to discriminant tests in terms of their interaction with the DBD and then analyzed for their effect on conformation, or it is possible that these two steps of discriminant tests are performed simultaneously taking advantage of a conformational change in the presence of the compound to also detect the interaction with the DBD. In this application the expression "specific interaction" is used to exclude nonspecific forms of binding, which include the type that is known to occur between hydrophobic compounds and proteins through non-selective hydrophobic interactions. The term "specific interaction" is also used to distinguish the properties of the compounds of this invention from the compounds that affect the thermostability of the proteins, by changing the chemical properties of the solvent in bulk. Therefore, molecules excluded from the scope of this aspect of the invention include heat stabilizing agents such as glycerol, trimethiamine oxide, and deuterated water. Compounds that interact specifically with a protein of the p53 family will show their effect at much lower concentrations than these bulk solvents or nonspecific hydrophobic interactions. For example, glycerol is effective at a 600 mM concentration.

However, the effects of compounds that interact specifically with a protein of the p53 family will be observed at compound concentrations of less than 1 mM, preferably less than 100 micromolar, and more preferably less than 10 micromolar, in in vitro assays or assays. based on cells. In relation to the practice of the invention, the following definitions will generally be valid. As used herein, unless otherwise indicated, the term "alkyl" includes saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties, or combinations thereof. Similarly, the terms "alkenyl" and "alkynyl" define hydrocarbon radicals having straight, branched or cyclic moieties, in which at least one double bond, or at least one triple bond, respectively, is present. These definitions are also valid when the alkyl, alkenyl or alkynyl group is present within another group, such as alkoxy or alkylamine. As used herein, the term "alkoxy" includes O-alkyl groups wherein "alkyl" is as defined above. As used herein, unless otherwise indicated, the term "halo" includes fluoro, chloro, bromo or iodo. For convenience of description, when used herein, the term "C3-C10 cycloalkyl" refers to both cycloalkyl groups and cycloalkenyl groups, which have zero or optionally one or more double bonds, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadiene, cycloheptyl, cycloheptyl, bicyclo [3.2.1 joctane, norbornyl, and the like. When used herein, the term "C3-C10 heterocycloalkyl" refers to pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydropyranyl, pyranyl, thiopyranyl, aziridinyl, oxiranyl, methylenedioxyl, chromenyl, isoxazolidinyl, 1,3-oxazolidin-3-yl , isothiazolidinyl, 1,3-thiazolidin-3-yl, 1,2-pyrazolidin-2-yl, 1,3-pyrazolidin-1-yl, piperidinyl, thymorpholinyl, 1,2-tetrahydrothiazin-2-yl, 1,3 -tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morphoininyl, 1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, tetrahydroazepinyl, piperazinyl, chromanium, etc. One skilled in the art will understand that the connection of said C3-C10 heterocycloalkyl rings is through a carbon or through a nitrogen heteroatom with sp3 hybridization. When used herein, the term "C5-C9 heteroaryl" refers to furyl, thienyl, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, 1,3,5-oxadiazolyl, , 2,4-oxadiazolyl, 1,3-oxadiazolyl, 1, 3,5-thiadiazolyl, 1,2,3-thiadiazolyl, 1,4-thiadiazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, 1, 2 , 4-triazinyl, 1, 2,3-triazinyl, 1, 3,5-triazinyl, pyrazolo [3,4-b] pyridinyl, cinnolinyl, pteridinyl, purinyl, 6,7-dihydro-5H- [1] pyridinyl, benzo- [b] thiophenol, 5,6,7,8-tetrahydroquinolin-3-yl, benzoxazole, benzothiazoyl, benzisothiazoyl, benzisoxazolyl, benzlmidazolyl, tianaphtenyl, isothianaphtenyl, benzofuranyl, isobenzofuranyl, isoindolyl, indolyl, indolizinyl, indazolyl, isoquinyl, quinolyl, phthalazinyl , quinoxalinyl, quinazolinyl, benzoxazinyl, and the like. One skilled in the art will understand that the binding of a C5-C9 heteroaryl group to the rest of a structure is generally carried out without limitation, that is, through a carbon atom or through a hetero atom with sp2 hybridization. Similarly, phenyl and naphthyl are representative of aryl When, in a drawing, a bond is drawn but no identification is made of the group located at the distal end thereof, it is understood that a methyl group will be conventionally recognized. In the absence of any drawn link, the position is occupied by hydrogen, if valence allows, as is easily understood in the art. Thus, the indication R-O- means R-O-CH3.

A. Compounds of the invention that favor wild-type activity in a protein of the p53 family The non-peptide organic compounds of the invention can be any type of compound that, when exposed to a wild-type or mutant protein of the p53 family, favor the activity of wild types of the protein. The preferred compounds are relatively small organic compounds (compared to typical proteins, with 50 to 150 kD). The present invention provides for the first time such compounds, which are not peptides or, more particularly, they are antibodies, but nevertheless interact specifically with p53, and thus stabilize a DBD wild type conformation of p53 or p53 protein. Organic compounds that are not peptides are particularly useful as pharmaceutical agents for various reasons. For example, non-peptide compounds are much less immunogenic than peptides, are more readily absorbed into the interior of the body through a mucosal barrier or other barrier of cell layers, and may be less labile. In one aspect, the active compounds discovered by the methods of the invention can be defined as a compound containing both a hydrophobic group (eg, a flat polycyclic) and a cationic group (preferably an amine) linked by a linker of length specific. Benzimidazole, benzoquinoline, phenothiazine, and styrylquinazoline are preferred in the hydrophobic position. Active cationic groups are both secondary and tertiary amines, including, but not limited to, dimethylamine, diethylamine, diethanolamine, methylamine, methylpiperazine, and morpholine. Certain larger amines were correspondingly more active when tested in the hydrophobic phenothiazine series; accordingly, a larger amine is preferred in this position. Positively charged groups in the cationic position are active and preferred (see table 1, below). With respect to this aspect of the invention, the spacing between the hydrophobic and cationic groups must be at least one propyl length; Linkers shorter than a propyl length were substantially less effective under the particular conditions of the assay (see Table 2 below). Thus, linkers having a length of about 3 to 5 carbon bonds (from 5 to 9 Angstroms, and more preferably 6 to 8 Angstroms) are preferred, although the most active are linker-containing compounds with the length of a linker. propyl (approximately 6.5 Angstroms). Linkers longer than the length of a butyl linker have resulted in compounds which, under the particular conditions of the assay, were less effective than the corresponding compounds with linkers with the length of a butyl linker (Table 2). Even more preferred are branched linkers that retain the correct distance; these linkers have generally been more active in this test than the corresponding linear linkers as long as they still maintain approximately the correct linker length of between 5 and 9 Angstroms (and optimally around 6.5 Angstroms). Accordingly, in one aspect, the compounds of the invention have the formula: F1-L-F2 and F1 is selected from the group consisting of: ; where R1, R2, R3 are the same or different, and are independently selected from the group consisting of hydrogen, halogen, methoxy and nitro; L is a straight chain or branched chain alkyl having a length of 5 to 9 Angstroms; and F2 is a secondary or tertiary amine. In other aspects, F2 is dimethylamine, diethylamine, diethanolamine, methylpiperazine or morpholine. For example, F2 can be an amine selected from the group consisting of: ; Y R4es-O-CH2-CH3oH. The chemical structures of various compounds of the invention are shown below. It has been found that each of these compounds significantly enhances the stability of the confirmation sensitive epitope for p53 in at least one mutant p53 DBD, at temperatures close to physiological. 1. Acridines 2. Quinazolines 3. Phenothiazoles In accordance with the general design principles described herein, the following groups of compounds are preferred in the practice of the present invention.

(I) (il) (lll) (IV) (V) where, for group I, R5 is -N-R18R19, wherein R18 is H, Ci-Cß alkyl, or phenyl, and R 19 is H, Ci-Cß alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) pNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21 or - (CH2) p- (CHR22) m- (CH2) n- NR20R21 'where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or CrCß alkyl, and R j20. and, D R21 are each independently selected from: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, C6-C10 aryl, C5-C9 heteroaryl, (C? -Ce alkyl) -C6-C12 aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C-Cß alkyl, Ci-Cß alkoxy, (CrC-β alkyl) -3-C10 -heterocycloalkyl, or (CrCe alkyl) ) -arilo Ce-Cío; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C? -Ce alkyl) -piperizine; R6 is (a) C -Cß alkyl, or C2-C8 alkenyl, each being optionally substituted with one or more phenyl groups, or (b) phenyl substituted with halo, Ci-Cß alkoxy; and R7 and R8 are identical or different, and are selected from H, nitro, Ci-C alco alkoxy, or halogen selected from fluoro, chloro, and bromo; where for group II, R is C Cß alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) pNR20R21. - (CH2) P- (CHR22) m- (CH2) n- NR20R21, or - (CH2) P- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5 , n is 0-5, R22 is hydroxy or Ci-Cs alkyl, and R20 and R21 are each independently selected from H, C1-C12 alkyl, C3-C2 cycloalkyl, C3-C10 heterocycloalkyl, aryl Ce Cι, Cs-Cg heteroaryl, (C?-Ce alkyl) -C6-C? 2aryl aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Cβ alkoxy , (C 1 -C 6 alkyl) -heterocycloalkyl C 3 -C 10, (C 1 -C 6 alkyl) -heteroaryl C 5 -C 9, or (C 1 -C 6 alkyl)-C 1 -C 4 -aryl; where, for group III, R 10 is -N-R 18 R 19, wherein R 18 is H, C C β alkyl, or phenyl, and R 19 is H, Ci-C β alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n- NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or C1-C6 alkyl and R20 and R21 are each independently selected from: (a) H, C-alkyl ? -C? 2, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, Ce-C14 aryl, Cs-Cg heteroaryl, (C? -Ce alkyl) C6-C? 2 -aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Cβ alkoxy, (C?-Ce) alkyl-C 3 -C 10 alkyl heterocycle, (C -Ce) alkyl-C 5 -C 9 heteroaryl, or (C-alkyl) ? -Ce) -C6-C10 aryl; or (b) NR20R21, taken together, represents hydrogen, morphoiin, or 4- (C? -Ce alkyl) -piperizine; A and B are the same or different, and each represents carbon or nitrogen; and R11 and R12 are the same or different, and are selected from H, nitro, C-C alco alkoxy, or halogen selected from fluoro, chloro, and bromine; where, for group VI, R 13 is -N-R 18 R 19, wherein R 18 is H, C -Ce alkyl, or phenyl, and R 19 is H, CrC 6 alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21 . wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cs alkyl and R20 and R21 are each independently selected from: (a) H, C1-C12 alkyl , C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, (CrCe alkyl) -5-C9 heteroaryl, C5-C9 heteroaryl, Ce-Cryl aryl, and (C6-C6 alkyl) -6-C10 alkyl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C-C6 alkyl, C1-C6 alkoxy, C3-C10 alkyl (C? -Ce) -cycloheterocycle, or alkyl (C? -C6) ) -aril C6-C? 0; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C? -C6 alkyl) -piperizine; A and B are the same or different, and each represents carbon or nitrogen; and R 14 and R 15 are the same or different, and are selected from H, nitro, Ci-Cβ alkoxy, or halogen selected from fluoro, chloro, and bromine; and where, for group V, A is carbon or nitrogen; R16 is -N-R18R19, wherein R > 18 is H, C? -C6 alkyl, or phenyl, and R19 is H, C? -C alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, cycloheteroalkyl C3-? C8, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or C C6 alkyl and R20 and R21 are each selected independently from: (a) H, C1-C12 alkyl, C3-C12 , C 3 -C 10 heterocycloalkyl, Ce-Cι aryl, C C-Cg heteroaryl, (C?-Ce alkyl) -C 6 -C 10 aryl, and C 5 -C 9 (C 1 -C 6 alkyl) heteroaryl, or wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Ce alkyl, Ci-Cβ alkoxy, (C 1 -Ce) alkyl-C 3 -C 10 heterocycle, (Ci-Ce alkyl) -heteroaryl C 5 -C 9 , or (C 1 -C 6 alkyl) aryl Ce-Cι; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (Ci-CeJ-piperizine alkyl, and R17 is selected from H, nitro, Ci-Cβ alkoxy, or halogen selected from fluoro, chloro, and Bromine The particularly preferred compounds of the invention include the following eleven compounds: (1-benzyl-piperidin-4-yl) - (3-phenotin-10-yl-propyl) -amine [2 (4-chloro-phenyl) -ethyl] - (3-phenothiazin-10-yl-propyl) -amine (3-Phenothiazin-10-yl-propyl) -thiochroman-4-yl-amine [1- eb -3- (2,6,6-trimethyl-cyclohex-2-phenyl) -allyl] -3- (-phenot-azln-10-yl-propyl) -amine (7-ethoxy-1, 2,3,4-tetrahydro-naphthalen-2-yl) - (3-phenothiazin-10-yl-propyl) -amine N '- (9-fluoro-benzo [c] acridin-7-yl) -N, N-dimethyl-propane-1,3-diamine N'-acridin-9-l-N, N-dimethyl-propane-1,3-diamine 2. { 4- [4- (benzo [g] quinolin-4-ylamino) -phenyl] -1-p-piperazin-1-yl} Ethanol N -. { 2- [2- (4-bromo-phenyl) -vinyl] -7-chloro-quinazolin-4-yl} -N 1 ', MN1 -diethyl-pentane-1,4-diamine N-benzo [g] quinolin-5-iI-N'-cyclohexyl-propane-1,3-diamine 2 - [(2-hydroxy-ethyl) -3-. { 2- [2- (4-methoxy-phenyl] -vinyl] -cynazolin-4-ylamino} -propyl) -amino] -ethanol.

The non-peptidic organic compounds of the present invention can be synthesized using conventional techniques. The compounds of the invention and which are used in the methods of the invention also include prodrugs of compounds that favor a wild-type activity of a protein of the p53 family. Prodrugs are compounds that, when administered to a mammalian subject (particularly a human being), are converted into the active molecule in significant and effective amounts. The compounds of the invention may be in the form of free acids, free bases or pharmaceutically effective salts thereof. These salts can be prepared easily by treating a compound with an appropriate acid. Such acids include, by way of example and not limitation, inorganic acids such as hydrohalic acids (hydrochloric, hydrobromic, etc.), sulfuric acid, nitric acid, phosphoric acid, etc .; and organic acids such as acetic acid, propanoic acid, 2-oxopropanoic acid, propanedioic acid, butanedioic acid, etc. Conversely, the salt can be converted to the free base form by alkali treatment.

B. Therapeutic endpoints and dosages The compounds identified by the methods of the invention are useful for the treatment of diseases associated with unstable or misfolded conformational proteins. Diseases associated with conformationally unstable or misfolded proteins are known to include cystic fibrosis (CFTR), Marfan syndrome (fibrillin), amyotrophic lateral sclerosis (superoxide dismutase), scurvy (collagen), maple syrup urine disease (alpha-ketoacid-dehydrogenase complex), osteogenesis imperfecta (pro-alpha pro-type I), Creutzfeld-Jakob disease (prion), Alzheimer's disease (beta-amyloid), familial amyloidosis (lysozyme), cataracts (crystalline), familial hypercholecterolemia (LDL receptor), α1-antitrypsin deficiency, Tay-Sachs disease (beta-hexosaminidase), retinitis pigmentosa (rhodopsin), and leprecaunismo (insulin receptor). Naturally, the methods and compounds described in the present specification are particularly useful in the treatment of cancers, and especially useful in the treatment of cancers associated with mutant p53 genes. One skilled in the art will appreciate that, from the point of view of a physician or a patient, virtually any relief or prevention of an undesired symptom associated with a morbid condition, and in particular a cancerous condition (eg, pain, tenderness) would be desirable. , weight loss, and the like). In addition, in relation to a cancerous state, any reduction in the mass or rate of tumor growth is desirable, as well as an improvement in the histopathological picture of the tumor. Therefore, for the purposes of this application, the terms "treatment", "therapeutic use" or "medicinal use", as used herein, will refer to any and all uses of the claimed compositions to remedy a morbid state or symptoms, or to prevent, prevent, delay or otherwise reverse the progression of the disease or other unwanted symptoms in any way. By conventional means, an effective dosage and treatment protocol can be determined, starting with a low dose in laboratory animals and then increasing the dosage while the effects are observed, and also varying the dosage regimen systematically. Animal studies, preferably studies with mammals, are commonly used to determine the maximum tolerable dose, or MTD, of bioactive agent by weight in kilograms. Those skilled in the art regularly extrapolate the doses in terms of efficacy and prevention of toxicity in other species, including humans. Before undertaking efficacy studies in humans, phase I clinical studies in normal subjects help to establish safe doses. A clinician can take many factors into account when determining an optimal dosage for a given subject. Among these, one of the main ones is the toxicity and half-life of the chosen heterologous gene product. Additional factors include the weight of the patient, the age of the patient, the general condition of the patient, the particular cancer disease being treated, the severity of the disease, the presence of other drugs in the patient, the in vivo activity of the product gene, and the like.

The test dosages should be chosen after considering the results of animal studies and clinical literature. As shown below by means of a real work embodiment, a dose of 200 mg / kg / day was highly effective in inhibiting and / or reversing tumor growth in an animal model of a human cancer. Based on this result, a typical human dose of Compound X compound for cancer treatment is 0.1 to 10 g / day, injected intravenously or directly into the tumor mass, or administered orally, depending on of the patient's condition. Naturally, for a compound with a different level of efficacy and / or toxicity, these values would be altered accordingly. In addition, dosages may be administered in two or more increments per day. The compounds for the use of the methods of the invention can also be formulated as an implantation device with delayed release, to achieve an extended and sustained administration. Examples of such sustained release formulations include biocompatible polymer compositions, such as poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., Polymers for Advanced Technologies 3: 279-292 (1992). An additional guide for selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (compilers), "Biodegradable Polymers as Drug Delivery Systems", volume 45 of "Drugs and the Pharmaceutical Sciences", M Dekker, New York, 1990, and US Pat. No. 5,573,528 to Aebischer et al. (granted November 12, 1996). In particular, when in vivo use is contemplated, the various biochemical components of the present invention are preferably of high purity, and are substantially free of potentially harmful contaminants (e.g., have at least the quality of National Formulary (NF), in generally at least analytical quality, and preferably at least pharmaceutical quality). To the extent that a given compound must be synthesized before use, such subsequent synthesis or purification should preferably result in a product that is substantially free of any potentially toxic agents that may have been used during the synthesis or purification. For use in the treatment of a cancerous condition in a subject, the present invention also provides in one of its aspects, a set or kit, in the form of a vial or ampoule filled in a sterile manner, containing a compound of which demonstrated the effectiveness in the methods of the invention. In one embodiment, the set contains a compound of the invention, for example Compound y, Compound X or Compound Z, as a ready-to-administer formulation, either in unit dose amounts or in multi-dose amounts, and in where the case incorporates a label that provides instructions on the use of its content for the treatment of cancer. Alternatively, and in accordance with another embodiment of the invention, the kit provides a sterile filled vial or ampoule containing said product.

C. Drug Discovery Methods Each or all of the steps of the discriminant evaluation for compounds that interact with a p53 family protein, and in particular a p53 DBD, and / or affect its wild-type activity , can be transferred to high capacity assays in search of candidate compounds. High capacity discriminant assays are well known in the art, and can be performed in any of several formats. Useful formats are, for example, ELISAs, proximity and scintillation technology, competitive fixation assays, and displacement fixation assays. Lab automation, which includes robotic technology, can greatly reduce the time needed to perform discriminant assays on a large number of compounds, and is commercially available, for example, from Tecan, Scitec, Rosys, Mitsubishi, CRS Robotics, Fanuk, and Beckman-Coulter Sagian, to name just a few companies. Once the candidate compounds have been identified (or concurrently with their identification), secondary discriminant assays can be performed to determine the cellular and / or in vivo effects of the compounds on the activity of a protein of the p53 family. 1. Proteins of the p53 family established as targets by the methods and compositions of the invention The p53 protein is ubiquitous in all eukaryotic organisms. Accordingly, the p53 proteins and p53 DBDs to be employed in the methods and compositions of the invention can proceed, or be derived, from any eukaryotic cell, which includes fungi (eg, Saccharomyces cerevisiae), insects ( for example Drosophila) and mammals (for example mice and / or humans), although human p53 proteins are preferred. Additional p53 homologs have been identified in mammals with related structure and function, notably p63 and p73; these proteins of the p53 family, and for example their respective DBDs, can also be used in the methods and compositions of the invention. In addition, proteins of the p53 family (as defined herein) but still to be discovered can also be used in the methods and compositions of the invention. As noted above, the p53 protein contains at least three different domains: a transcriptional activation domain located at the amino-terminal end; the central DBD; and an oligomerization domain at the carboxyl-terminal end. Furthermore, a negative regulatory domain appears at the carboxyl-terminal end of the protein. Most missense mutations of p53 associated with human cancers occur in the DBD. The methods and compounds of the invention are directed to stabilize the conformation of such mutations in the wrong sense. Particularly preferred targets are mutant p53s which contain one or more of the so-called "hot spots" for mutation at the positions of residues 175, 245, 248, 249, 273 and 282 (all positions of residues are indicated with respect to to the sequence of human p53, the position of the analogous residue in p53 proteins of other organisms can easily be determined by homology alignment with the human sequence). Other common mutations in p53 occur in 132, 135, 138, 141, 143, 146, 151, 152, 154, 157, 158, 159, 163, 173, 176, 179, 186, 194, 196, 213, 220, 237, 238, 241, 242, 258, 266, 272, 278, 280, 281, 285 and 286; these are also objects of the invention. In addition, the invention is illustrated below by way of working examples showing the conformational stabilization of the following mutant p53 proteins: 143A, 173A, 175S, 241 D, 249S and 273H. Cancers associated with missense mutations in the p53 proteins, particularly in the DBP of the p53 protein, include, but are not limited to, colorectal carcinoma, bladder carcinoma, hepatocellular carcinoma, ovarian carcinoma, lung carcinoma, breast carcinoma, squamous cell carcinoma of the head and neck, esophageal carcinoma, thyroid carcinoma, and neurogenic tumors such as astrocytoma, ganglioblastoma, and neuroblastoma. The above cancers, and others, are treatable by the methods and compounds of the invention. The DBD of p53 is located approximately in the remains of amino acids 100-300. It has been shown that a nucleus with residues 102 to 292, resistant to proteolysis, is sufficient for DNA binding, and the crystal structure of the DBD of p53 has been resolved for residues 94 to 312 (Cho et al., 1994, Science 265, 346; Friend, 1994, Science 265, 334). Accordingly, for use in the methods of the invention, the N-terminal end of the DBD of p53 may start from the remainder 50 to the remainder 110, and preferably start somewhere between the residues 94 and 102. The C-terminal end of the DBD of p53 may end up between the remainder 286 and the remainder 340, and preferably ends between residue 292 and residue 312. "Thermodynamically destabilized mutants of p53" are mutants that do not retain one or more of the functional properties of p53, such as DNA binding at physiological temperatures (ie, around at 37 ° C), but recover this or these functions at reduced temperatures. For example, all mutants that are commonly found retain the ability to fix DNA in vitro at low temperature (Friedlander et al., 1996, see above). 2. Trial formats to. Fixation Assay Formats The principle of the assays employed to identify compounds that are simply fixed to the DBP of p53 involves preparing a reaction mixture of the DBD protein of p53 and the test compound, under conditions, and for a time, sufficient to allow the two components to interact and bind, thus forming a complex that can be extracted and / or detected in the reaction mixture. The DBD species used may vary depending on the objective of the discriminant assay. For example, when looking for compounds that interfere with a particular binding domain, the full length protein of the p53 family containing the binding domain, the DBD itself, or a fusion protein containing DBP of p53 can be used. fused to a protein or polypeptide that provides advantages in the assay system (eg, in labeling, in the isolation of the resulting complex, etc.). The DBD-derived peptides for use in this technique must comprise at least 6 consecutive amino acids, preferably 10 consecutive amino acids, more preferably 20 consecutive amino acids, and even more preferably 30 or even 50 consecutive amino acids, or more, of the DBD. Discriminant tests can be carried out in different ways. For example, a method for carrying out such a test would involve anchoring the protein, polypeptide, peptide or p53 DBD fusion protein, or the test substance, on a solid phase, and detecting the DBD / test compound complexes , anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the DBP reactant of p53 can be anchored on a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly. Any of a variety of suitable labeling systems can be employed, including, but not limited to, radioisotopes such as 125 I and 32 P, enzyme labeling systems that generate a detectable colorimetric signal or light when exposed to a substrate. , and fluorescent brands. In another embodiment of the invention, a DBD protein anchored to the solid phase is complexed with labeled antibody. A test compound could then be tested for its ability to break the association of the DBD / antibody complex. In practice, microtiter plates can be conveniently used as a solid phase. The anchored component can be immobilized by non-covalent bonds or by covalent bonds. Non-covalent binding can be achieved by simply coating the solid surface with a solution of the protein, and drying it. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and preserved. To carry out the test, the non-immobilized component is added to the coated surface containing the anchored component. Once the reaction has been completed, the unreacted components are removed (for example by washing) under conditions such that any complexes formed remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be achieved in various ways. When the non-immobilized component has previously been marked, the detection of the immobilized mark on the surface indicates that complexes have formed. When the previously immobilized component has not been previously marked, an indirect mark can be used to detect complexes anchored on the surface; for example by using an antibody specific for the component not previously immobilized (in turn, the antibody can be labeled directly or indirectly with a labeled anti-Ig antibody). In other modalities, the fixation can be detected without making use of a direct or indirect mark. For example, a biophysical property that varies when fixation occurs can be determined. A particularly advantageous solid support system for this discriminant assay is the BIAcore 2000 ™ system, commercially available from BIAcore, Inc. (Piscataway, NJ, USA). The BIAcore ™ instrument (http://www.biacore.com) uses the optical phenomenon of surface plasmon resonance (SPR) to observe biospecific interactions in real time. The SPR effect is essentially an evanescent electric field that is affected by local changes in the refractive index in a metal-liquid interface. The sensor chip is made with a sandwich of gold film between glass and a matrix of carboxy methyldextran to which the ligand or protein to be tested is chemically bound. This sensor pellet is mounted in a fluidic cartridge that forms flow cells through which the analyte compounds can be injected. The iigand-analyte interactions on the sensor chip are detected as changes in the angle of a polarized beam of light reflected on the tablet surface. Fixation of any mass to the pellet affects the SPR in the gold / dextran layer. This change in the electric field in the gold layer interacts with the beam of reflected light and alters the angle of reflection proportionally to the amount of mass fixed. The reflected light is detected in a diode array and translated into a fixation signal that is expressed as response units (RU). Since the response is directly proportional to the fixed mass, the kinetic and equilibrium constants for protein-protein interactions can be measured. Alternatively, a reaction in a liquid phase can be carried out, the reaction products can be separated from the unreacted components, and the complexes detected. b. Methods for measuring the conformation of a protein of the p53 family The conformation of the p53 protein can be measured in different ways. For example, antibodies can be used to probe the conformation of the pDD of p53. Preferred methods of the invention employ monoclonal antibodies which are specific for p53 conformations of p53 and / or DBD of active (for example, DNA binding) or inactive (thermodynamically destabilized, misfolded, or unfolded) conformations. For example, mAb1620 recognizes an epitope on DBD of p53 that is closely associated with the tumor suppressor activity of the p53 protein. Ball et al., 1984, EMBO J. 3: 1485-1491; Gamble et al., 1998, Virology 162: 452-458. Thus, mAb1620 will not set a DBD of p53 when it adopts an inactive conformation. Conversely, the epitope recognized by mAb240 is exposed when p53 is inactivated by mutation or wild-type p53 is denatured (Bartek et al., 1990, Oncogene 5, 893-899, Stephen et al., 1992, J. Mol. 225, 577-83). Other monoclonal antibodies, known or still to be discovered, which are specific for shaping can also be used in the methods of the invention. Such antibodies are useful because they can be easily adapted to high capacity discriminant assays. Methods for preparing antibodies, including monoclonal antibodies, are well known in the art. Other methods for measuring the conformation of a protein of the p53 family such as p53 or a DBP of p53 include, but are not limited to, dye uptake, spectroscopic methods (e.g., circular dichroism, NMR), size exclusion chromatography, ultracentrifugation, specific DNA binding (for example at physiological temperatures as opposed to low temperatures), and protein specific binding (for example, SV40 large T antigen is only fixed to the active conformation of type wild, and not inactive conformation). As noted above, many of the p53 mutations commonly found can not bind DNA at physiological temperatures, but they fix DNA at reduced temperatures. Therefore, one aspect of the measurement of the conformation of the protein of the p53 family in the presence of the test compounds is the temperature dependence. Preferably, the shaping is measured at physiological temperatures (around 38 ° C), an appropriate range is between 20 and 50 ° C, and more preferably between 35 and 42 ° C. The conformation of the target protein can also be measured over time, from a few minutes to several hours or more. When a wild type p53 or wild type p53 DBD protein is used in the discriminant assays, the heating is generally carried out for a longer time and at higher temperatures than when a mutant p53 DBD is employed. One skilled in the art can easily determine the appropriate temperature using the information provided herein. In addition, both the binding of a compound and any change in the conformation of a protein of the p53 family can be determined simultaneously. In this assay, a change in the conformation of a protein of the p53 family in the presence of a test compound is scored as a hit. As non-limiting examples, high-capacity discriminant assays evaluating compounds that interact with DBP of p53 to cause a conformational change are illustrated below. These high capacity discriminant assays have been able to identify a class of compounds for use in the methods of the invention. At temperatures close to the physiological, these compounds enhance the stability of the epitope for mAb1620, sensitive to conformation, in wild-type p53 proteins and in various mutants. Low micromolar concentrations of compound have transiently intensified the conformational stability of the epitope within living cells and have allowed p53 mutant to activate transcription. As described in more detail later, a prototype compound has modulated the conformation and function of p53 when administered to mice harboring tumors with mutant p53, and significantly inhibited the growth of human tumor xenografts with naturally mutated p53. c. Animal-based cell-based assays Once candidate compounds have been identified using the above-described primary discriminant assay (s), cell-based assays and animal-based assays are generally carried out to determine the effect of the candidate compounds in these systems. Initial assays may involve cell lines derived from tumors that possess a mutant gene encoding a p53 family protein, or cell lines engineered to express a mutant protein of the p53 family. The effect of the candidate compounds on any one (or all) of the wild type activities of p53 is evaluated. For example, the induction of WAF1 in the presence of the candidate compound indicates that the compound retains the function in the mutant p53 by favoring the specific properties of DNA binding rather than the indiscriminate binding properties. Any gene regulated upstream or downstream by p53, or other members of the p53 family, can be examined. Other activities of p53 include growth suppression and apoptosis. Growth suppression is readily determined in cells from tissue cultures by microscopy or by means of a colony formation assay. Apoptosis can be visualized by TUNNEL staining or staining with propium iodide and flow cytometry. In addition, animal-based models can be used to perform discriminant assays for both the toxicity and the efficacy of candidate compounds. For example, tumors possessing mutant p53 can be induced in an animal model and candidate compounds administered to the animal. The toxicity and growth or regression of the tumor are evaluated. A working example of one of these discriminant tests is given below. 3. Sources of compounds for discriminant assays Compounds that can be subjected to discriminant assays according to the invention include, but are not limited to, small organic molecules that are capable of entering a cell and affecting the activity of a protein from the family of p53. Several libraries of compounds are commercially available from companies such as Pharmacopoeia, Arqule, Enzymed, Sigma, Aldrich, Maybridge, Trega and PanLabs, to name but a few sources. Discriminant assays can also be performed on libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, in search of compounds that interact with the DBP of p53. However, the preferred compounds are not proteins or peptides (ie, a chain of three or more amino acids joined by peptide bonds). Antibodies are peptides that are immunoglobulins or fragments of an immunoglobulin that bind to an antigen; therefore, the preferred compounds are also not antibodies. Specific classes and examples of compounds for use in the methods of the invention are described below. Once a compound favoring a wild-type activity of a protein of the p53 family has been identified, molecular modeling techniques can be used to design variants of the compounds that are most effective. Examples of molecular modeling systems are the CHARM programs (Polygen Corporation, Waltham, Ma, USA) and QUANTA (Molecular Simulations Inc., San Diego, CA, USA). CHARM performs the functions of energy minimization and molecular dynamics. QUANTA performs the interactive construction, modification, visualization, and analysis of the behavior of the molecules among themselves. For example, once a compound favoring a wild-type activity of a protein of the p53 family has been identified, the compound can be used to generate a hypothesis. As will be further detailed below, a preferred hypothesis is that of a flat polycyclic hydrophobic group separated by about 5 (five) to 9 (nine) Angstroms, and more preferably 6 (six) to 8 (eight) Angstroms, of a polar amine. This hypothesis can be generated from any one of the preferred compounds of the present invention using the Catalyst program (Molecular Simulations Inc., San Diego, CA, USA). In addition, Catalyst can use the hypothesis to search the proprietary database, the small molecule database of Cambridge (Cambridge, England), as well as other databases mentioned above, to identify additional examples of the compounds of this invention. The compounds of the present invention can also be used to design more efficient variants using modeling packages such as Ludi, Insight II; C2-Minimizer and Affinity (Molecular Simulations Inc., San Diego, CA, USA). A particularly preferred modeling package is MacroModel (Columbia University, NY, NY, USA). The compounds of the present invention can also be used as the basis for developing a rational combinatorial library. A similar library can be explored for more effective compounds. Although the nature of the combinatorial library depends on factors such as the particular compound selected from among the preferred compounds of the present invention to form the base of the library, and the desire to synthesize the library using a resin, it will be recognized that the compounds of The present invention provides the required data suitable for combinatorial design programs such as C2-QSAR (Molecular Simulations Inc., San Diego, CA, USA). Having described the invention, the following examples are offered by way of illustration, and not as limitation.

Saw. EXAMPLE 1 DBD Thermostabilization Test of p53 A high capacity assay using DBD of wild-type p53 was developed. Pharmacological compounds were tested discriminant using the assay, and compounds that stabilized the active conformation of DBD were scored as successes.

A. Materials and Methods Thermostabilization test. Recombinant DBD (residues 94-312) was prepared in the manner described from wild-type and mutant p53 proteins, and DBD from p53-labeled FLAG (Pavletich et al., 1993, Genes and Dev. 7, 2556-2564; Bullock and others, 1997, see above). The mutant proteins used were 143A, 173A, 175S, 249S, and 273H. Various organic compounds with small molecules were tested. The products were dissolved in DMSO to prepare stock solutions, at a concentration of 10 mg / ml, and diluted before use. The proteins (0.25-1.0 ng / well) were diluted in a buffer containing 23 mM HEPES, pH 6.8, 150 mM KCl, 10 mM dithiothreitol, and were bound, in a volume of 50 μl, to ReactiBind microtiter plates (Pierce ) for 35 minutes on ice. The wells were rinsed with 25 mM HEPES, pH 6.8, 150 mM KCl, the diluted DMSO compound or vehicle was added, and the plates were incubated at the indicated temperatures. The incubation was stopped by placing the plates on ice; ELISA assays were performed while maintaining the plates on ice, to avoid additional alterations of the epitopes. The wells were blocked for 1 hour with 5 percent skim milk (Difco), and cold, in HEPES / KCl buffer, before the addition of the primary antibodies. 1: 100-1: 250 monoclonal antibodies mAb1620, mAb240 (Calbiochem) and anti-FLAG M2 (Eastman Kodak Company) were diluted in HEPES / KCl, and added at 100 μl / well for 30 minutes. The plates were rinsed twice with cold HEPES / KCl buffer, and incubated with horseradish peroxidase conjugated anti-mouse IgG (HRP) (Boehringer Mannheim) for another 30 minutes. The HRP signal was revealed using TMB developer (Pierce), and the optical density of the signal was read on a BioRad microplate reader set at 450 nm.

B. Results The conformation of DBD of p53 is thermolabile. The epitope recognized by mAb1620 is conformation dependent, and its presence on p53 is closely associated with the tumor suppressor activity of the protein (Ball et al., 1984, see above, Gamble and Milner, 1988, see above). Conversely, the epitope recognized by mAb240 is a linear epitope that is exposed when p53 is inactivated by mutation or when wild-type p53 is denatured (Bartek et al., 1990, Oncogene 5, 893-899, Stephen and Lane, 1992). , J. Mol. Biol. 225, 577-583). The DBD of recombinant human p53 (residues 94-312) underwent a transition in vitro from the active to the inactive conformation, gradually losing the epitope of 1620 while accumulating the epitope of 240. It was heated to temperatures close to the physiological DBD of p53 purified protein that had been immobilized on microtiter plates, and probed with mAb1620 in an ELISA format. The epitope of 1620 was lost in a manner dependent on temperature and time (Figure 1A). The loss of the 1620 epitope was specifically related to the loss of conformation, since a FLAG epitope that was bound to the DBD remained completely stable (Figure 1B). Furthermore, loss of the epitope of 1620 occurred in concert with the intensified appearance of the epitope 240, ensuring that the epitope loss of 1620 reflected a conformational change in the DBP of p53, and not a loss of the immobilized protein. The epitope half-life of 1620 in DBD of wild-type p53 was approximately 35 minutes at 23 ° C, and progressively decreased at higher temperatures until it was less than 5 minutes at 45 ° C (Figure 1A). In parallel, the DBD DNA binding capacity of p53 in gel displacement assays was reduced by being heated in solution (data not shown). The epitope half-life of 1620 in DBD of wild-type p53 was approximately double that of mutant DBD in position 143 at 37 ° C (Figure 1C). This finding is consistent with previous reports of reduced thermodynamic stability for several other mutant p53 proteins, and states that the epitope of 1620 can be used to observe the DBD conformation of p53 (Bullock et al., 1997, see above). The compounds stabilize the conformation of p53. The ELISA assay was used to identify compounds that stabilized the conformation of active p53 and allowed mutant proteins to retain better wild-type functions. Several compounds suppressed loss of epitope for mAb1620 at physiological temperature (for examples see Figure 2A). The relative potency of the compounds was established in titration experiments by determining the concentration required to stabilize 50% of the epitope for mAb1620. The active compounds stabilized the epitope in a dose-dependent manner (Figure 2B). The DMSO solvent and various analogs of the active compounds did not result in stabilization (Figure 2B, see Tables 1 and 2). It was also stabilized by the full-length wild-type p53 compounds, as well as DBD from various mutant p53 proteins (data not shown, figure 2C). In the presence of compound, the mutant proteins were as stable as the wild-type protein in the absence of compound.

Although the compounds preserved the epitope for mAb1620, they did not rescue p53 that had already lost the epitope. For example, there was no increase in the reactivity of mAb1620 when DGD of p53 was heated before the addition of Compound Y. Although the rate of epitope loss was reduced when the compound was present, prolonged heating resulted in eventual loss. of the positive conformation for 1620. In addition, it did not appear that the compound had irreversibly bound to p53, since the addition and washing of Compound Y before incubation at 37 ° C did not prevent the loss of epitope (data not shown). These findings are consistent with a model in which the DBD interaction of p53 with compound allows the protein to retain more stable functional conformation, as recognized by mAb1620. Structure of the active compounds. All the active compounds identified bind a hydrophobic group (flat polycyclic) and a cationic group (often an amine) through a linker with a specific length. Benzimidazole, benzoquinoline, phenothiazine, and styrylquinazoline in the hydrophobic position (R1) were active, while subtle changes in these groups, and bicyclic or monocyclic simple groups were not active in the particular conditions tested (Table 1). The "active" compounds in this trial were named if there was a difference greater than a factor of 10 between the two pairs compared (see Table 1) in the amount of compound needed to stabilize 50% of the epitope for mAb1620. Thus, it should be noted that the so-called inactive compounds according to this test were not absolutely inactive, only relatively inactive. Accordingly, the active cationic groups (R2) included dimethiamine, diethylamine, diethanolamine, methylamine, methylpiperazine, and morphoiin (Table 1). Certain larger amines were correspondingly more active when tested in the phenothiazine series. The negatively charged or uncharged groups such as carboxyl or benzene in the R2 position were inactive (Table 1). Also the spacing between the groups R1 and R2 critical for the activity of the compound, since the linkers shorter than a propyl suppressed the activity of the compound (Table 2). The butyl linkers were slightly less potent than the propyl linkers, while longer linkers abolished the activity of the compound (Table 2 and data not shown). Branched linkers that retained the correct distance were generally more active than the corresponding linear linkers. These general observations do not limit the scope of the invention, but may be employed in the practice of the invention to design additional molecules.

TABLE 1 Dependence of the activity according to the structural characteristics of the compounds R2 LINKER R1 * Active and inactive denote a difference > 10 times in the potency of the pairs of compounds confronted. The relative potency was determined by means of the amount of compound required to stabilize 50% epitope for mAb1620 in titration experiments.

TABLE 2 Dependence of the activity as a function of the spacing between the groups R1 and R2 COMPOSITE SC50 (Um) COMPOSITE SC50 (Um) * The concentration of compound required to maintain 50% epitope for mAb1620 in 0.5 ng DBP of p53 by heating at 45 ° C for 30 minutes.

C. Discussion The results demonstrate the proof of principle for a new strategy of restoring the mutant p53 function and the development of anticancer therapies. This example describes the discovery of the first family of compounds capable of acting on isolated DBD to favor its conformational stability.

VII: EXAMPLE 2 Determination of the conformation of p53 in cells and tumors In this example and in the examples that follow, it is shown that prototype compounds function at low mutant p53 micromolar concentrations in living cells and tumors, and suppressing tumor growth with naturally mutated p53.

A. Materials and methods Cell culture. All cell lines were obtained from the ATCC, and were cultured in the recommended media, with 10 percent bovine fetal leather (Gibco BRL). Determination of the conformation of p53. Approximately 1 x 10 7 H1299 cells / reporter + mutant p53 were treated overnight, washed three times with Tris-buffered saline, and cold, and used in 1.5 ml of hypotonic lysis buffer (20 mM HEPES, pH 7.4 , 10 mM NaCl, 20 percent glycerol, 0.2 mM EDTA, 0.1 percent Triton-X 100, 10 mM dithiothreitol, with protease inhibitors). The cells were pelleted in microcentrifuge tubes at 2,000 r.p.m. for 5 minutes at 4 ° C, and nuclear extracts were prepared by resuspending the peliets in the same buffer with 0.5 M NaCl. Tumor samples were homogenized in a Dounce homogenizer, using three volumes of the above buffer with 0.5 M NaCl. The lysates were clarified by centrifugation at 10,000 rpm for 10 minutes at 4 ° C. The nuclear extracts were normalized in terms of p53 content according to the quantification derived from Western stains with mAbDO-1 antibody, and p53 was captured on wells of MaxiSorp F96 plates (Nunc) that had been coated overnight at 4 ° C with mAbDO -1 at a concentration of 1 μg / ml in 0.05 M carbonate buffer, pH 9.6. The wells were washed with cold phosphate buffered saline (PBS), blocked for 3 hours at 4 ° C using 4% skimmed milk in PBS, and probed by using mAb1620 antibody conjugated with HRP, in skimmed milk . Incubation with antibody was carried out for one hour on ice, after which the wells were washed three times with PBS containing 0.05 percent Tween 20, and the TMB substrate was used to develop the signal. A standard curve was established using the lysate of H1299 / reporter + p53 mutant temperature changers (32 ° C), which expressed large amounts of p53 positive for 1620. The quantification of the samples was within the linear range of the standard curve , and was corrected with respect to the total p53 in each sample, as well as with respect to the p53 fraction positive for 1620 in the lysates that had not been treated.

B. Results Stabilization of the conformation in the cells. The ability of the compounds to stabilize the positive conformation for 1620 of cellular p53 was assayed, using live cells expressing exclusively mutant p53. H1299 cells, which were null with respect to p53, were transfected with a mutant p53 derived from tumor (position 173), and an antibody for p53 (mAbDO-1) not sensitive to conformation was used in Western stains to select a clone that expressed abundant amounts of the mutant protein. In the extracts from the transfectant, low stationary levels of p53 were detected that presented the epitope for mAb1620, confirming that a small fraction of mutant p53 can retain the active conformation (Chen et al., 1993, Oncogene 8, 2159-2166). Low micromolar concentrations of Compound X increased in the cells approximately 5 times the steady-state fraction of positive versus 1620 (Figure 3A). The maximum levels of epitope enrichment were reached at 4 or 6 hours after treatment. The total amount of p53 had not changed, as measured by reactivity with mAbDO-1, which is directed against an epitope not sensitive to conformation, located at the amino-terminal end of the protein.

C. Discussion The results show that the stabilizing compounds of the conformation, identified by the methods of the invention, can stabilize the active conformation of p53 in living cells. Compounds that restore mutant p53 in tumors may be targeted either to the total set of nonfunctional p53, or to the subset of p53 that presents the epitope for mAb1620. The key objective for the compounds described herein appears to be freshly synthesized p53 mutant which still retains the active conformation. Actually, the compounds intensified epitope persistence for 1620, but were unable to restore the 1260 epitope that had been lost due to previous in vitro heating. Compounds that enhanced the stability of the newly synthesized active p53 conformation would allow the accumulation of steady-state levels of functional p53 in a time-dependent manner. The four-hour delay observed in reaching the maximum epitope intensification for 1620 in the cells is consistent with this hypothesis (Figure 3A).

VIII; EXAMPLE 3 Restoring the p53 function A. Materials and methods Transactivation assays. Cells were transfected with expression plasmids encoding mutant p53 proteins (173A, 249S) and a selectable marker by neomycin, using cationic liquid-based transfection reagent DOTAP (Boehringer Mannheim) or calcium phosphate. The cells were also transfected with a plasmid encoding the hygromycin resistance marker and a p53 reporter gene composed of four copies of a p53 binding sequence corresponding to a p53-binding sequence in the promoter region of the thymidine gene. -Kinase of Herpes Simplex virus (base numbers 26 to 58 of the entry number in GenBank S57428 thymidine-kinase, which starts with the sequence GCCTTGCCT and ends with the sequence TGCCTTTTC) located upstream of the SV40 basal promoter carrying the gene of luciferase. A confronted cell pair was prepared by transfecting a clone of carrier cells of the reporter construct with an additional construct for the expression of mutant p53. The transfected clones were selected for growth in media containing hygromycin or G418, as appropriate. They were treated with compound monolayers of cells in 96-well tissue culture plates (Costar), and the luciferase activity was determined using a substrate conversion assay (Promega) and quantified with a luminometer for Dynatech micro-pools. Expression of WAF1 and p533. Cultured cells were treated for 21 hours, rinsed 3 times with cold Tris-buffered saline, scraped and pelleted at 10,000 rpm for 30 seconds before being resuspended in 50 mM HEPES, pH 7.5, 0.1 percent NP-40 , 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM DTT, 50 μg / ml aprotinin, 1 mg / ml Pefabloc (Boehringer Mannheim). Protein concentrations were determined using Bradford reagent (BioRad), and 5 or 10 μg of cell lysate was loaded on polyacrylamide / SDS gels with 8.16 percent gradient (Novex). The proteins were transferred onto Immobilon P membrane (Millipore) in Towbin's buffer (Towbin et al., 1979, Proc. Nat. Acad. Sci. USA 76, 4350) with 20 percent methanol. The membranes were bisected between the molecular weight markers of 32.5 and 47.5 KDa, and blocked for 1 hour at room temperature in SuperBIock (Pierce) plus 3 percent skimmed milk. The lower half of the stain was probed for WAF1 expression using the monoclonal antibody EA10 clone (Calbiochem WAF1 Ab-1), and the upper half of the stain was probed for total p53 expression using mAbDO-1 ( Calblochem p53 Ab-6), The stains were washed for one hour in three changes of Tris-buffered saline, containing 0.1 percent Tween 20, before the addition of the secondary antibody, anti-mouse IgG conjugated with HRP. Bands were visualized using Renaissance ECL (DuPont) and exposure to Hyperfilm ECL (Amersham Life Science).

B. Results Restoration of p53 function in cells. To determine whether stabilization of the p53 conformation could result in better retention of the wild-type functions, the authors of the present invention examined the transcription activity of p53 specific to the sequence. H1299 cells were transfected with a p53 transducible luciferase reporter gene, and a stable clone (H1299 / reporter) was transfected secondarily with mutant p53, in order to obtain a paired clone that expressed both the reporter gene and mutant p53 in the position 173 (H1299 / reporter + p53 mutant). The compounds enhanced the transcription activity of the mutant p53, as measured by the induction of the reporter gene (Figure 3B). Low levels of transcription activation were observed in H1299 / reporter cells, which may be due to the presence of a p53 homolog, p73 (data not shown). Although the authors of the present invention have not yet established whether these compounds can enhance the activity of p73, the large increase, dependent on p53, in the induction of reporter gene suggests that p53 is the primary target in these cells. The activation of the reporter gene, dependent on p53, occurred within a relatively small interval, since the efficacy of the compounds at high doses was limited by the detachment of the cells. The intensification of transcription activity peaked at 12-16 hours after treatment (data not shown). This observation is consistent with the expression of reporter gene occurring as a secondary event after stabilization of the functional conformation of p53, which occurred after 4-6 hours after treatment. Compound Y was superior to Compound X in the reporter induction assays. This can be attributed to a side effect of Compound Y, which involves damage to the DNA and leads to high levels of p53 proteins (Figure 3B). Compound Y, but not Compound X, intensified total p53 protein levels at concentrations required for cellular activity. To ensure that DNA damage is not solely responsible for the induction of p53 reporter gene by Compound Y, the authors of the present invention have tested the effects of the DNA damaging agent, adriamycin. Adriamycin did not induce the reporter gene within a wide range of concentrations (0.4 to 40 μg / ml), despite its ability to induce accumulation of mutant p53 in the cells (data not shown). These results demonstrate that conformational stabilization, but not the accumulation of mutant p53, may favor specific transcription activity. In particular, Compound X, which does not raise the steady-state levels of total p53 protein, seems to restore the transcription function of p53 only through the stabilization of the conformation.

Compound X over-regulated WAF1, a cellular gene product responsive to p53, in the presence of mutant p53. Saos-2 osteosarcoma cells, which did not express p53, were transfected with mutant p53 expression vectors and clones expressing any one of two mutants (at position 173 or position 249). The clones expressed lower basal levels of WAF1, compared to parental Saos-2 cells, possibly reflecting the selection of faster growth clones that the authors of the present invention had made. These cells were treated with Compound X for 16 hours, and analyzed in Western stains with respect to p53 and WAF1, lysates representing equal amounts of protein. The cells that had expressed one of the two p53 mutant proteins, but not the parental Saos-2 cells, had high levels of WAF1 expression after treatment (FIG. 4). The total amount of p53 protein in these lysates had not changed substantially. Adriamycin did not induce WAF-1 expression in Saos-2 cells with mutant p53, although high expression of WAF1 was given in U20S cells expressing wild-type p53 (data not shown).

C. Discussion The mode of action of the stabilizing agents of the conformation described herein is clearly different from that observed for traditional cytotoxic antineoplastic agents. Cytotoxic agents that are used in cancer chemotherapy are generally ineffective in cells with mutant p53 (Lowe et al., 1993, Nature 362, 847-849, O'Connor et al., 1977, Cancer Res. 57, 4285-4300). . In fact, the DNA-damaging agent, adriamycin, did not restore mutant p53 for transcription activity in the assays performed. Cytotoxic compounds are known to produce a pronounced induction of total p53 protein in normal and tumor cells. Compound X did not induce total p53 protein levels in cells or tumors. Since the induction of p53 is a sensitive measure of damage to cellular DNA, it is unlikely that Compound X could damage DNA at effective concentrations. Taken together, the findings of the authors of the present invention indicate that the stabilization of the positive conformation with respect to 1620, and the functional restoration of mutant p53 activity, can occur through a mechanism independent of DNA damage. Several lines of evidence allow to discard a non-specific effect on the stabilization of the protein. Gliceroi, a non-specific inhibitor of protein denaturation that works by displacing water and creating a more hydrophobic microenvironment around protein molecules, can restore the nuclear localization of a mutant mouse p53 in cells at a 600 mM concentration ( Brown et al., 1997, J. Clin. Invest. 99, 1432-1444). Compound X was active at a 0.03 mM concentration in this assay, which suggests a much more precise interaction involving specific contacts between the compound and p53 (data not shown). In addition, the observation that Compound X can affect the conformation of p53 in the presence of a large excess of other proteins in the culture and in vivo (see below) is consistent with a selective recognition of p53. Moreover, the nature of the interaction of the compound with p53 may not imply a tight binding to the structure of the native protein. A strong interaction with a small subset of the protein molecules that are in a transition state can serve to block any further deviation outside the active conformation, or facilitate reversion to the native conformation.

EXAMPLE 4 Tumor growth assay A. Materials and methods Tumor growth assay. Cultured cells were washed with PBS, and 1 x 10 6 cells A375.S2 or bin 5 x 10 6 DLD1 cells were inoculated in Matrigel (Becton Dickinson) at 90%, unilaterally, on the left side of NU / NU-mice. NuBR females, 20 grams (Charles River Laboratories). Compound X was administered intraperitoneally in a saline solution with Pluronic P-105 (BASF) at 0.1%. The diameter of the tumor was measured in two dimensions using the compass, and it was transformed into tumor volume (Euhus et al., 1986, J. Surg. Oncol., 31, 229-234).

B. Results Modulation of p53 in vivo. Compound X intensified the steady-state levels of the p53 fraction presented by the epitope for mAb1620 in tumors with mutated p53. The compound was administered intraperitoneally at a rate of 100 mg / kg to mice that were carriers of subcutaneous tumors derived from mutant H1299 / reporter-p53 cells injected. The animals were sacrificed after a single dose of the compound, and the tumor lysates were analyzed for the expression of total p53 and p53 positive for 1620. Total p53 levels had not changed, as measured in Western stains with mAbDO -1. The lysates were normalized for minor variations in the total p53 content. The epitope had increased in the space of 3 to 5 hours after treatment (Figure 5). The time course of the in vivo response was similar to that of the cultured cells (Figure 3A). To evaluate the functional restoration of mutant p53 in vivo, the authors of the present invention evaluated the expression of the luciferase reporter gene in tumors from treated and untreated animals. After 8 hours after dosing, a maximum induction of the reporter gene was observed 4.5 times (FIG. 5). The time lag between the conformational and functional responses may reflect the time required for the translation of the luciferase transcript and the accumulation of the protein. The maximum plasma concentration of compound in mice was approximately 10 μg / ml, which is below that which would be required for maximal induction of the reporter gene in the cells (data not shown). Therefore, lower levels of reporter gene induction in tumors, compared to cultured cells, may be due to suboptimal exposure.

C. Discussion The results show that compounds that stabilize the conformation can functionally restore a number of mutants chosen at random. Thus, the methods and compounds of the invention are broadly applicable to different mutants of p53. For example, the mutation at position 241 in DLD-1 cells, which affects a secondary DNA contact site, can be functionally supplemented through the stabilizing activity of Compound X. Thus, many of the mutants of p53, including some in the DNA contact sites can be restored after the stabilization of the active conformation. Compound X demonstrated therapeutic selectivity in vivo despite stabilizing in vivo the conformation of p53 both wild-type mutated mutant. In fact, the compound appeared safe, and no mortality was observed when the mice were administered at doses of 200 mg / kg / day (100 mg / kg b.i.d.) for 14 consecutive days (data not shown). The selectivity may be due to such low steady state levels of p53 in normal cells, compared to much higher levels present in tumor cells (Lassus et al., 1996, EMBO J., 15, 4566-4573). On the other hand, specific stresses for the tumor, such as DNA damage and deprivation of oxygen or nutrients, may preferentially favor tumor cells for the apoptotic effects of p53 (Chen et al., 1996, Genes and. Dev. 10, 2438- 2451). If so, it may be possible by combining p53 stabilizing compounds to achieve antitumor synergistic effects with radiation or genotoxic therapies. The above written description is sufficient to enable a person skilled in the art to practice the invention. Indeed, various modifications of the means described above for carrying out the invention, which are obvious to those skilled in the field of molecular biology, medicine, or related fields, are intended to be within the scope of the following claims.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for promoting a wild type activity in a mutant form of a human protein of the p53 family, in which one or more functional activities of said protein are at least partially diminished by the inability of said protein to maintain a conformation functional under physiological conditions, said method comprising the steps of: (a) contacting said mutant protein with a non-peptide organic compound that is capable of binding to one or more domains of said mutant protein under physiological conditions and stabilizing a functional conformation of the same, and (b) allow said stabilized protein to interact with one or more macromolecules participating in said wild-type activity. 2. The method according to claim 1, wherein said protein is selected from the group consisting of p53, p63 and p73. 3. The method according to claim 2, wherein said protein is p53. 4. The method according to claim 1, wherein said non-peptidic organic compound is selected from the group consisting of: where, for group I,

R5 is -N-R18-R19, wherein R18 is H, C? -C6 alkyl, or phenyl, and R19 is H, Ci-C? Alkyl, C3-C10 cycloalkyl, or phenyl wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p (CHR22) m- (CH2) n-NR20R21, or - (CH2) p (CHR22) m- (CH2) n -NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Ce alkyl, and R20 and R21 are each independently selected from: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocyclic alkyl, CQ-C10 aryl, C5-C9 heteroaryl, (C? -Ce alkyl) C6-C? 2 alkyl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C-Cß alkyl, Ci-Ce alkoxy, (C?-Ce) alkyl-C 3 -C 10 alkylheterocycle, or (C?-Ce) alkyl-Ce-Cι alkyl; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C, -C6 alkyl) -piperazine; R6 is (a) Ci-Cß alkyl or C2-C8 alkenyl, each being optionally substituted with one or more phenyl groups, or (b) phenyl substituted with halo, Ci-Cß alkoxy; and R7 and R8 are identical or different, and are selected from H, nitro, Ci-C alco alkoxy, or halogen selected from fluoro, chloro, and bromo; where, for group II,

R9 is C1-C6 alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted by hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21. - (CH2) P (CHR22) m- (CH2) n-NR20R21, or - (CH2) P (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or CrCß alkyl, and R20 and R21 are each independently selected from H, alkyl, C1-C12, C3-C2 cycloalkyl, C3-C10 heterocycloalkyl, Ce-Cryl aryl, heteroaryl C5-C9, (Ci-CβJ-aryl Ce-C ?2 alkyl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-C alco alkoxy, (C alquilo alkyl? -Ce) -C3-C10 heterocycloalkyl, (C? -Ce) -heteroaryl C5-C9 alkyl, or (C? -Ce) alkyl-Ce-Cylearyl ether, where, for group III,

R 10 is -N-R 18 R 19, wherein R 18 is H, C Ce alkyl, or phenyl, and R 19 is H, Ci-Ce alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) P- (CHR22) m- (CH2) n-NR20R21, or - (CH2) P- (CHR22) m- (CH2) n- NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R20 and R21 are each independently selected from: (a) H, alkyl C? -C? 2, C3-C? 2 cycloalkyl, C3-C10 heterocycloalkyl, C6-C? Aryl, C6-C10 aryl, C5-C9 heteroaryl, (C? -Ce) alkyl-C6-C12-alkyl, in wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, C-β alkoxy, (C?-Ce) alkyl -heterocyclic C 3 -C 10 alkyl, (C CC alquilo alkyl) -heteroaryl C C- C9, or

(Ci-CβJ-C6-C aralkyl alkyl or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C?-C6 alkyl) -piperazine; A and B are the same or different, and each represents carbon or nitrogen, and R11 and R12 are the same or different, and are selected from H, nitro, Ci-Ce alkoxy, or halogen selected from fluoro, chloro and bromo, wherein, for group IV,

R 13 is -N-R 18 R 19, wherein R 18 is H, C 1 -C 6 alkyl, or phenyl, and R 19 is H, C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or alkyl, and R20 and R21 are each independently selected from: (a) H, C1- alkyl C12, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, (C6-C6 alkyl) -C5-C9 heteroaryl, C5-C9 heteroaryl, Ce-Cryl aryl and (C? -Ce) alkyl-heteroteroium Ce-Cio, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Cß alkoxy, (C?-C6) alkyl, C3-C10 heterocycloalkyl, or alkyl (Ci-CβJ-aryl Ce-Cι) ! or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (CrCe alkyl) -piperazine; A and B are equal or different s, and each represents carbon or nitrogen; and R14 and R15 are the same or different, and are selected from H, nitro, Ci-Ce alkoxy, or halogen selected from fluoro, chloro and bromo; and where, for group V,

A is carbon or nitrogen; R16 is -N-R18R19, wherein R18 is H, CrC6 alkyl, or phenyl, and R19 is H, Ci-Cß alkyl, C3-C10 cycloalkyl. or phenyl wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - ( CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R20 and R21 are each independently selected from: (a) H, C? -C? 2 alkyl, C3-C? 2 cycloalkyl, C3-C10 heterocycloalkyl, Ce-C14 aryl, Cs-Cg heteroaryl, (C? -Ce alkyl) -arro Ce-Cio, and (C 1 -Ce) alkyl-C 5 -C 9 heteroaryl, or wherein said groups are optionally substituted with one more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Cβ alkoxy, (C C-Ce) alkyl-C 3 -C 10 -heterocycloalkyl, (C?-Ce) alkyl-C 5 -C 9 heteroaryl, or (C?-Ce) alkyl-C 6 -C 6 -aryl; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (C6-alkyl) -piperizine; and R 17 is selected from H, nitro, Ci-Cβ alkoxy, or halogen selected from fluoro, chloro and bromo. 5. The method according to claim 1, wherein said non-peptidic organic compound is fixed to the DNA binding domain, residues 94 to 312, of human p53 protein. 6. The method according to claim 5, wherein the DNA binding domain of said p53 protein comprises a missense mutation at an amino acid position selected from the group consisting of residues 143, 173, 175, 241 and 249 7. The method according to claim 1, wherein the operations (a) and (b) are carried out simultaneously.

8. The method according to claim 1, wherein the operations (a) and (b) are carried out sequentially.

9. - The use of a non-peptidic organic compound that is capable of binding to one or more domains of the mutant protein under physiological conditions and stabilizing a functional conformation thereof, for the manufacture of a medicament for treating a human subject in connection with a morbid condition associated with the possession of a mutant protein of the p53 family having decreased one or more wild-type activities, wherein said stabilized protein contained in said drug interacts with one or more macromolecules participating in the wild-type activity.

10. The use as claimed in claim 9, wherein said protein is selected from the group consisting of p53, p63, and p73.

11. The use as claimed in claim 10, wherein said protein is p53.

12. The use as claimed in claim 10, wherein said non-peptidic organic compound is attached to the DNA binding domain, residues 94 to 312 of human p53 protein.

13. The use as claimed in claim 12, wherein the DNA binding domain of said p53 protein comprises a missense mutation at an amino acid position selected from the group consisting of residues 143, 173, 175, 241 and 249.

14. The use as claimed in claim 9, wherein the interaction of said stabilized protein contained in said medicament with one or more macromolecules participating in said wild-type activity is brought to out simultaneously.

15. The use as claimed in claim 9, wherein the interaction of said stabilized protein contained in said medicament with one or more macromolecules participating in said wild type activity is carried out sequentially.

16. The use as claimed in claim 10, wherein said morbid condition is cancer.

17. The use of a non-peptidic organic compound that is capable of binding to one or more domains of a human protein of the p53 family under physiological conditions, and stabilizing a functional conformation thereof, for the manufacture of a medicament for treating cancer in a human being wherein said stabilized protein contained in said medicament interacts with one or more macromolecules participating in a wild-type activity of said protein.

18. The use as claimed in claim 17, wherein said protein is selected from the group consisting of p53, p63, and p73.

19. The use as claimed in claim 17, wherein said protein is p53.

20. The use as claimed in claim 17, wherein said non-peptidic organic compound is selected from the group consisting of: where, for group I, R5 is -N-R18R19, wherein R18 is H, C6 alkyl, or phenyl and R19 is H, Ci-Ce alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, - (CH2) n-NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R20 and R21 are selected each, independently, of: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, aryl Cedo, C5-C9 heteroaryl, (C? -Ce) alkyl-C6-Ci2 -aryl, in wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, Ci-Cß alkyl, Ci-Ce akoxy, (Ci-Cß alkyl) -3-C10 -heterocycloalkyl, or (CrC 6 alkyl) aryl Ce-Cι; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (aiquiloCrCe) -piperizine; R6 is (a) d-Cß alkyl or C2-Cd alkenyl, each being optionally substituted with one or more phenyl groups, or (b) phenyl substituted with halo, Ci-Cß alkoxy; and R7 and R8 are the same or different, and are selected from H, nitro, Ci-Ce alkoxy, or halogen selected from fluoro, chloro and bromo; where, for group II, R is Ci-Cß alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) P- (CHR22 ) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or Ci-Cß alkyl, and R 20 and R 21 are each independently selected from H, C 1 -C 12 alkyl, C 3 -C 12 cycloalkyl, C 3 -C 10 heterocycloalkyl, Ce-Cι aryl, C 5 -C 9 heteroaryl, (Ci alkyl -Cβ) -C6-C12 aryl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C-C-alkyl, C1-C6-akoxy, (Ci-Ce alkyl) -3-C10 -heterocycloalkyl, ( Ci-Cß alkyl) -heteroaryl C5-Cg, or (C-C alquilo alkyl) -aryl Ce-Cι alkyl; where, for group III, R10 is -N-R18R19, wherein R18 is H, d-C6 alkyl, or phenyl, and R19 is H, C1-C6 alkyl, C3-C10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, cycloheteroalkyl C3-C8, -CONR18 (CH2) PNR20R21. - (CH2) P- (CHR22) m- (CH2) nNR20R21, or -ÍCH2) P- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or C1-C6 alkyl, and R20 and R21 are each independently selected from: (a) H, C? -C? 2 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, aryl Ce-Cι, C 5 -C 9 heteroaryl, (C 1 -C 6 alkyl) -C 6 -C 12 alkyl, wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C 1 -C 6 alkoxy, (C C β alkyl) ) -C3-C10 heterocycloalkyl, (Ci-Ce alkyl) -heteroaryl C5-C9, or (C6-alkyl) -6-C-aryl or, or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (alkyl d-Cß) -piperizine; A and B are the same or different, and each represents carbon or nitrogen; and R11 and R12 are the same or different and are selected from H, nitro, d-C alco alkoxy, or halogen selected from fluoro, chloro, and bromo; where, for group IV, R 3 is -N-R 18 R 19, wherein R 18 is H, C 1 -C 6 alkyl, or phenyl, and R 19 is H, C 1 -C 7 alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group it is optionally substituted by hydroxy, cycloheteroalkyl C3-C8, -CONR18 (CH2) PNR20R21. - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n-NR20R21, where p is 0-5, m is 0-5 , n is 0-5, R22 is hydroxy or Ci-Ce alkyl, and R20 and R21 are each independently selected from: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, ( Ci-Cβ alkyl) -5C9 heteroaryl, C5-C9 heteroaryl, C6-C6 aryl, and (Ci-Cß alkyl) -C6-C10aryl, wherein said groups are optionally substituted with one or more hydroxy, halo , amino, trifluoromethyl, Ci-Ce alkyl, C 1 -C 6 alkoxy, alkyl (d-Cß) -heterocycle C 3 -C 10 alkyl, or (C 6 -C 6) alkyl-Ce-Cιaryl; or (b) NR ^ R21, taken together, represents hydrogen, morpholine, or 4- (Ci-Cß alkyl) -piperizine; A and B are the same or different, and each represents carbon or nitrogen; and R14 and R15 are the same or different, and are selected from H, nitro, alkoxy CI-CT, or halogen selected from fluoro, chloro, and bromo; and where, for group V, A is carbon or nitrogen; R 16 is -N-R 18 R 19, wherein R 18 is H, C 1 -C 6 alkyl, or phenyl, and R 19 is H, C 1 -C 7 alkyl, C 3 -C 10 cycloalkyl, or phenyl, wherein said alkyl, cycloalkyl or phenyl group is optionally substituted with hydroxy, C3-C8 cycloheteroalkyl, -CONR18 (CH2) PNR20R21, - (CH2) p- (CHR22) m- (CH2) n-NR20R21, or - (CH2) p- (CHR22) m- (CH2) n -NR20R21, wherein p is 0-5, m is 0-5, n is 0-5, R22 is hydroxy or C1-C6 alkyl, and R20 and R21 are each independently selected from: (a) H, C1-C12 alkyl, C3-C12 cycloalkyl, C3-C10 heterocycloalkyl, Ce-C14 aryl, C5-C9 heteroaryl, (Ci-Cß alkyl) -aryl Ce-Cι alaryl, and (C Cß) alkyl-C5-C9 heteroaryl, or or wherein said groups are optionally substituted with one or more hydroxy, halo, amino, trifluoromethyl, C1-C6 alkyl, C6-C6 alkoxy, (C6-C6 alkyl) -3-C10 -heterocycloalkyl, (dCS alkyl) -heteroaryl C5-C9, or (C-C-alkyl) -aryl Ce-Cyl; or (b) NR20R21, taken together, represents hydrogen, morpholine, or 4- (Ci-Ce alkyl) -piperizine; and R17 is selected from H, nitro, CrC6 alkoxy, or halogen selected from fluoro, chloro, and bromine.

21. The use as claimed in claim 17, wherein said non-peptidic organic compound is attached to the DNA binding domain, residues 94 to 312, of human p53 protein.

22. - The use as claimed in claim 17, in which the protein of the p53 family that constructs the target of said non-peptide organic compound is of the wild type.

23. The use as claimed in claim 17, wherein the protein of the p53 family that constitutes the target of said non-peptide organic compound is a mutant encoded by an allelic variant.

24. The method according to claim 1, wherein said non-peptidic organic compound is selected from the group consisting of: (1-Benzyl-piperidin-4-yl) - (3-phenothiazin-10-yl-propyl) -amine [2- (4-chloro-phenyl) -ethyl] - (3-phenothiazin-10-yl-propyl) -amine (3-phenothiazin-10-yl-propyl) -thiochroman-4-yl-amine [1-methyl] -3- (2,6,6-Trimethoxy-cyclohex-2-enyl) -al-1] - (3-phenothiazin-10-yl-propyl) -amine (7-ethoxy-1, 2,3,4-tetrahydro-naphthalen-2-yl) - (3-phenothiazin-10-yl-propyl) -amine N '- (9-fluoro-benzo [c] acridin -7-yl) -N, N-dimethyl-propane-1,3-diamine N'-acridin-9-yl-N, N-dimethyl-propane-1,3-diamine ' 2-. { 4- [4- (benzo [g] quinolin-4-ylamino) -phenyl] -1-piperazin-1-yl} -ethanol N -. { 2- [2- (4-bromo-phenyl) -vinyl] -7-chloro-quinazolin-4-yl} -N 1, MN 1 -diethyl-pentane-1, 4-diamine N-Benzo [g] quinolin-5-yl-N'-cyclohexyl-propane-1,3-diamine 2 - [(2-hydroxy-ethyl) - (3- {2- [2- (4-methoxy-phenyl) -vinyl] -quinalozin-4-ylamino} -propyl) -aminoj-ethanol The use as that claimed in claim 17, wherein said non-peptidic organic compound is selected from the group consisting of: (1-Benzyl-piperidin-4-yl) - (3-phenothiazin-10-ii-propyl) -amine [2- (4-chloro-phenyl) -ethyl-J- (3-phenothiazin-10-yl-propyl) - amine (3-Phenothiazin-10-yl-propyl) -thiochroman-4-yl-amine [1-methyl-3- (2,6,6-trimethyl-cyclohex-2-enyl) -allyl] - (3-phenothiazine- 10-yl-propyl) -amine (7-ethoxy-1, 2,3,4-tetrahydro-naphthalen-2-yl) - (3-phenothiazin-10-yl-propyl) -amine N '- (9-fluoro-benzo [c] acridin-7-yl) -N, N-dimethyl-propane-1,3-diamine N'-acridin-9-yl-N, N-dimethyl-propanol -1, 3-diamine 2-. { 4- [4- (Benzo [g] quinolin-4-ylamino) -phenyl] -1-piperazin-1-yl-) ethanol N4-. { 2- [2- (4-bromo-phenyl) -vinyl] -7-cyoro-quinazolin-4-yl} -N1, N1-diethyl-pentane-1,4-diamine N-benzo [g] quinolin-5-yl-N'-cyclohexyl-propane-1,3-diamine 2 - [(2-Hydroxy-ethyl) - (3- {2- [2- (4-methoxy-phenyl) -vinyl] -quinazolin-4-ylamino} -propyl) -aminoj- ethanol