MX2007008931A - Quinoxaline derivatives as antitumor agents - Google Patents

Abstract

The invention relates to methods of screening for binding partners, especially binding partners essential for the biological activity of erastin (e.g. VDACs such as VDAC3) . The invention also provides reagents and methods for effective killing of cancer cells with erastin and related compounds or derivatives, such like the compounds (1).

Description

ERASTINE AND PROTEINS THAT LINK THE ERASTINE, AND USES THEMSELVES FINANCING OF GOVERNMENT The work described herein is based, in general or in part, by the National Cancer Institute Grant (IRC) CA01-7061 -01. The government of the United States has certain rights in the invention. Background of the Invention Many drugs administered in the treatment of a disease are intended for general differences between an affected cell and a normal cell. For example, paclitaxel, which is used to treat ovarian and breast cancer and inhibits microtubule function, is thought to exhibit tumor cell specificity based on the increased rate of proliferation of tumor cells relative to normal cells (Miller et al. and Ojima, Chem. Rec. 1: 195-211, 2002). However, despite this consensus view, the in vitro activity of paclitaxel varies widely across cell lines (Weinstein et al., Science 275: 343-349, 1997), which indicates that genetic factors can modify the sensitivity of tumor cells with paclitaxel and that the receptivity of tumor cells is not simply determined by their rate of proliferation.

Molecularly targeted therapeutics represent a promising new approach to discovering the anti-cancer drug (Shawver et al., Cancer Cell 1: 117-23, 2002). Using this approach, small molecules are designated to directly inhibit highly oncogenic proteins that are mutated or overexpressed in specific tumor cell types. By directing specific molecular defects found within tumor cells, this approach can ultimately produce therapies adapted to each genetic elaboration of the tumor. Two recent examples of molecularly targeted or targeted anti-cancer therapeutic agents are Gleevec (imatinib mesylate), an inhibitor of the abelsen kinase oncoprotein-clustered region at the borderline (BCR-ABL) found in chronic positive myeloid leukemia-chromosome of Philadelphia (Capdeville et al., Nat Rev Drug Discov 1: 493-502, 2002) and Herceptin (trastuzumab), a monoclonal antibody directed against the HER2 / NEU oncoprotein found in metastatic breast cancers (Mokbel and Hassanally, Curr Med Res. Opin 17: 51-9, 2001). A complementary strategy involves scrutinizing for selective anti-tumor agents of the genotype that become lethal or tumor cells only in the presence of specific oncoproteins or in the absence of specific tumor suppressors. Such selective, genotype compounds could direct oncoproteins or could direct other critical proteins involved in signaling networks linked with oncoprotein. Compounds that have been reported to present synthetic lethality include (i) the rapamycin analogue CCI-779 in myeloma cells lacking PTEN (Shi et al., Cancer Res 62: 5027-34, 2002), (ii) Gleevec in cells transformed with BCR-ABL (Druker et al., Nat Med 2: 561-6, 1996) and (iii) a variety of fewer well-characterized compounds (Stockwell et al., Chem Biol 6: 71-83, 1999; Torrance et al., Nat Biotechnol 19: 940-5, 2001). Despite the search discussed above, there remains a significant need to develop and / or identify compounds that selectively target tumor cells. Brief Description of the Invention Using a synthetic lethal projection method, particularly a totally high synthetic lethal projection method, which is useful for identifying agents or drugs to treat or prevent conditions or diseases such as the presence or development of tumors or other characterized conditions by hyperproliferation of cells (e.g., leukemia), the Applicants have identified a variety of comp "these / agents / drugs useful for treating or preventing cancer (e.g., tumors or leukemia) in an individual, such as a human being who needs of the treatment or prevention The invention also provides cellular proteins that bind directly or indirectly certain compounds / agents identified as having therapeutic value. Such cellular proteins provide additional methods for treating diseases or conditions characterized by hyperproliferation of cells (e.g., leukemia). As used herein, the terms "agent" and "drug" are used interchangeably, they can be compounds or molecules. In one embodiment, the present invention is directed to a compound described herein, including salts thereof.

In another embodiment, the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound described herein. In yet another embodiment, the invention is a method of stimulating cell death, including administering to the cell an effective amount of a compound described herein. In one aspect, the present invention is a method for identifying a candidate anti-tumor agent, including the steps of: a) contacting a cell with a sufficient amount of a test agent under suitable conditions; and b) determining whether the test agent improves or inhibits the level of a protein that binds erastin or a nucleic acid that encodes a protein that binds erastin. The method may further include the steps of: a) contacting the test agent with a tumor cell (in vitro or in vivo); and b) determining whether the test agent inhibits the growth of the tumor cell. In another aspect, the present invention is a method for identifying a candidate anti-tumor agent, which includes: a) contacting a protein that binds erastin or a cell that expresses a protein that binds erastin to a test agent, wherein the protein that binds the erastin or the test agent is optionally labeled with a detectable label; and b) determining whether the test agent binds the protein that binds the erastin. The method may further include: a) contacting the test agent with a tumor cell (in vivo or in vitro); and b) determining whether the test agent inhibits the growth of the tumor cell. The two methods immediately described above can be repeated using a library of different test agents. In a further aspect, the present invention relates to projection methods for identifying compounds that kill or inhibit the growth of tumorigenic cells, such as tumorigenic cells genetically modified or genetically engineered, but not their normal cellular isogenic counterparts. The method has been used to identify known and novel compounds with selective genotype activity, including the known compounds doxorubicin, daunorubicin, mitoxantrone, canfothecin, sangivamycin, equinomycin, bouvardine, NSC146109 and a novel compound referred to herein as erastin. These compounds generally have increased activity in the presence of one or more of the following: hTERT oncoprotein, SV40 large oncoprotein SV (LT), small T oncoprotein (ST), human papillomavirus 16 (HPV) oncoprotein E6, E7 oncoprotein of HPV, and H RAS, N RAS and KRAS oncogenes. The applicants determined that overexpression of hTERT and either E7 or LT decreases the expression of topoisomerase 2a and that overexpression of RASV12 and ST in cells expressing hTERT both decrease the expression of topoisomerase 1 and sensitized cells to a process of non-apoptotic cell death initiated by erastin. The invention relates to a method for identifying agents (eg drugs) that are selectively toxic to (e.g., death or inhibition of growth of) tumorigenic cells, such as genetically modified tumorigenic cells, including tumorigenic cells human (eg, genetically modified human tumorigenic cells and / or tumor cells). In one embodiment, the invention relates to a method for identifying an agent (eg, drug) that selectively destroys or eliminates or inhibits the growth of genetically modified human tumorigenic cells (is toxic a), which comprises contacting test cells , which are genetically modified human tumorigenic cells, with a candidate agent; determining the viability of the test cells connected to the candidate agent; and compare the viability of the test cells with the feasibility of an appropriate control. In all modalities, viability is assessed by determining the ability of an agent (e.g., drug) to eliminate or inhibit the growth / proliferation of the cells, or both. If the viability of the test cells is less than that of the control cells, then an agent (eg, drug) is identified that is selectively toxic to (remove or inhibit the growth of) human tumorigenic cells genetically modified. An appropriate control is a cell that is the same type of cells as the test cells, except that the control cell is not genetically modified because it is tumorigenic. For example, the control cells may be the primary parental cells from which the test cells are derived. The control cells are connected to the candidate agent under the same conditions as the test cells. An appropriate control can be tested simultaneously, or it can be pre-established (for example, a standard or pre-established reference). In one embodiment, the method for identifying a selectively toxic agent to tumorigenic cells comprises further assessing the toxicity of an agent identified as a result of projection on human tumorigenic cells genetically modified in an appropriate animal model or on a cell-based system or assay. additional or based without cell. For example, an agent or drug thus identified can be assessed for its toxicity to cancer cells such as cells with tumor or cells with leukemia of individuals or their toxicity to one (one or more) cell lines with cancer (tumor). For example, the method may comprise further assessing the selective toxicity of an agent (e.g., drug) to tumorigenic cells in an appropriate mouse or non-human primate model. The invention further relates to a method for producing an agent (e.g., drug) that is identified by the method of the present invention such as an agent (e.g., drug) that is selectively toxic to genetically modified human tumorigenic cells. An agent (eg, drug) that is shown to be selectively toxic to tumorigenic cells is synthesized using known methods.

The invention further relates to a method for identifying agents (eg, drugs) that are toxic to genetically modified tumorigenic cells, such as genetically modified human tumorigenic cells. In one embodiment, the invention relates to a method for identifying an agent (e.g., drug) that eliminates or inhibits the growth of (is toxic to) human tumorigenic cells that are genetically modified, comprising contacting the test cells, the which are human tumorigenic cells genetically modified, with a candidate agent; determining the viability of the test cells contacted with the candidate agent; and compare the viability of the test cells with the feasibility of an appropriate control. If the viability of the test cells is less than that of the control cells, then an agent (eg, drug) that is toxic to (remove or inhibit the growth of) human tumorigenic cells is identified genetically. Here, an appropriate control is a cell that is the same type of cell (e.g., genetically modified human tumorigenic cells) as the test cells, except that the control cell is not connected to the candidate agent. An appropriate control can be run or tested simultaneously, or it can be pre-established (for example, a standard or pre-established reference). For example, an agent or drug thus identified can be assessed for its toxicity to cancer cells such as cells with tumor or cells with leukemia obtained from individuals or their toxicity to one (one or more) cell lines with cancer (tumor). In one embodiment, the method for identifying a toxic agent to genetically modified tumorigenic cells comprises further assessing the toxicity of an agent identified as a result of projection on human tumorigenic cells genetically modified in an appropriate animal model or in a system or assay based on additional cell or based without cell. For example, the method may further comprise assessing the toxicity of an agent (eg, drug) to tumorigenic cells in an appropriate mouse or non-human primate model. The invention further relates to a method for producing an agent (e.g. drug) that is identified by the method of the present invention, such as an agent (e.g., drug) that is toxic to human, genetically modified tumorigenic cells. An agent (e.g., drug) that is shown to be toxic to tumorigenic cells is synthesized using known methods. In another embodiment, the present invention is a method for reducing the growth rate of a tumor, which comprises administering an amount of a therapeutic agent, sufficient to reduce the growth rate of the tumor, in The therapeutic agent is: (a) an agent that increases or inhibits the level of a VDAC protein; (b) an agent that increases or inhibits the activity of a VDAC protein; (c) an agent that binds to a VDAC protein; (d) an agent that binds and / or modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (e) an agent comprising a VDAC polypeptide or functional variants thereof; or (f) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof. Suitable agents can have the aforementioned activity in the existing form or after complete or partial metabolism.

In one aspect, the invention is a method for treating a patient suffering or suffering from a cancer, comprising administering to the patient a therapeutic agent selected from: (a) an agent that increases or inhibits the level of a VDAC protein; (b) an agent that increases or inhibits the activity of a VDAC protein; (c) an agent that binds to a VDAC protein; (d) an agent that binds and / or modulates a complex of protein comprising at least one VDAC and optionally one or more other proteins; (e) an agent comprising a VDAC polypeptide or functional variants thereof; and (f) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof. Suitable agents can have the aforementioned activity in the existing form or after complete or partial metabolism. Agents suitable for use in reducing the growth rate of a tumor and in treating a patient suffering from cancer include, but are not limited to, small organic molecules, peptides, proteins, peptidomimetics, nucleic acids, antibodies and combinations of the same. Such agents are typically formulated with a pharmaceutically acceptable carrier, and can be administered intravenously, orally, buccally, parenterally, by a nebulizer by inhalation, by topical or transdermal application. An agent can also be administered by the local administration. An agent can additionally be administered in conjunction with at least one additional anti-cancer chemotherapeutic agent that inhibits the cancer cells in an additive or synergistic manner. In another aspect, the invention is a method for increasing the sensitivity of a tumor cell with an agent chemotherapy, where a tumor cell is contacted with a compound that increases or decreases the abundance of a protein that binds erastin. In a related aspect, the invention is a method for reducing the sensitivity of a normal cell with a chemotherapeutic agent, where a normal cell is contacted with a compound that decreases or increases the abundance of a protein that binds erastin. In certain embodiments of the invention, a candidate agent is identified by projecting a commented compound library, a combinatorial library, or another library comprising unknown or known compounds (e.g., agents, drugs) or both. In certain embodiments, the invention is a method for identifying a candidate therapeutic agent for inhibiting unwanted cell proliferation, including: a) mixing a test agent and a VDAC protein or a protein complex comprising at least one VDAC protein and optionally one or more other proteins; b) determining whether the test agent binds to the VDAC protein; and c) if the test agent binds to the VDAC protein, contact the test agent with a cell (in vivo or in vitro) and determine whether the Test alters the proliferation of the cell. Linking the VDAC protein with the test agent can be detected, for example, by a physical binding assay, such as an immunological binding assay, two-hybrid yeast assay, fluorescence polarization assay, surface plasmon resonance or assay fluorescence resonance energy transfer (FRET). In certain modalities, the invention relates to the erastin compound and a class of erastin-related compounds (e.g., the compounds of the present invention). In further embodiments, the invention relates to the compound, erastin B and its related compounds. In further embodiments, the invention relates to the compound, erastin A and its related compounds. In further embodiments of the invention, the invention relates to erastin analogs that selectively eliminate or inhibit the growth of (are toxic to) human tumorigenic cells that are genetically modified. Optionally, these compounds of the invention are formulated with a pharmaceutically acceptable carrier as pharmaceutical compositions. The invention also relates to methods for identifying cellular components involved in tumorigenesis. Cellular components include, for example, proteins (eg example, enzymes, receptors), nucleic acids (e.g., DNA, RNA), and lipids (e.g., phospholipids). In one embodiment, the invention relates to a method for identifying (one or more) cellular components involved in tumorigenesis wherein (a) a cell, such as a genetically modified human tumorigenic cell, is contacted with erastin; and (b) a cellular component that interacts with erastin is identified, either directly or indirectly. The cellular component that is identified is a cellular component involved in tumorigenesis. In a further embodiment, the invention relates to a method for identifying a (one or more) cellular components that interact with erastin wherein (a) a cell, such as a genetically modified human tumorigenic cell, a tissue, an organ, an organism or a Used or an extract of one of the above, is put in contact with the erastina; and (b) a cellular component that interacts with erastin is identified, either directly or indirectly. The cellular component that is identified is a cellular component that interacts with erastin; either directly or indirectly. The invention further relates to methods of treating or preventing cancer. In one embodiment, the invention relates to a method for treating or preventing cancer in which a therapeutically effective amount of a compound, such as, for example, erastin or its analog, or a compound of Formulas l-V below, is administered to an individual in need of cancer treatment. In certain embodiments, the cancer is characterized by cells in which the RAS sequence is activated. In certain additional embodiments, the cancer is characterized by cells expressing small SV40 oncoprotein T, or are phenotypically similar to cells expressing ST, and / or oncogenic HRAS. In certain preferred embodiments, the cells express substantially the wild-type level of Rb (eg, at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, or 150). %, etc.). The invention also relates to methods for identifying agents (eg drugs) that interact with one or more cellular components that interact, directly or indirectly, with erastin. In one embodiment, the invention relates to a method for identifying an agent that interacts with a cellular component that interacts with erastin, comprising (a) contacting a cell, a tissue, an organ, an organism, or a Used or an extract of one of the above with erastin; (b) identify a cellular component that interacts (directly or indirectly) with erastin; (c) contacting a cell, a tissue, an organ, an organism or a Used or an extract of one of the foregoing with a candidate agent, which is an agent or drug to be valued for its ability to interact with a component cell that interacts with erastin; and (d) determining whether the agent interacts (directly or indirectly) with the cellular component in (b). If the agent interacts with the cellular component in (b), it is an agent that interacts with a cellular component that interacts with the erastin. In a related aspect, the invention also relates to methods for identifying agents (e.g., drugs) that interact with one or more cellular components that are known to interact, directly or indirectly, with erastin, the method comprising: (a) contacting a cell, a tissue, an organ, an organism or a lysate or an extract of one of the foregoing with a candidate agent, which is an agent or drug that is valued for its ability to interact with the cellular component that is known to interact with the erastina; and (b) determining whether the agent interacts (directly or indirectly) with the cellular component in (a). If the agent interacts with the cellular component in (a), it is an agent that interacts with the cellular component that interacts with the erastin. In certain embodiments, the cell is a genetically modified human tumorigenic cell. In further embodiments, the invention relates to compounds that interact, directly or indirectly, with one (one or more) cellular components that interact with erastin. In certain modalities, the cellular component that interacts with Erastin is involved in tumorigenesis. An agent (e.g., drug) that is shown to interact with a cellular component that interacts with erastin is synthesized using known methods. The invention further relates to a method for identifying an agent (e.g., drug) that induces death in tumor cells, such as by an apoptotic or non-apoptotic mechanism. In one embodiment, a method for identifying an agent that induces death in tumor cells by a? The non-apoptotic mechanism comprises (a) contacting test cells, which are tumor cells (or an organ or tissue containing tumor cells) with a candidate agent that induces death in tumor cells; (b) assessing whether the agent in (a) induces apoptosis in test cells; and (c) comparing the induction of apoptosis in the cells in (b) with an appropriate control. If apoptosis is induced in the control cells but not in the test cells, then an agent (e.g., drug) is identified that induces death in tumor cells by a non-apoptotic mechanism. An appropriate control is a cell that is the same type of cells as that of the test cells except that the control cell is contacted with a known agent to induce apoptosis in the cell. An appropriate control can be tested or run simultaneously, or it can be pre-established (for example, a prerequisite standard or reference). established). In certain embodiments, the test cells are human tumorigenic cells that are genetically modified. As used herein, "a" and "an" refer to one or more of the matters or matters referred to. In certain aspects, the present invention provides methods for conducting a drug discovery business. In one embodiment, the invention relates to a method for conducting a drug discovery business, comprising (a) identifying an agent (eg, drug) that is selectively toxic to human tumorigenic, genetically modified cells; (b) assessing the efficacy and toxicity of an agent identified in (a), or analogs thereof, in animals; and (c) formulating a pharmaceutical preparation that includes one or more agents rated in (b). The assessed efficacy may be the ability of an agent to selectively induce cell death in tumorigenic cells in an animal. In a further embodiment, the method for conducting a drug discovery business comprises establishing a distribution system for distributing the pharmaceutical preparation for sale. Optionally, a sales group is established for marketing the pharmaceutical preparation. In a further embodiment, the invention relates to a method for conducting a proteomimetics business, which comprises identifying an agent (eg, drug) that is selectively toxic to human tumorigenic cells. genetically modified and confer, to a third party, the rights of development of the additional drug of agents that are selectively toxic to human tumorigenic cells genetically modified. In another embodiment, the invention relates to a method for conducting a drug discovery business, comprising: (a) identifying a (one or more) agent (e.g., drug) that is toxic to human, genetically modified tumorigenic cells; (b) assessing the efficacy and toxicity of an agent identified in (a), or analogs thereof, in animals; and (c) formulating a pharmaceutical preparation that includes one or more agents rated in (b). For example, the identified agent is erastin. The assessed efficacy may be the ability of an agent to selectively induce alterations in cell growth, toxicity or cell death in tumorigenic cells in an animal. In a further embodiment, the method for conducting a drug discovery business comprises establishing a distribution system for distributing the pharmaceutical preparation for sale. Optionally, a sales group is established for marketing the pharmaceutical preparation. In a further embodiment, the invention relates to a method for conducting a proteomimetics business, which comprises identifying an agent (e.g., drug) that is toxic to human tumorigenic, genetically modified cells and confer, to a third party, the development rights of the additional drug of agents that are selectively toxic to human tumorigenic cells modified genetically. In a further embodiment, the invention relates to a method for conducting a drug discovery business, comprising: (a) identifying a (one or more) agent (e.g., drug) that interacts with a cellular component that interacts with the erastina; (b) assessing the efficacy and toxicity of an agent identified in (a), or analogs thereof, in animals; and (c) formulating a pharmaceutical preparation that includes one or more agents rated in (b). The efficacy assessed may be its ability to selectively induce cell death in tumorigenic cells in an animal. In a further embodiment, the method for conducting a drug discovery business comprises establishing a distribution system for distributing the pharmaceutical preparation for sale. Optionally, a sales group is established for marketing the pharmaceutical preparation. In a further embodiment, the invention relates to a method for conducting a proteomics business, which comprises identifying an agent (e.g., drug) that interacts with a cellular component that interacts with the erastin and concessioning, to a third party, the Additional drug development rights of agents that interact with a cellular component that interacts with erastin.

In still another embodiment, the invention is a method for conducting a pharmaceutical business, which includes: (a) identifying a candidate therapeutic agent to inhibit cell proliferation, wherein the candidate therapeutic agent is: (i) an agent that increases or inhibits a level of a VDAC protein; (ii) an agent that increases or inhibits the activity of a VDAC protein; (iii) an agent that binds to a VDAC protein; (iv) an agent that binds and / or modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (v) an agent comprising a VDAC polypeptide or functional variants thereof; (vi) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof; or (vii) a compound described herein, (b) conducting a therapeutic profiling of the candidate therapeutic agent identified in step (a) for efficacy and toxicity in animals; and (c) formulating a pharmaceutical preparation that includes one or more of the candidate therapeutic agent identified in step (b) having a profile acceptable therapeutic Instead of or in addition to one or both of steps (b) and (c), the method may include concessioning to a third party the additional development rights of the candidate therapeutic agent. In a further embodiment, the method for conducting a drug discovery business comprises establishing a distribution system for distributing the pharmaceutical preparation for sale. Optionally, a sales group is established for marketing the pharmaceutical preparation. In a further embodiment, the invention is a method for conducting a pharmaceutical business that includes: (a) identifying a candidate therapeutic agent to inhibit cell proliferation, wherein the therapeutic agent (-anuidate is: (i) an agent that increases or inhibits a protein of VDAC; or (ii) an agent that increases or inhibits the interactions between a VDAC protein and a second protein, and (b) concessioning to a third party the additional development rights of the candidate therapeutic agent.

In a further embodiment, the method for conducting a drug discovery business involves establishing a distribution system to distribute the preparation. pharmaceutical for sale. Optionally, a sales group is established for marketing the pharmaceutical preparation. Another aspect of the invention is a method for conducting a pharmaceutical business that includes one or more of marketing, producing, concessioning to a third party the rights to market or concession to a third party the rights to produce a device, wherein the equipment comprises (a) one or more reagents to determine the levels of a protein that binds the erastin, the activity of a protein that binds the erastin, or both in a biological sample; and (b) instructions for interpreting the test results. In general, the instructions indicate whether the levels and / or activity of the protein that binds erastin are normal, increased or decreased relative to their desired level and / or activity, such that one can determine whether the level and / or or activity should be altered or predicted if a therapy (partially) dependent on the level and / or activity (for example, cancer chemotherapy) would be successful. In certain embodiments, the instructions include guidance with respect to one or more of the normal, decreased or elevated levels or activity of a protein that binds the erastin. In certain modalities, the instructions include a guide with respect to subsequent treatment with one or more (i) an agent that increases or inhibits the level of a VDAC protein; (ii) an agent that increases or inhibits the activity of a VDAC protein; (iii) an agent that binds to a VDAC protein; (iv) an agent that binds, modulates, or binds and modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (v) an agent comprising a VDAC polypeptide or functional variant thereof; (vi) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof; and (vii) a compound described herein, based on the level of a protein that binds erastin, the activity of a protein that binds erastin, or both. In certain embodiments, the instructions include a guide considering whether treatment with one or more of the following was successful: (i) an agent that increases or inhibits a level of a VDAC protein; (ii) an agent that increases or inhibits the activity of a VDAC protein; (Mi) an agent that binds to a VDAC protein; (iv) an agent that binds, modulates, or binds and modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (v) an agent comprising a VDAC polypeptide or functional variant thereof; (vi) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof; and (vii) a compound described herein, based on the level of a protein that binds erastin, the activity of a protein that binds erastin, or both. In certain modalities, the instructions include a guide considering the probability of events of a cancer therapy based on the level of a protein that binds the erastin, the activity of a protein that binds the erastin, or both. Identifying genetic alterations that increase the sensitivity of human cells to specific compounds can finally allow the mechanistic dissection of oncogenic signaling networks and chemotherapy to adapt to specific tumor types. Applicants have developed a systematic process to discover small molecules with increased activity in cells that harbor genetic changes specific. Using this system, it is determined that various clinically used anti-tumor agents are more potent and more active in the presence of specific genetic elements. In addition, a novel compound is identified that selectively removes cells expressing small oncoprotein T and oncogenic RAS. These small genetically engineered molecules can serve as guides for the development of anti-cancer drugs with a favorable therapeutic index. The present invention also provides packaged pharmaceuticals. In one embodiment, the packaged pharmaceutical comprises: (i) a therapeutically effective amount of an agent that is selectively toxic to human, genetically modified tumorigenic cells; and (ii) instructions and / or a label for the administration of the agent for the treatment of patients having cancer. In a particular embodiment, the agent is erastin. In another embodiment, the packaged pharmaceutical comprises: (i) a therapeutically effective amount of an agent that is toxic to human tumorigenic, genetically modified cells; and (ii) instructions and / or a label for the administration of the agent for the treatment of patients having cancer. In another related embodiment, the packaged pharmaceutical comprises: (i) a therapeutically effective amount of an agent that interacts with a cellular component that interacts with the erastin; and (ii) instructions and / or a label for the administration of the agent for the treatment of patients who have cancer. The instruction or label can be stored in an electronic medium such as CD, DVD, floppy disk, memory card, etc., which can be read by a computer. The present invention further provides the use of any agent identified by the present invention in the manufacture of a medicament for the treatment of cancer, for example, the use of erastine or its analogues in the preparation of the drug for the treatment of cancer. In certain embodiments, the methods of the invention further comprise co-administering one or more agents, such as chemotherapeutic agents that typically kill the cells through an apoptotic mechanism. Agents suitable for use in reducing the growth rate of a tumor and in the treatment of a patient suffering or suffering from cancer include but are not limited to small organic molecules, peptides, proteins, peptidomimetics, nucleic acids, antibodies, and combinations thereof. It is contemplated that all embodiments of the invention may be combined with one or more other modalities. In another aspect, the present invention relates to projection methods for identifying compounds that suppress the cellular toxicity of a protein in cells genetically modified, but not their isogenic normal cell counterparts. These methods have been used to identify known and novel compounds with genotype-selective activity. Optionally, these compounds have increased activity in the presence of a mutant protein. The invention relates to a method for identifying agents (e.g., drugs) that selectively suppress cellular toxicity in genetically modified cells. In one embodiment, the invention relates to a method for identifying an agent (e.g., drug) that suppresses the cellular toxicity of a mutant protein in genetically modified cells, comprising contacting test cells (e.g., genetically modified cells). expressing a mutant protein) with a candidate agent; determining the viability of the test cells contacted with the candidate agent; and compare the viability of the test cells with the feasibility of an appropriate control. If the viability of the test cells is more than that of the control cells, then an agent (eg, drug) that selectively expresses cellular toxicity is identified. An appropriate control is a cell that is the same type of cells as that of the test cells except that the control cell is not genetically modified to express a protein that causes toxicity. For example, control cells they may be the primary parental cells from which the test cells are derived. The control cells are contacted with the candidate agent under the same conditions as the test cells. An appropriate control can be tested or run simultaneously, or it can be pre-established (for example, a standard or pre-established reference). In certain aspects, the present invention provides methods for treating a condition in a mammal, which comprises administering to the mammal a therapeutically effective amount of a erastin analog, for example, a compound represented by the general formula I: (D wherein the condition is characterized by cells with enhanced Ras signaling activity and altered (eg, reduced or increased) activity of a cellular target protein of the small SV40 t antigen; and optionally a substantially wild-type level of Rb activity; and wherein: R1 is selected from H, -Z-Q-.Z, -alkyl Ci-8-N (R2) (R4), -C1-8-OR3 alkyl, carbocyclic or heterocyclic of 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; ' R2 and R4 are each independently selected from H, C1_alkyl, C1_4alkyl aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, provided that when both of R2 and R4 are in the same atom of N and not both in H, are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, alkyl C1-8, aryl, C1-4 aralkyl, and heteroaryl; R3 is selected from H, C- alkyl, C1- aralkyl, aryl, and heteroaryl; W is selected from ; Q is selected from O and NR2; and Z is each independently selected from C 1-6 alkyl, C 2-6 alkenyl. and C2-6 alkynyl- When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not at the group end (thus excluding, for example, enol ethers, alkynol ethers, enamines and / or or inamines).

In certain modalities, W is selected from In certain such embodiments, R1 is selected from -Z-Q-Z, -C 1-8 alkyl-N (R2) (R4), -C1-8 -OR3 alkyl, aryl, heteroaryl, and C -4 aralkyl.

In certain embodiments, W In certain such embodiments, R1 is selected from -ZQZ, -alkyl of Ci-8-N (R2) (R4), -alkyl of C -8-OR3, aryl, heteroaryl, and aralkyl of C i -4. In certain modalities, R1 is selected from -Z-Q-Z, -alkyl of Ci-8-N (R2) (R4), -alkyl of Ci-8-OR3, aryl, heteroaryl, and aralkyl of C-i-4. In certain embodiments, R 4 is selected from C 4 aralkyl and acyl. In certain such embodiments, R4 is acyl. In certain embodiments wherein R 4 is acyl, R 4 is -C (O) -alkyl of Ci-3-Y, and Y is selected from H, alkyl, alkoxy, aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl. In certain such embodiments, Y is selected from aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl. In such preferred embodiments, Y is selected from aryloxy and heteroaryloxy. In such more preferred embodiments, C1-3-Y alkyl is -CH20-phenyl, wherein phenyl is optionally substituted with halogen, preferably chloro. In certain preferred embodiments where Y is -CH20-phenyl, the rest of the values are selected in such a way that the erastin is excluded from the modality. In certain embodiments, aryl is optionally substituted with a group selected from C 1-6 alkyl, CF 3, hydroxyl, C 1-4 alkoxy, aryl, aryloxy, halogen, -NR 2 R 4, nitro, carboxylic acid, carboxylic ester, and sulfonyl. Suitable agents can have the aforementioned activity in the existing form or after complete or partial metabolism.

In certain embodiments the condition is characterized by cells with substantially wild-type level of Rb activity. In certain such embodiments, the cells are further characterized by enhanced Ras signaling activity and / or altered (eg, reduced or increased) activity of a small cell SV40 t antigen cell target protein. In certain embodiments, the compound removes or kills the cells by a non-apoptotic mechanism. In certain embodiments, the compound kills the cells by a mechanism other than a non-apoptotic mechanism. In certain embodiments, the cells have improved Ras sequence activity (for example RasV12), overexpress the SV40 small T antigen, have activity substantially reduced phosphatase PP2A, and / or levels or modulated VDAC activity (eg, increase or inhibit), such as VDAC2 or VDAC3. In certain modalities, the condition is cancer. In certain embodiments, the cells are induced to express the small SV40 t antigen, for example, by infecting the cells with a viral vector overexpressing the small SV40 t antigen, such as a retroviral vector or an adenoviral vector. In certain embodiments, the viral vector is a retroviral vector or an adenoviral vector. In certain embodiments, the method further comprises co-administering to the mammal an agent, such as a chemotherapeutic agent, that removes the cells through an apoptotic mechanism. In certain embodiments, the agent co-administered is selected from: an EGF receptor antagonist, arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine, carminomycin, mechlorethamine chloride, pentamethylmelamine, thiotepa, teniposide, cyclophosphamide, chlorambucil, demethoxy hypocreclin A, melphalan, ifosfamide, trofosfamide, treosulfan, podophyllotoxin or podophyllotoxin derivatives, etoposide phosphate, teniposide, etoposide, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, metopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine (BCNU) , lomustine (CCNU), lovastatin, 1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin, phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin, deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin, epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate, actinomycin D, safracins, saframycin, quinocarcin, discodermolides, vincristine, vinblastine, vinorelbine tartrate, vertoporphine, paclitaxel, tamoxifen, raloxifene, thiazofuran, thioguanine, ribavirin, EICAR, estramustine, estramustine sodium phosphate, flutamide, bicalutamide, buserelin, leuprolide, pteridines, enedin, levamisole, aflacone, interferon , interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, betamethasone, hydrochloride Gemcitabine, verapamil, VP-16, altretamine, tapsigargine, oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin, docetaxel, deoxygenated paclitaxel, TL-139, 5'-syn- anhydrovinblastine (hereafter: 5'-without-vinblastine), camphenocin, irinotecan, (Camptosar, CPT-11), topotecan (Hycamptine), BAY 38-3441, 9-nitrocamptothecin (Oretecin, rubitecan), exatecan (DX- 8951), lurtotecan (GI-14721 C), gimatecan, diflomotecan of homocanptothecins (BN-80915) and 9- aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), indolocarbazoles (for example, NB-506), protoberberins, intoplicins, idenoisoquinolones, benzo-phenazines or NB-506. Another aspect of the invention provides a method for removing a cell, promoting cell death or inhibiting cell proliferation, which comprises administering to the cell: (1) an effective amount of a compound represented by the general formula I: wherein: R1 is selected from H, -ZQ-Z, -alkyl Ci-8-N (R2) (R4), -alkyl-C1-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl , and aralkyl of Ci-4; R2 and R4 are each independently selected from H, Ci-4 alkyl, Ci-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, provided that when both R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selects from H, C 1-8 alkyl, aryl, C 1-4 aralkyl heteroaryl; R3 is selected from H, Ci-4 alkyl, C aryl aralkyl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently each one that is selected from Ci-6 alkyl, C2-6 alkenyl. and C2-6 alkynyl (when Z is an alkenyl or alkynyl group, the double or triple bond or bonds are not preferably at the group end); and (2) an agent that increases the abundance of VDAC (e.g., VDAC2, VDAC3) in the cell. Another aspect of the invention provides a method for removing a cell, comprising administering to the cell: (1) an effective amount of a compound represented by the general formula I: wherein: R1 is selected from H, -ZQ-Z, -alkyl Ci-8-N (R2) (R4), -alkyl-C1-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl , and C1-4 aralkyl; R 2 and R 4 are each independently selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, heteroaryl, acyl, at least one ion, and arylsulfonyl, with the proviso that that when both of R2 and R4 are in the same N atom and not both in H, they are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C -8 alkyl, aryl, C -4 aralkyl) and heteroaryl; R3 is selected from H, C1-4 alkyl, C1-4 aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently selected from C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl (when Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not the term of the group); Y (2) an agent that increases the abundance of VDAC (eg, VDAC2, VDAC3) in the cell. In another embodiment, the invention is a method for promoting cell death that includes administering to the cell an effective amount of a compound of formula (I). In certain embodiments, the compound is as described above. In certain modalities, the cell is a cancer cell. In certain embodiments, the agent comprises a polynucleotide that encodes a VDAC, such as VDAC3. In certain embodiments, the agent is a VDAC protein (e.g., VDAC3) adapted to be transported in the cell, eg, fused to a heterologous internalization domain. In certain embodiments, the agent is a liposome preparation comprising a VDAC protein (e.g., VDAC3). In certain embodiments, the agent enhances or inhibits the expression of endogenous VDAC (VDAC3), stimulates or suppresses the expression of VDAC (e.g., VDAC3), or enhances or inhibits the function of a VDAC inhibitor (e.g., VDAC3). In certain aspects, the method also involves administering an agent that increases the abundance of VDAC (e.g., VDAC 1, VDAC2, VDAC3) in the cell. In certain aspects, the The method also involves administering an agent that decreases the abundance of VDAC (e.g., VDAC 1, VDAC2, VDAC3) in the cell. In another aspect, the invention is a method for increasing the sensitivity of a tumor cell to a chemotherapeutic agent (eg, additively or synergistically), wherein a tumor cell is contacted with a compound described herein. In a related aspect, the invention is a method for reducing the sensitivity of a normal cell to a chemotherapeutic agent, wherein a normal cell is contacted with a compound described herein. In one embodiment, the invention is a method for identifying patients who are likely to respond to a treatment with compounds of the invention. Using standard characterization methods known in the art, identified patients possessing neoplasias presenting one or more of the following attributes could be expected to be responsive: activity of the atypical Ras signaling sequence as characterized by the activation of one or more members of sequence (e.g., phosphorylated Erk1 / 2, phosphorylated MEK, etc.), and / or VDAC protein expression (1, 2 or 3) and / or sensitivity of a cell line of similar or identical genotype for compound exposure of the invention either in vi tro or ¡n vivo.

Another aspect of the invention provides a compound represented by the general formula I: where: R1 is selected from H, -ZQ-Z, -alkyl from C1.8-N (R2) (R4), -alkyl from C1-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl , and aralkyl of Ci-; R2 and R4 are each independently selected from H, Ci-4 alkyl, Ci-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both R2 and R4 are in the same atom of N and not both in H, they are different (except in certain modalities where R2 and R4 are both H), and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci.sub.8 alkyl, aryl, Ci-4 aralkyl, and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-10 aralkyl aryl, and heteroaryl; Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl. and when Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not at the group end, or a pharmaceutically acceptable salt thereof. Another aspect of the invention provides a compound represented by general formula II: wherein Ar is a substituted phenyl; R 1 is selected from H, C 1-8 alkyl, -ZQ-Z, C 8 -N alkyl (R 2) (R 4), C 8 -OR 3 alkyl, carbocyclic or 3- to 8-membered heterocyclic. , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently each present selected from H, C -4 alkyl, C1-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both R2 and R4 are on the same N atom and either R2 or R 4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, alkyl of d. 8, aryl, C1- aralkyl, aryl, and heteroaryl; R3 is selected from H, C4 alkyl, C1- aralkyl) aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2; and Z is independently selected from C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl- When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not the term of the group. Another aspect of the invention provides a compound represented by general formula III: (??) wherein Ar is a substituted or unsubstituted phenyl; R is selected from H, C-β-alkyl, -ZQ-Z, -C 1-8 alkyl-N (R2) (R4), -C 1-8 alkyl-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, C1-4 alkyl, Ci-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci alkyl. 8, aryl, C1.4 aralkyl, and heteroaryl; R3 is selected from H, C- alkyl, C1- aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; W is selected from Q is selected from O and NR2; and Z is independently selected from C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl- When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not the term of the group. Another aspect of the invention provides a compound represented by general formula IV: (IV) wherein Ar is a substituted or unsubstituted phenyl; R1 is C1-8 alkyl; R2 and R4 are each independently selected from H and Ci-8 alkyl; R5 represents 0-4 substituents on the ring to which they are United; Q is selected from O and NR2. Another aspect of the invention provides a compound represented by the general formula V: wherein R is selected from H and Ci-8 alkyl; R2 is selected from H and C1-8 alkyl; R3 is selected from halogen, d-8 alkoxy and alkyl from R 4 is selected from H, halogen, Ci-8 alkoxy and C 1-8 alkyl; R5 is selected from H, halogen and nitro; and n is 1 or 2. It is contemplated that any of the compounds represented by the formulas IV above may be used for a method of 1) treating a condition in a mammal comprising administering to the mammal a therapeutically effective amount of the compound, 2) removing a cell which comprises administering to the cell a) an effective amount of the compound, and b) an agent that increases the abundance of VDAC (eg, VDAC2, VDAC3) in the cell, or 3) eliminating a cell comprising administering to the cell a) an effective amount of the compound, and b) an agent that decreases the abundance of VDAC (e.g., VDAC2, VDAC3) in the cell. It is contemplated that all embodiments of the invention may be combined with one or more other embodiments, even those described under different aspects of the invention. In certain embodiments of the invention, a compound or agent is not a compound described in Table 2. Brief Description of the Drawings Figure 1 is a schematic representation showing the relationships between experimentally transformed human cells. The BJ cells are fibroblasts of the primary human prepuce. BJ-TERT cells are derived from BJ cells and express hTERT, the catalytic subunit of the telomerase enzyme. J-TERT / LT / ST B cells are derived from BJ-TERT cells by introducing a genomic construct that encodes both oncoproteins of large simian viruses (LT) and small T (ST). BJ-TERT / LT / ST / RASV12 tumor cells are derived from BJ-TERT / LT / ST cells by the introduction of an oncogenic HRAS allele (RASV12) (Hahn et al., 1999, Nat Med 5, 1164-70 ). BJ-TERT / LT / RASV12 cells are derived from BJ cells by the introduction of cDNA constructs encoding TERT, LT, RSV12 and a control vector (Hahn et al., 2002, Nat Rev Cancer 2, 331-41). BJ-TERT / LT / RASV12 / ST cells are derived from BJ / TERT / LT / RASV12 cells by the introduction of a cDNA encoding ST (Hahn et al., 2002, Nat Rev Cancer 2, 331-41). TIP5 cells are fibroblasts of the primary human prepuce. Cell lines derived from TIP5 are prepared by introducing vectors encoding hTERT, LT, ST, RAS, or the E6 or E7 proteins of papilovirus, as shown. E6 and E7 can together replace for LT (Lessnick et al., 2002, Cancer Cell 1, 393-401). Figure 2 shows the chemical structures of nine genotype-selective compounds. Figure 3 shows graphic representations of the effect of equinomycin and camptothecin in genetically modified cells. The indicated cells were treated with equinomycin (A) or camptothecin (B, C) in 384-well plates for 48 hours. The percent inhibition of cell viability, measured using calcein AM, is shown. Error bars indicate a standard deviation. (A) BJ, BJ-TERT, BJ-TERT / LT / ST and BJ-TERT / LT / ST / RASV12 cells treated with equinomycin; (B) BJ, BJ-TERT, BJ-TERT / LT / ST and BJ-TERT / LT / ST / RASV12 cells treated with camptothecin; and (C) BJ-TERT / LT / RASV12, BJ-TERT / LT / RASV12 / ST and BJ cells.

TERT / LT / ST / RASV12 treated with camptothecin. Figure 4 shows graphic representations of the effect of erastin on genetically modified cells. The indicated cells were treated with erastin in 384-well plates for 48 hours. The percent inhibition of cell viability, measured using calcein AM, is shown. Error bars indicate a standard deviation. (A) BJ, BJ-TERT, BJ-TERT / LT / ST and BJ-TERT / LT / ST / RASV12 cells treated with erastin; (B) BJ-TERT / LT / RASV12 cells (lacking ST), BJ-TERT / LT / RASV12 / ST (tumorigenic cells) and BJ-TERT LT / ST / RASV12 (tumorigenic cells) treated with erastin; and (C) TI cells P5 / TERT, TI P5 / TERT / E6, TI P5 / TERT / LT, TI P5 / TE RT / LT / ST and TI P5 / TERT / LT / ST / RASV12 independently derived. Figure 5 shows that the protein directions of Selective tumor compounds are activated in genetically modified tumorigenic cells. Western blot analysis of Used from BJ, BJ-TERT, BJ-TERT / LT / ST, BJ-TERT / LT / ST / RASV12, BJ-TERT-LT / RASV12 and BJ-TERT / LT / RASV cells 2 / ST with an antibody directed against topoisomerase II (A) or TOPI (B, C). In panel (C), the cells were transfected with a siRNA directed against TOPI, alamic A / C or with a DNA double-strand duplex control of the same length (TOPI dsDNA). In each case, the blot analysis was tested with an antibody against F-4E to identify differences in the amount of loaded protein. The relative quantity is quantified below each band. (D) A TOPI siRNA prevents cell death caused by camptothecin in genetically modified tumor cells. The cell number was determined after transfection with a siRNA directed against TOPI and treatment with the indicated concentrations of camptothecin. (E) Okadaic acid, an inhibitor of PP2A and other cellular phosphatases, sensitizes primary human cells to camptothecin. The BJ primary cells were treated simultaneously with the indicated concentrations of both camptothecin and okadaic acid and the effect on the viability of calcein AM staining was determined. Although okadaic acid destroys BJ cells at the highest tested concentrations, at 3.4 nM it has no effect on its own concentrations, but it does have BJ cells sensitive to camptothecin. (F) Okadaic acid stimulates the expression of TOP1. The BJ primary cells were treated with the indicated concentrations of okadaic acid and the level of expression of TOPI is determined by Western blot analysis. The relative quantity is quantified below each band.

Figure 6 shows that erastin induces rapid cell death in a ST / RASV12 dependent or mode. (A) The time-dependent effect of erastin on BJ-TERT and BJ-TERT / LT / ST / RASV12 cells. The cells were seeded in 384 well plates in the presence of the indicated concentrations of erastin. Inhibition of cell viability is determined after 24, 48 and 72 hours using calcein AM. (B) The effect of erastin on the viability staining of Blue Alamar in BJ-TERT cells (red) and B J-TERT / LT / ST / RASV12 (blue). (C) Photograph of BJ-TERT / LT / ST / RASV12 and BJ primary cells treated with erastin. The cells were allowed to bind overnight, then treated with 9 μm erastin. for 24 hours and they were photographed. Figure 7 shows that camptothecin, but not erastin, induces apoptosis characteristics. (A) BJ-TERT / LT / ST / RASV12 cells treated with camptothecin, but not treated with erastin, have fragmented nuclei (10-20% of total nuclei, red and blue arrows) as shown. (B) BJ-TERT / LT / ST / RAS 12 cells treated with camptothecin but not treated with erastin show Annexin V. staining The percentage of cells in the indicated M1 region were 6%, 6% and 38% in untreated cells, treated with erastin (9 μ?) And treated with camptothecin (1 μ?), Respectively. (C) BJ-TERT / LT / ST / RASV12 cells treated with camptothecin, but not treated with erastina harbor caspase 3 activated. Lysates from samples treated with camptothecin and erastin were analyzed by Western Blot Analysis with an antibody directed against the dissociated, active form of caspase 3. The blot analysis was failed with an antibody directed against the F4E for the control of loading levels. Figure 8 shows the chemical structures of erastin and erastin B. Figure 9 shows that the nuclei remain intact in the tumor cells treated with erastin. Figure 10 shows that the erastin induces the formation of reactive oxygen species. Figure 11 shows the chemical structure of erastin A. Figure 12 indicates that the expression of VDAC3 is significantly elevated in tumorigenic BJELR cells relative to that in non-tumorigenic BJEH cells. Figure 13 shows the relative, expression levels of the VDAC isoforms in target cells using the level of VDAC-1 set at 100%. Figure 14 shows proteins identified by Western Blot Analysis and SDS-PAGE of extruded experiments using mitochondrial extract with Erastin derivatives (B1) inactive and (A6) active, immobilized. Figure 15 shows compounds 12, 13 and 5 in MCL assays in (a) HCT116 cells, (b) DLD-1 cells, (c) OVCAR-3 cells and (d) BT549 cells. Figure 16 samples compounds 12, 13 and 5 in MCL assays in (a) MiaPaca2 cells, (b) DU145 cells, (c) SK-Mel 28 cells and (d) Malm3M cells. Figure 17 samples compounds 12, 13 and 5 in MCL assays in (a) BT549 cells, (b) MCF-7 cells, (c) HOP-92 cells and (d) HOP-62 cells. Figure 18 shows the induction of inhibition of tumor growth in HT-1080 xenografts by compound 6. Figure 19 shows the induction of resistant tumor regression in HT-1080 xenografts by compound 5.

Figure 20 shows body weight trends in HT-1080 xenografts for compound 5. Figure 21 shows the induction of tumor growth inhibition in PANC-1 xenografts by compound 6. Figure 22 shows the induction of tumor regression in PANC-1 xenografts by compound 5. Detailed Description of the Invention The ability of genotype selective compounds to serving as molecular probes is based on the premise of genetic chemistries, that small molecules can be used to identify fundamental biological effects of proteins and sequences (Schreiber, 1998, Borgorg, Med Chem 6, 1127-1152, Stockwell, 2000, Nat Rev Genet 1, 116-25; Stockwell, 2000, Trends Biotechnol 18, 449-55). For example, the observation that the natural product of rapamycin slows cell growth made possible by the discovery of the target mammal Rapamycin (mTOR) as a protein that regulates cell growth (Brown et al., 1994, Nature 369, 756-758; Sabatini et al., 1994, Cell 78, 35-43). The applicants have combined these two approaches, chemistry and molecular genetics, to discover the sequence affected by mutations assted with human diseases such as cancer. Applicants have genetically modified a series of human tumor cells with defined genetic elements for use in identifying those critical sequences cjyc disruption leads to a tumorigenic phenotype (Hahn er al., 1999, Nat Med 5, 1164-70; Hahn er al., 2002, Nat Rev Cancer 2, 331-41; Lessnick et al., 2002, Cancer Cell 1, 393-401). The postulated applicants of those experimentally transformed cells could make it possible to identify genotype selective agents from both sources of the known and novel compound that presents synthetic lethality in the presence of specific alleles. related to cancer. Compounds with selective genotype lethality can serve as molecular probes for signaling networks present in tumor cells and as lead for the subsequent development of clinically effective drugs with a favorable therapeutic index and / or as an effective drug. Using this approach, the Requesters have conducted several high-throughput screening studies of libraries of natural and synthetic compounds, and have identified compounds that exhibit strong activities that destroy cancer. Among these compounds are erastin and erastin analogs such as Erastin A and Erastin B. However, a wide variety of test agents or compounds can be used in projection studies (eg, methods to identify anti-tumor candidates). ) described herein. Such test agents include, but are not limited to, small organic molecules, peptides, peptidomimetics, proteins (including antibodies), nucleic acids, carbohydrates. In this way, the invention provides compounds of formula I that kill cancer cells, especially cancer cells of specific genotype, such as those with high Ras signaling activity, altered small SV40 t antigen target activity, and / or Rb activity substantially intact.

The Applicants have also identified various cellular proteins that directly or indirectly link erastin and / or its analogues. These proteins include: Voltage-Dependent Anion Channels (VDAC1, VDAC2 and VDAC3), Prohibitin, Riboforin, Sec61 and Sec22b. Quantitative RT-PCR also suggests that destructive erastin-sensitive cells have elevated levels (eg, 2-6 times higher, typically 2-2.5 times higher) than VDAC3 expression. While not wishing to be bound by any particular theory, these experiments suggest that high levels of VDAC, particularly VDAC3 and VDAC2, the destructive cell mediated by erastin (and its analogue) improve expression, and may still be required for efficacy. Thus, one aspect of the invention provides a method for selectively killing cancer cells, especially those with high Ras activity, altered small SV40 t antigen target activity, and preferably Rb and / or p53 activity substantially intact. , the method comprising administering to a mammalian patient in need of treatment a therapeutically effective amount of a compound represented by the general formula I: wherein: R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from Ci.8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, Ci-4 alkyl, Ci-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C 1-8 alkyl, aryl, C 4 aralkyl, and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not term of the group. In certain embodiments, the compounds of formula I do not include erastin or erastin A. As is well known in the art, the constitutive activation of Ras appears to be an important factor for the malignant growth of human cancer cells. Mutations of the RAS proto-oncogenes (H-RAS, N-RAS, K-RAS) are frequent genetic aberrations found in 20% to 30% of all human tumors, although the incidences in tumor type vary greatly (Bos, Cancer Res. 49: 4682-4689, 1989). The highest rates of RAS mutations were detected in adenocarcinomas of the pancreas (90%), colon (50%), and lung (30%). In follicular and undifferentiated carcinomas of the thyroid, the incidence of RAS mutations is also considerable (50%). The majority of commonly observed RAS mutations arise at sites critical for Ras regulation-namely, codons 12, 13, and 61. Each of these mutations results in the abrogation of normal GTPase activity. The activation of Ras is also frequently observed in Hematological malignancies such as myeloid leukemias and multiple myelomas. In approximately one third of the myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), RAS genes are mutationally activated. RAS mutations occur in approximately 40% of patients with newly diagnosed multiple myeloma, and the frequency increases with the progression of the disease. On the other hand, polyomaviruses infect a wide variety of vertebrates (12 members now known). Murine polyomavirus was isolated by Ludwig Gross in 1953 while he was studying leukemia in mice and named because it causes solid tumors in multiple sites. The second member of the family, Simian Vacuolization Virus 40 (Simian Vacuolating Virus 40) (SV40) was isolated by Sweet and Hilleman in 1960 in cultures of primary monkey kidney cells being used to develop Sabin OPV (Hilleman, Dev Biol Stand 94: 183-190 , 1998). Two human polyomaviruses were isolated in 1971, cBK virus (BKV) and JC virus (JCV). The polyomaviruses encode three proteins involved in the cellular transformation of qualified large tumor antigen (LT), medium T antigen (mT) and small tumor antigen (sT). These three proteins result from differential splicing of the transcript from the early region and contain homologous sequences. The large polyoma T antigen interacts with the tumor suppressor protein, pRb and is able to immortalize primary fibroblasts in cultures. The Dna J domain located at its N-terminus, particularly the HPDKYG sequence found between residues 42 and 47, is critical for functional inactivation of Rb family proteins, as also the case with the SV40 large T antigen. LT expression is not sufficient to produce a completely transformed cell phenotype - this requires mT, which is the major transforming protein of the polyomavirus. The mouse medium polyoma T consists of 421 amino acids and can be divided into at least three domains, some of which are divided with LT and sT. The amino terminal domain comprises the first 79 amino acids and is also present in LT and sT. Adjacent to this, between residues 80-192, is a domain that is also present in the polyoma sT and contains two rich regions of cysteine, Cys-X-Cys-XX-Cys, which has also been identified in small SV40 t . The mutation of these cisterns nullifies the ability of mT to transform cells. The remaining 229 amino acids are unique to mT and contain the major tyrosine phosphorylation site of mouse mT and a hydrophobic region (approximately 20 amino acids in the carboxy terminus) involved in the membrane location of this protein that is necessary for its transforming activity . The SV40 small t antigen comprises 174 amino acids. The region between residues 97-103 interacts with the protein phosphatase 2A (PP2A). This interaction reduces the ability of PP2A to inactivate the protein kinases ERK1 and MEK1, resulting in the stimulation of proliferation of quiescent monkey kidney flukes. The t-antigen-dependent assays also identify other regions that had the ability to improve cell transformation. These regions are located in the N-terminal part that is divided by the small and large SV40 T antigens and can potentially function as a Dna J domain. The small t antigen can also be assted with tubulin and it has been suggested that it plays a role in its biological function. The applicants discovered that cells with both the activated Ras activity and the expression of the small t antigen (and thus the activity of the small t antigen bank protein, for example decreased PP2A, etc., or ERK1 and improved MEK1) are decreased. be selectively removed by erastin and its analogs, probably via a non-apoptotic mechanism. In a preferred embodiment, the cell expresses a substantially wild type level of Rb and / or p53 (or other protein D6 / E7 ratios). Thus, in certain embodiments, cancer cells of certain specific genotypes can be selectively removed by the compounds of the invention. These may include cancers that constitutively harbor active Ras mutations or mutations of Ras signaling sequence, and ERK1 activity, improved MEK1 or reduced PP2A activity. In certain distinct embodiments, the genotype of the target cells can be selectively altered (for example, to express the small t antigen of SV40, express ERK1 or MEK1, or inhibit PP2A, etc.), so that the target cells previously not susceptible to the destruction of erastina and erastina analog are now susceptible for such destruction. Specifically, the invention provides a method for selectively destroying cancer cells having high Ras activity and expression of small t antigen (or altered small t antigen target protein activity, such as PP2A activity, enhanced ERK1 or MEK1 activity or a mechanism that resembles the effects of sT, including but not limited to mutations in the regulatory subunit of PP2A), while protecting relatively normal cells that do not have elevated Ras activity, even when these cells also express the small t antigen. This may be useful since many cancers harbor somatic RasV12 or other similar mutations that lead to elevated Ras signaling activity, whereas normal cells in the same patient / individual normally do not have the same RasV12 or other sequence mutations. Ras. The orastine and its analogues can be used to selectively kill these cancer cells, if the cancer cells also express the small t antigen (or have altered activity of the target protein of the small t antigen). Even though other normal cells in the individual / patient also express the small t antigen, the object of the method would still be effective in killing cancer cells since normal cells probably do not have elevated Ras signaling activity. Even if the individual does not express the small t antigen, the small t antigen can be delivered to the patient (either as a protein or as DNA encoded by the vector) to confer the erastin / erastin analogue that destroys it on the cancer cells (but not normal). Since the small t antigen by itself is not understood to be sufficient to induce adverse effects in the patient, the side effects of the treatment (which provides the small t antigen to the patient) would be minimal or non-existent. In fact, no less than 30 million Americans are considered to have been exposed to SV40 despite polio vaccinations between 1955 and 1963. The SV40 found its way into the vaccine through macaque kidney cells used to develop the vaccine. polio virus. This method is not used any longer and polio vaccines have been free of the virus since 1963. DNA studies in the 90s found SV40 in some human tumors. However, the association of virus DNA with dividing cells in tumor tissue does not prove that the virus causes the formation of the tumor. In October 2002, a scientific panel from the US Institute of Medicine concluded that there is a way to determine whether the widespread use of polio vaccine contaminated with SV40 simian virus decades ago led to estimates of increased cancer in humans. In some embodiments, the high Ras activity is manifested by a constitutively active Ras mutation (N-, H-, or K-Ras) at 12, 13 and / or 61 amino acid positions. In some other embodiments, the elevated Ras activity is manifested by enhanced activity of one or more components upstream of the Ras sequence proteins, including but not limited to Raf, MEK, MAPK, etc. In still other embodiments, expression of the small t antigen can be accomplished by infection of target cells with vectors, such as adenoviral or retroviral vectors expressing the SV40 small t antigen (see below).

Alternatively, the small t antigen can be provided directly to the target cells. For example, the small t antigen can be introduced into target cells using various methods known in the art (see details below). In one embodiment, the t antigen small can be provided to the target cell by trapping it in liposomes carrying positive charges on its surface (e.g., lipofectins) and which are optionally labeled with antibodies against cell surface antigens of the target tissue, e.g., antibodies against a cell surface antigen cancerous In another embodiment, the small t antigen can be provided to target cells by transcytosis, using any of the "internalization peptides" capable of mediating this effect, including but not limited to the N-terminal domain of the HIV Tat protein ( for example, residues 1-72 of Tat or a smaller fragment thereof that can promote transcytosis), all or a portion of the protein of Drosophila antenopedia III, a sufficient portion of mastoparan, etc. (see below). In other embodiments, decreased PP2A (and / or other small t-antigen target proteins) can be achieved by providing an antibody, NAi (siRNA, short hairpin RNA, etc.), antisense sequence, or small molecule inhibitor specific for such protein White. Providing such antagonists of a protein to a target cell is well known in the art. See, for example, WO04078940A2, EP1439227A1, WO04048545A2, US20040029275A1, WO03076592A2, WO04076674A1, W09746671 A1, all incorporated herein by reference.

Another aspect of the invention provides a bound therapeutic method using erastin / erastin analogs and one or more agents or therapies (eg, radiotherapy) that removes cells via an apoptotic mechanism. Such agents include many chemotherapeutic drugs described below. It is believed that certain proteins have high expression levels in erastin-sensitive cells. One of such protein, VDAC3 is elevated 2-2.5 times in abundance when exposed to erastin, for example, and while Applicants do not wish to be bound by theory, their presence or even increased abundance is thought to be essential for Erastine-mediated destruction. . Thus in another aspect of the invention, a method is provided to kill or decrease the proliferation rate of cells having a high level of a VDAC such as VDCA2 or VDAC3, which comprises contacting the target cells with erastin and / or erastin analog of formulas I-IV. In certain embodiments, target cells are engineered to express a higher level of a VDAC such as VDAC2 or VDAC3 to increase the susceptibility to destroy or decrease the rate of proliferation by erastin and its functional analogues. For example, a VDAC protein can be introduced into the target cells using various methods known in the art (see details below). In one embodiment, the VDAC protein can be provided to the target cell by trapping it in liposomes bearing positive charges on its surface (e.g., lipofectins) and which are optionally labeled with antibodies against cell surface antigens of the target tissue, e.g. antibodies against a cell surface antigen with cancer. In another embodiment, the VDAC protein can be delivered to the target cells by transcytosis, using any of the "internalization peptides" capable of mediating this effect, including but not limited to the N-terminal domain of the HIV Tat protein (e.g. , residues 1-72 of Tat or a smaller fragment thereof that can promote transcytosis), all or a portion of the Drosophila Antenopedia III protein, a sufficient portion of mastoparan, etc. (see below). Alternatively, nucleic acids encoding a functional VDAC can be introduced into such target cells, for example, adenoviral or retroviral vectors expressing VDAC. In addition, the endogenous VDAC activity (eg, VDAC3) can be stimulated by an agent that either stimulates the expression of VDAC, or suppresses the activity of a VDAC inhibitor (transcription or translation inhibitor, or an inhibitor that promotes folded VDAC). in the cell).

In certain aspects, the method of the invention also involves administering an agent that increases the abundance of VDAC (e.g., VDAC 1, VDAC2, VDAC3) in the cell. The agent for increasing the abundance of VDAC may, for example, include a polynucleotide encoding VDAC, such as VDAQ3; be a VDAC protein (eg, VDAC3) adapted to be transported in the cell, for example fused to a heterologous internalization domain or formulated in the liposome preparation. In certain aspects, the method of the invention also involves administering an agent that decreases the abundance of VDAC (eg, VDAC 1, VDAC2, VDAC3) in the cell. The agent for decreasing the abundance of VDAC can, for example, inhibit the expression of endogenous VDAC (for example VDAC3), suppress VDAC expression (for example VDAC3) or improve the function of a VDAC inhibitor (for example VDAC3). The following sections describe certain exemplary embodiments of the invention, which are contemplated as being capable of combining with each other. In addition, the modalities are for illustrative purposes only, and should not be constructed to be limiting in any respect. Genetically Modified Cell Lines In one aspect, the present invention relates to genetically modified tumorigenic cell lines.

Previous reports have indicated that it is possible to convert primary human cells to tumorigenic cells by introducing hTERT expressing vectors and oncogenic RAS proteins as well as others that interrupt the function of p53, RB and PP2A (Hahn et al., 2002, Mol Cell Biol 22, 2111-23, Hahn et al., 1999, Nature 400, 464-8, Hahn and Weinberg, 2002, Nat Rev Cancer 2, 331-41, Lessnick er al., 2002, Cancer Cell 1, 393- 401). The Applicants make use of a series of genetically modified human tumorigenic cells and their precursors, which were created by introducing specific genetic elements into fibroblasts of the primary human foreskin (Figure 1). A variety of characteristics of these genetically modified tumorigenic cells have been previously reported, including their bending time, their resistance to replicative senescence and crises in culture, their response to gamma irradiation, their ability to grow in an independent manner of anchoring and its ability to form tumors in immunodeficient mice (Hahn et al., 1999, supra; Hahn er al., 2002, supra; Lessnick et al., 2002, supra). In a series of genetically modified cells, the following genetic elements were sequentially introduced into primary BJ fibroblasts: the human catalytic subunit of the telomerase enzyme (hTERT), a genomic construct that encodes the virus oncoproteins of simian 40 large (LT) and small T (ST), and an oncogenic allele of HRAS (RASV1Z). The resulting transformed cell lines were named, respectively: BJ-TERT, BJ-ERt / LT / ST, and BJ-TERT / LT / ST / RASV12. In a second series, cell lines were created in which complementary DNA constructs (cDNA) encoding LT and ST were used in place of the SV40 genomic construct that encodes both of these viral proteins. In this last series, ST is introduced in the last stage, which allows the Applicants to test compounds in the presence or absence of ST. This last genetically modified human tumorigenic cell line was named BJ-TERT / LT / RASV12 / ST. In a third series, cell lines derived from independently prepared human TIP5 foreskin fibroblasts created by introducing cDNA constructs encoding hTERT, LT, ST and RASV12 (Lessnick et al., 2002, Cancer Cell 1, 393-401) were used. These cell lines were called, respectively: TIP5 / TERT, TI P5 / TERT / LT, TI P5 / TERT / LT / ST, and TI P5 / TERT / LT / ST / RASV12. In a fourth series, cell lines derived from TIP5 fibroblasts were used when introducing cDNA constructs that encode hTERT, E6, E7, ST and RASV12. These cell lines were named, respectively: TIP5 / TERT / E6, TIP5 / TERT / E6 / E7, TI P5 / TERT / E6 / E7 / ST, and TIP5 / TERT / E6 / E7 / ST / RASV12. In this series, HPV E6 and E7, which inactivates p53 and RB, respectively, serve a similar function as LT in the previous series. However, using HPV E6 and E7, the Applicants were able to observe the effects of inactivation, separately and independently, p53 and RB. The results of a large-scale screening for compounds exhibiting selective destruction of these genetically modified tumorigenic cell lines are described in the following examples. Projection Methods for Selective Genotype Compounds In certain embodiments, the invention relates to large-scale projections for compounds that exhibit selective destruction of or inhibition of the growth of (are selectively toxic to) genetically modified tumorigenic cell lines. As used herein, the terms "agent" and "drug" are used interchangeably. As used herein, the term "is toxic to" refers to the ability of an agent or compound to eliminate or inhibit the growth / proliferation of tumorigenic cells. Large-scale projections include projections where hundreds or thousands of compounds are projected in a high-throughput format for selective toxicity to genetically modified tiimorigen cells. In one embodiment of the invention, selective toxicity is determined by comparing the cell viability of test cells, which are genetically modified tumorigenic cells, and control cells with a candidate agent. An appropriate control is a cell that is the same type of cell as that of the test cells except that the control cell is not genetically modified because it is tumorigenic. For example, the control cells may be primary parental cells from which the test cells are derived. The control cells are contacted with the candidate agent under the same conditions as the test cells. An appropriate control can be run simultaneously, or it can be preset (for example, a standard or pre-established reference). In certain embodiments, the candidate agent is selected from a library of the compound, such as a combinatorial library. Cell viability can be determined by any of a variety of means known in the art, including the use of dyes such as calcein acetoxymethylester (calcein AM) and Alamar Blue. In certain embodiments of the invention, a dye such as calcein AM is applied to the test and control cells after treatment with a candidate agent. In live cells, calcein AM is dissociated by intracellular esterases, forming calcein derived from anionic fluorescent, which can not be diffused outside living cells. Since, living cells show a green fluorescence when they are incubated with Calcein AM, while the dead cells do not. The green fluorescence that is presented by living cells can be detected and can thus provide a measurement of cell viability. In certain embodiments of the invention, an agent that has been identified as one that selectively induces cell death in a genetically modified tumorigenic cell is further characterized in an animal model. Animal models include mice, rats, rabbits, and monkeys, which may be non-transgenic animals (eg, wild type) or transgenic animals. The effect of the agent that selectively induces cell death in genetically modified tumorigenic cells can be assessed in an animal model for any number of effects, such as its ability to selectively induce cell death in tumorigenic cells in the animal and its general toxicity to the animal. For example, the method may further comprise evaluating the selective toxicity of an agent (drug) to tumorigenic cells in an appropriate mouse model. The effect of the agent that induces death in genetically modified tumorigenic cells can be assessed in an animal model for any number of effects, such as its ability to induce death in tumorigenic cells in the animal and its general toxicity to the animal. For example, the method may further comprise assessing the toxicity of a agent (drug) to tumorigenic cells in an appropriate mouse model. To illustrate, an agent can be further evaluated using a tumor growth assay that assesses the ability of the test agent to inhibit the growth of established solid tumors in a mouse. The assay can be performed by implanting tumor cells in the adipose balls of nude mice. The tumor cells are then allowed to grow to a certain size before the agents are administered. Tumor volumes are monitored or verified for a fixed number of weeks, for example, three weeks. The general health of the test animals is also verified during the course of the trial. In further embodiments of the invention, an agent that has been identified as one that selectively kills or inhibits the growth / proliferation of genetically modified tumorigenic cells is further characterized in cell-based assays to assess its mechanism of action. For example, the agent can be tested in apoptosis assays to assess its ability to induce cell death by means of a pro-apoptotic sequence. In further embodiments of the invention, an agent that induces death in tumor cells is assessed for its ability to induce death in tumorigenic cells by a non-apoptotic sequence. For example, the agent can be tested in apoptosis assays to assess its ability to induce cell death by means of of a pro-apoptotic sequence. In other embodiments, the invention relates to a method for identifying agents (drugs) that selectively suppress cellular toxicity in genetically modified cells. In one embodiment, the invention relates to a method for identifying an agent (drug) that suppresses cellular toxicity, comprising contacting test cells with a candidate agent; determining the viability of the test cells contacted with the candidate agent; and compare the viability of the test cells with the feasibility of an appropriate control. If the viability of the test cells is more than that of the control cells, then an agent (drug) is identified that selectively suppresses cellular toxicity. An appropriate control is a cell that is the same type of cells as that of the test cells except that the control cell is not genetically modified to express a protein that causes toxicity. For example, the control cells may be the primary parental cells from which the test cells are derived. The control cells are contacted with the candidate agent under the same conditions as the test cells. An appropriate control can be run simultaneously, or it can be pre-established (for example, a standard or pre-established reference). In certain modalities, the selective compounds of The genotype of the invention (anti-tumor agents) can be any chemical (element, molecule, compound, drug), whether synthetically made by recombinant techniques, or isolated from a natural source. For example, these compounds may be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules (such as antisense nucleic acid molecules or RNAi). In addition, these compounds can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled in libraries. These libraries may comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other kinds of organic compounds. These compounds can also be genetically modified or genetically modified products isolated from Used or cell growth media - bacterial, animal or plant - or can be used Cells or growth media themselves. The presentation of these compounds to a test system can be in either an isolated form of or as mixtures of compounds, especially in initial projection stages. Selective Compounds of the Genotype of the Invention Results of the Applicants demonstrate that it is possible to identify compounds with increased potency and activity in the presence of specific genetic elements. Although previous reports indicated that it may be possible to identify compounds Selective genotypes in the case of a genetic element of interest (Simons et al., 2001, Genome Res 11, 266-73, Stockwell et al., 1999, Chem Biol 6, 71-83, Torrance et al., 2001, Nat Biotechnol 19, 940-5), the work described herein provides a systematic test of synthetic lethality using more than 23,000 compounds and one or more genetic elements related to cancer. The nine identified selective compounds help to define consequences of introducing TERT and one or more of LT, ST, E6, E7 and oncogenic RAS into normal human cells. One effect of these genetic changes is to increase the rate of cell proliferation and allow sensitivity to small molecules that inhibit DNA synthesis. Although it is well established that such white preferential agents rapidly replicate tumor cells, they reassure to understand this principle that arises from this unbiased projection approach. In addition, the methodology made it is possible to easily distinguish between compounds that have a clear base of genetic selectivity and those that do not. The results show that the expression of hTERT and either E7 or LT sensitize topoisomerase II poisons cells. Since the loss or inactivation of RB (Sellers and Kaelin, 1997, J Clin Oncol 15, 3301-12; Sherr, 2001, Nat Rev Mol Cell Biol 2, 731-7) and telomerase activation (Hahn and Weinberg, 2002, Nat Rev Cancer 2, 331-41; Harley, 1994, Pathol Biol (Paris) 42, 342-5) are found in the majority of human cancers, these observations may explain, in part, the activity of these agents in a diverse range of human tumor types. The Applicants discovered that camptothecin is selectively lethal to cells harboring both ST and oncogenic RAS due to the combined effect of these two genes on topoisomerase I expression. Rapidly dividing tumor cells use topoisomerase I to uncoiled superhelical DNA to effect cell division continuous and fast. When these two sequences are altered simultaneously, topoisomerase I is stimulated, perhaps indirectly, and such tumor cells are produced sensitive to topoisomerase I poisons. These observations suggest that one aspect of the ability of ST to transform human cells together with RASV12, LT and hTERT may be the effect of ST and RASV12 on the expression of topoisomerase I. Mutations in HRAS and KRAS have been described in many types of human cancers. In addition, the inactivation of PPP2R1 B, a component of PP2A, has recently been reported in colon and lung tumors (Wang et al., 1998, Science 282, 284-7), whereas mutations in a different subunit of PP2A have been reported. described in cancers of melanoma, lung, breast and colon (Calin et al., 2000, Oncogene 19, 1191-5, Kohno et al., 1999, Cancer Res 59, 4170-s4; Ruediger et al., 2001, Oncogene 20, 1892-9; Ruediger er al., 2001, Oncogene 20, 10-5). At present, it remains unclear whether a simultaneous alteration of these two sequences occurs at high frequency in human tumors or if cancers in which both of these sequences are disturbed, show increased susceptibility to these compounds. In addition, the Applicants identified a novel compound, called erastin (see Figure 8), which is lethal to cells that express both ST and RASV12. The treatment of cells with this compound fails to eliminate cells lacking RASV12 and ST, even when it is used in concentrations eight times higher than what is required to observe an effect in cells that express both RASV12 and ST, which indicate a degree of specificity. The lethal effect of erastina is fast and irreversible once obtained. Erastin can be used to induce cell death in any tumor cell where the contact of the tumor cell with erastin results in cell death. Tumorigenic cells in which the lethality can be produced by the erastin activity include not only genetically modified tumorigenic cells, such as genetically modified cells expressing both ST and RASV12, but also tumorigenic cells comprising an activated RAS sequence independent of the expression of ST and RASV12. Additionally, the Applicants tested 135 erastin analogues for activity and selectivity in tumor cells against normal cells. 134 of these analogs were inactive. One was active and selective, but less powerful than erastin. This compound was named erastin B (see Figure 8). In certain embodiments of the invention, the invention relates to the compound, erastin. In further embodiments, the invention relates to analogs of the compound, erastin, which analogs exhibit selective toxicity to genetically modified tumorigenic cells, such as human tumorigenic cells modified genetically. In one embodiment, the erastin analog, which exhibits selective toxicity to genetically modified human tumorigenic cells, is erastin B. In certain embodiments, the invention relates to a racemic mixture of a compound of the invention, the mixture of which has selective cell toxicity. Tumorigenic genetically modified. For both camptothecin (CPT) and erastin, Applicants identified synergy between altered sequences by expression of RASV12 and ST. The expression of RASV12 leads to the activation of several well characterized signaling sequences, including the RAF-MEK-MAPK signaling cascade, the phosphatidylinositol 3-kinase (PI3K) signaling sequence and the Ral-dissociation factor sequence. guanine (Ral-GDS). Each of these sequences has been implicated in human cancers, and recent work demonstrates that these sequences work in concert in this cell transformation system (Hamad et al., 2002, Genes Dev 16, 2045-57). In addition, ST binds inactive PP2A, a widely expressed serine-threonine phosphatase. Although the specific enzymatic targets of PP2A that are perturbed with ST expression are not yet known, substantial overlap exists between sequences altered by PP2A and RAS (Millward et al., 1999, Trends Biochem Sci 24, 186-91). Further understanding of the mechanism by which erastin induces death in cells harboring alterations of these two signaling sequences may provide clues to the nature and functional overlap point between these two sequences. Erastin analogues of the invention, other than erastin B and erastin A, are represented by the general formula I: 00 wherein: R1 is selected from H, -Z-Q-.Z, -alkyl of Ci-8-N (R2) (R4), -C3-alkyl-3-alkyl, carbocyclic or 3- to 8-membered heterocyclic, aryl, heteroaryl, and C -4 aralkyl; R 2 and R 4 are each independently selected from H, C 1-4 alkyl, C 1 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R 2 and R 4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, alkyl of d. 8, aryl, Ci-4 aralkyl, and heteroaryl; R3 is selected from H, Ci-4 alkyl, C-i- aralkyl, aryl, and heteroaryl; Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C2-6 alkenyl and C2-6 alkynyl- When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not the term of the group. In certain preferred embodiments when both of R2 and R4 are the same N atom they are either both H or are different. In certain embodiments, R1 is H.

In certain modalities, W is In certain embodiments, R 4 is selected from H or substituted or unsubstituted lower alkyl.

In certain modalities R1 is H, W is , and selects from H or substituted or unsubstituted lower alkyl Exemplary compounds of formula I include: Additional erastin analogues of the invention are represented by general formula II: wherein Ar is a substituted phenyl; R1 is selected from H, C1-8 alkyl, -ZQ-Z, -Ci-8-Nalkyl (R2) (R4), -C 8 alkyl-OR3, carbocyclic or 3- to 8-membered heterocyclic , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently each occurring selected from H, Ci-4 alkyl, Ci- aralkyl, ary I, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are on the same N atom and either R2 or R4 are acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected of H, alkyl of 8, aryl, aralkyl of Ci-4, aryl, and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl- When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not the term of the group. In certain embodiments, R 5 represents 1-4 substituents, such as halogen or nitro. In certain embodiments R 5 represents a substituent, such as halogen or nitro, especially chlorine, located in the para position with the carbonyl of the quinazoline ring. In other modalities, R5 does not represents substituents on the ring (ie, all substituents are hydrogen atoms). In certain modalities, Ar is mono-substituted wherein the substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, Ar has a substituent in the ortho position wherein the substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, Ar is 2,6-disubstituted such that one substituent is halogen, lower alkoxy, or lower alkyl and the second substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, the compounds of formula II do not include those in which the substituent on Ar is ethoxy at an ortho position with the nitrogen bond of the quinazolinone ring. In further embodiments, the compounds of the formula II do not include those in which Ar does not have a lower alkoxy substituent or lower alkyl in the ortho position with the nitrogen bond of the quinazolinone ring. In certain embodiments of the compounds of the formula II, Ar have at least one halogen substituent. In certain embodiments, Ar has a halogen substituent in the ortho position. In preferred embodiments, the compounds of formula II include those wherein Ar is a 2,6-disubstituted phenyl ring wherein the substituents are halogen atoms. Exemplary compounds of formula II include: Additional erastin analogues of the invention are presented by general formula III: wherein Ar is a substituted or unsubstituted phenyl; R1 is selected from H, C1-8 alkyl, -ZQ-Z, -Ci-8-Nalkyl (R2) (R4), -C1-8 -OR3 alkyl, carbocyclic or 3- to 8-membered heterocyclic , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently each occurring selected from H, C1-4 alkyl, C1- aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci alkyl. a, aryl, C1- 1 aralkyl and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2; and Z is each independently selected from C-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. When Z is an alkenyl or alkynyl group, the double or triple bond or bonds are preferably not at the group end. In certain embodiments, R2 and R4 are either both H or are different. In certain embodiments, R 5 represents 1-4 substituents on the ring to which they are attached, such as halogen or nitro. In certain embodiments, R 5 represents a substituent, such as halogen or nitro, especially chlorine, located in the para position with the carbonyl of the quinazolinone ring. In another embodiment, R5 does not represent substituents on the ring (ie, all substituents are hydrogen atoms). In certain embodiments, the compounds of formula III do not include those in which the substituent on Ar is ethoxy at an ortho position with the bond to the nitrogen of the quinazolinone ring. In further embodiments, the compounds of formula III do not include those in which Ar does not have a substituent of lower alkoxy or lower alkyl in the ortho position with the nitrogen bond of the quinazolinone ring. In preferred embodiments of the present invention, Ar is a substituted phenyl. In certain embodiments of the compounds of formula III, Ar has at least one halogen substituent. In certain embodiments, Ar has a halogen substituent in the ortho position. In preferred embodiments, the compounds of formula III include those wherein Ar is a 2,6-disubstituted phenyl ring wherein the substituents are halogen atoms. Exemplary compounds of formula III include: Additional erastin analogues of the invention are represented by general formula IV: wherein Ar is a substituted or unsubstituted phenyl; R1 is C1-8 alkyl; R2 and R4 are each independently selected from H and Ci-8 alkyl; R5 represents 0-4 substituents on the ring to which they are attached; W is selected from Q is selected from O and NR2. In certain embodiments, R 5 represents 1-4 substituents on the ring to which they are attached, such as halogen or nitro. In certain embodiments, R 5 represents a substituent, such as halogen or nitro, especially chloro, located in the para position with the carbonyl of the quinazolinone ring. In another embodiment, R5 does not represent substituents on the ring (ie, all substituents are hydrogen atoms). In certain preferred embodiments of the present invention, Ar is a substituted phenyl. In certain embodiments, Ar is mono-substituted wherein the substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, Ar has a substituent in the ortho position wherein the substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, Ar is 2,6-disubstituted such that one substituent is halogen, lower alkoxy, or lower alkyl and the second substituent is halogen, lower alkoxy, or lower alkyl. In certain embodiments, the compounds of formula IV they do not include those in which the substituent on Ar is ethoxy in an ortho position with the nitrogen bond of the quinazolinone ring. In further embodiments, the compounds of formula IV do not include those in which Ar does not have a substituent of lower alkoxy or lower alkyl in the ortho position with the nitrogen bond of the quinazolinone ring. In certain embodiments of the compounds of formula IV, Ar has at least one halogen substituent. In certain embodiments, Ar has a halogen substituent in the ortho position. In preferred embodiments, the compounds of formula IV include those wherein Ar is a 2,6-disubstituted phenyl ring wherein the substituents are halogen atoms. Exemplary compounds of formula IV include: Additional erastin analogues of the invention are represented by general formula V: wherein R1 is selected from H and Ci-8 alkyl; R2 is selected from H and Ci-8 alkyl; R3 is selected from halogen, d-8 alkoxy and alkyl from R 4 is selected from H, halogen, C 1-8 alkoxy and -8 alkyl; R5 is selected from H, halogen and nitro; and n is 1 or 2. Exemplary compounds of formula V include: Any of the compounds of the formulas I-V can be used for any of the methods described herein for erastin and erastin analogues. The compounds included in the invention include enantiomers and diastereomers of the compounds described herein. The invention also includes salts, particularly pharmaceutically acceptable salts of the compounds described herein. In addition, the invention includes solvates, hydrates and polymorphic crystalline forms of the compounds described herein.

Suitable agents can have the aforementioned activity in the existing form or after complete or partial metabolism.

The invention also provides the synthesis or processing of a compound of the invention. In certain embodiments, the present invention provides the preparation of a compound A, A, wherein R5 and R1 are as described for structures ll-V. In certain embodiments a stage of the synthesis of a compound A is the reaction of a compound B, B, with a compound C, In certain embodiments the reaction of compound B with compound C is carried out in a polar aprotic solvent such as acetonitrile, DMSO, diethyl ether, butanone, cyclohexanone, acetophenone, tetrahydrofuran, acetone, dichloromethane, sulfolane, or dimethylformamide. In preferred embodiments the solvent is dichloromethane or dimethylformamide. In certain embodiments, the reaction is carried out under an atmosphere of nitrogen. In certain embodiments, an organic base, such as pyridine, diisopropylamine, 2,6-lutidine, trialkylamines (e.g., triethylamine), pyrrolidine, imidazole or piperidine, is added to a solution of a compound B followed by the addition of a compound C to the resulting solution. In preferred embodiments, the organic base is an amine base such as a trialkylamine such as triethylamine. In preferred embodiments the reaction is carried out at a range of 0-10 ° C. The invention further provides the preparation of a compound of the structure D, D, wherein R5, R1 and Ar are as described in structures 11-V. In certain embodiments, a step in the synthesis of D is the reaction of a compound A with a compound E, Ar-NH2. In certain embodiments the reaction of compound A with compound E is carried out in a polar aprotic solvent such as acetonitrile, DMSO, diethyl ether, butanone, cyclohexanone, acetophenone, tetrahydrofuran, acetone, dtchloromethane, sulfolane, or dimethylformamide. In preferred embodiments the solvent is acetonitrile. In certain embodiments, the reaction is carried out under a nitrogen atmosphere. In certain embodiments, the reaction is carried out in the presence of trichlorophosphine. In certain embodiments, the reaction is maintained in a range of 40-60 ° C for a period of time such as 5-15 hours. In other embodiments phosphoryl trichloride is added to the stirred solution of A and E and the resulting mixture is heated to reflux for a period of time, such as 1-5 hours. The invention also provides the preparation of a compound of the structure F, F, wherein R5, R1, Ar and W are as described for structures 11-V. In certain embodiments, a step in the synthesis of compound F is the reaction of compound D with HNR2 where HNR2 is equivalent to HW. In certain embodiments the reaction is carried out in the presence of potassium carbonate and a source of iodide, such as copper iodide, potassium iodide, cesium iodide, sodium iodide or tetrabutylammonium iodide, in a polar aprotic solvent. In certain embodiments compound D and potassium carbonate are combined, and HNR2 is added, followed by the iodide source. In preferred embodiments the solvent is acetonitrile. In certain embodiments, the iodide reagent is tetrabutylammonium iodide; in certain embodiments, the iodide reagent is sodium iodide. In certain embodiments, the mixture is maintained in the range of 50-70 ° C for a period of time such as 5-15 hours.

In certain embodiments HNR2 (HW) includes a second nitrogen atom on which an amine protecting group exists. In some embodiments, the protecting group may be tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2- (trimethylsilyl) ethoxycarbonyl, or 2,2,2-trichloroethoxycarbonyl. In certain embodiments the reaction is carried out in the presence of a base, such as potassium carbonate, sodium carbonate, pyridine, diisopropylamine, 2,6-lutidine, triethylamine, pyrrolidine, imidazole, or piperidine, and a source of iodide in a solvent aprotic polar. In certain embodiments, the base is potassium carbonate or triethylamine. In certain embodiments compound D and base combine and HNR2 is added followed by the iodide source. In preferred embodiments the solvent is acetonitrile or acetone. In certain embodiments, the iodide reagent is tetrabutylammonium iodide, in certain embodiments the sodium iodide iodide reagent. In certain embodiments the mixture is maintained in a range of 70-90 ° C for a period of time such as 1-10 hours. After the termination of the addition reaction, the protecting group can be removed from the resulting product by an appropriate deprotection reaction. For example, when the protecting group is tert-butoxycarbonyl, the protecting group can be removed by adding an acid to a solution of the compound (for example adding a solution of 4N HCl in dioxane to a solution of the product. in dioxane). In certain embodiments the reaction is then diluted with water and an organic solvent before neutralizing the mixture.

In certain embodiments the mixture is made basic by the addition of a saturated aqueous solution of sodium carbonate. It is contemplated that all embodiments of the invention may be combined with one or more embodiments, even those described under different aspects of the invention. The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbylC (O) -, preferably C (O) - alkyl. The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC (0) NH-. The term "acyloxy" is recognized in the art and refers to a group represented by the general formula hydrocarbylC (0) 0-, preferably C (0) 0- alkyl. The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, which has an oxygen attached thereto. Representative alkyl groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl. The term "alkenyl", as used herein, is refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl", the latter of which refers to alkenyl portions having substituents that replace a hydrogen in one or more carbons of the alkenyl group. Such substituents may be present in one or more carbons that are included or not included in one or more double bonds. In addition, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl or heteroaryl groups is contemplated. The term "alkyl" refers to the radical of saturated aliphatic groups, which includes straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and alkyl groups substituted with cycloalkyl. In preferred embodiments, a straight or branched chain alkyl has 30 or fewer carbon atoms in its backbone or structure (eg, Ci-C30 for linear chains, C3-C30 for branched chains), and most preferably 20 or some. Likewise, preferred cycloalkyls have 3-10 carbon atoms in their ring structure, and most preferably have 5, 6 or 7 carbons in the ring structure.

In addition, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl having substituents that replace a hydrogen on one or more carbons of the hydrocarbon skeleton. Such substituents may include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), a alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, a mine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl , a sulfonamide, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the substituted portions in the hydrocarbon chain can themselves be substituted, if appropriate. For example, substituents of a substituted alkyl can include substituted and unsubstituted forms of amino, azido, imino, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Alkylated substitutes are exemplary later. Cycloalkyls can also be substituted with alkyls, alkenyls, alkoxyls, alkylthios, aminoalkyls, alkyls substituted with carbonyl, -CF3 > CN, and similar. The term "Cx-y" when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, means that it includes groups containing x and y carbons in the chain. For example, the term "Cx-y alkyl" refers to substituted or unsubstituted hydrocarbon groups, including straight and branched chain alkyl groups containing x and y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2.2, 2-trifluoroethyl, etc. Alkyl of C0 indicates a hydrogen where the group is in a terminal position, an internal link. The terms "C2-y alkenyl" and "C2-y alkynyl" refer to unsaturated, unsubstituted or substituted aliphatic groups, analogous in length and possible substitution to the alkynes described above, but containing at least one double bond or triple, respectively. The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group. The term "alkylthio", as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylis. The term "alkynyl", as used herein, is refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl", the latter of which refers to alkynyl portions having substituents that replace a hydrogen on one or more carbons of the alkynyl group. Such substituents may be present in one or more carbons that are included or not included in one or more triple bonds. In addition, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated. The term "amide", as used herein, refers to a group wherein R9 and R10 each independently represents a hydrogen or a hydrocarbyl group, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted or substituted salts and amines thereof, for example, a portion that can be be represented by wherein R9, R10, and R10 each independently represents a hydrogen or a hydrocarbyl group, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. The term "aminoalkyl", as used herein, refers to an alkyl group substituted with an amino group. The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group. The term "aryl" as used herein includes substituted or unsubstituted single ring aromatic groups in which each ring atom is carbon. Preferably the ring is a ring of 5 to 7 members, more preferably a ring of 6 members. The term "aryl" also includes polycyclic ring system having two or more cyclic rings in which two or more carbons are common to two adjacent rings wherein at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The term "carbamate" is recognized in the art and is refers to a group wherein R9 and R0 independently represent hydrogen or a hydrocarbyl group. The terms "carbocycle", "carbocyclyl", and "carbocyclic", as used herein, refer to a non-aromatic, saturated or unsaturated ring in which each atom of the ring is carbon. Preferably a carbocycle ring contains from 3 to 10 atoms, more preferably from 5 to 7 atoms. The term "carbocyclylalkyl", as used herein, refers to an alkyl group substituted with a carbocycle group. The term "carbonate" is recognized in the art and refers to a group -OC02-R9, wherein R9 represents a hydrocarbyl group. The term "carboxy", as used herein, refers to a group represented by the formula -C02H. The term "ester", as used herein, refers to a group -C (0) OR9 wherein R9 represents a hydrocarbyl group. The term "ether", as used herein, refers to a hydrocarbyl group bonded through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. The ethers they can be either symmetrical or non-symmetric. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. The ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl. The terms "halo" and "halogen" as used herein mean halogen and include chlorine, fluorine, bromine, and iodine. The terms "hetaralkyl" and "heteroaralkyl", as used herein, refer to an alkyl group substituted with a hetaryl group. The terms "heteroaryl" and "hetaryl" include aromatic ring structures, substituted or unsubstituted, preferably rings of 5 to 7 members, more preferably rings of 5 to 6 members, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings wherein at least one of the rings is heteroaromatic, for example, other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur. The terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to non-aromatic, substituted or unsubstituted ring structures, preferably 3 to 10 membered ring, more preferably 3 to 7 membered rings, whose ring structures they include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings wherein at least one of the rings is heterocyclic, e.g. other celiac rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. The term "heterocyclylalkyl", as used herein, refers to an alkyl group substituted with a heterocycle group. The term "hydrocarbyl", as used herein, refers to a group that is attached through an atom of carbon that does not have a substituent = 0 or = S, and typically has at least one carbon-hydrogen bond and a carbon skeleton mainly, but can optionally include heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl and trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (having one carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof. The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with a hydroxy group. The term "lower" when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups where there are ten or fewer atoms without hydrogen in the substituent, preferably six or less. A "lower alkyl", for example, refers to an alkyl group containing ten or fewer carbon atoms, preferably six or less. In certain embodiments, substituents of acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, if they occur alone or in combination with others substituents, such as in hydroxyalkyl and aralkyl recitations (in which case, for for example, the atoms within the aryl group are not counted when the carbon atoms in the alkyl substituent are counted). The terms "polycyclyl", "polycyclic", and "polycyclic" refer to two or more rings (for example, cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and / or heterocyclyls) in which two or more atoms are common to two. adjacent rings, for example, the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7. The term "substituted" refers to portions having substituents that replace a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit one provided that such substitution is in accordance with the permitted valency of the substituted atom and the substituent, and that the substitution results in a stable compound, for example, which does not undergoes transformation spontaneously such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include substituents of acyclic and cyclic organic compounds, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of heteroatoms. The substituents may include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic portion. It will be understood by those skilled in the art that the substituted portions in the hydrocarbon chain can themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted", references for chemical moieties herein are understood to include substituted variants. For example, the reference for a group "a r i I o" or a portion implicitly includes both substituted and unsubstituted variants. The term "sulfate" is recognized in the art and refers to the group -OS03H, or a pharmaceutically acceptable salt thereof. The term "sulfonamide" is recognized in the art and refers to the group represented by the general formulas wherein R9 and R10 independently represent hydrogen or hydrocarbyl. The term "sulfoxide" is recognized in the art and refers to the group -S (0) -R9, wherein R9 represents hydrocarbyl.

The term "sulfonate" is recognized in the art and refers to the group S03H, or a pharmaceutically acceptable salt thereof. The term "sulfone" is recognized in the art and refers to the group -S (0) 2 -R9, wherein R9 represents hydrocarbyl.

The term "thioalkyl", as used herein, is -3 to an alkyl group substituted with a thiol group. The term "thioester", as used herein, refers to a group -C (0) SR9 or -SC (0) R9 wherein R9 represents a hydrocarbyl. The term "thioether", as used herein, is equivalent to an ether, where oxygen is replaced with a sulfur. The term "urea" is recognized in the art and may be represented by the general formula wherein R9 and R10 independently represent hydrogen or a hydrocarbyl. The term "small organic molecules" refers to a non-polymeric compound having a molecular weight of less than 2000 amu. Typically, such molecules have a molecular weight of less than 1000 amu, such as less than 500 amu. Methods for Identifying Targets for Selective Genotype Compounds In certain embodiments, the invention relates to the use of the genotype-selective composite subject, also referred to as "ligand" (e.g., erastin), to identify targets (also referred to herein as " "cellular components" (eg, proteins, nucleic acids or lipids) involved in conferring the desired cell phenotype In one embodiment, the invention provides a method for identifying cellular components involved in tumorigenesis, whereby a tumorigenic cell, such as a genetically modified human tumorigenic cell, tissue, organ, organism or a Used or an extract thereof is already in contact with an anti-tumor composite subject; and after putting them in contact, cellular components were identified that interact (directly or indirectly) with erastin, resulting in the identification of cellular components involved in tumorigenesis. In another embodiment, the invention provides a method for identifying cellular components involved in tumorigenesis. In this method, (a) a tumorigenic cell, such as a genetically modified human tumorigenic cell, tissue, organ, organism or a lysate or an extract thereof is contacted with a erastin inhibitor and placed in contact with the erastina; and (b) cellular components are identified that interact (directly or indirectly) with the erastin inhibitor, whose cellular components are involved in tumorigenesis. The cell can be contacted with erastin and the erastin inhibitor sequentially or simultaneously. The cellular components that interact with arastine or any agent of the present invention can be identified by known methods. As described herein, the subject compound (or ligand) of these methods can be created by any chemical method. Alternatively, the subject compound may be a biomolecule that occurs naturally synthesized in vivo or in vitro. The ligand can be optionally derivatized with another compound. An advantage of this modification is that the derivatizing compound can be used to facilitate the collection of the target ligand or ligand collection complex, for example, after ligand and target separation. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxigenin, green or crude fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase, photoactivatable crosslinkers or any combinations thereof. Derivatizing groups can also be used in conjunction with targets (eg, protein that binds erastin) to facilitate their detection. According to the present invention, an objective (cellular component) can be a biomolecule that occurs naturally synthesized in vivo or in vitro. An objective may be comprised of amino acids, nucleic acids, sugars, lipids, natural products or any combinations thereof. An advantage of the present invention is that no prior knowledge of the identification or function of the objective is necessary. The interaction between the ligand and the target can be eovalent or non-covalent. Optionally, the ligand of a ligand target pair may or may not have affinity for other targets. The objective of a ligand target pair may or may not have affinity for other ligands.

For example, the binding between a ligand and a target can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabelled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1). Alternatively, small molecules can be immobilized on a suitable solid support or affinity matrix such as an agarose matrix and used to project extracts from a variety of cell types and organisms. Similarly, small molecules can be contacted with the cell, tissue, organ, organism or Used or extract thereof and the solid support can be added later to recover the small molecules and associate target proteins. Expression cloning can be used to test the target within a small combination of proteins (King RW et al., 1997, Science 277: 973). Peptides (Kieffer et al., 1992, PNAS 89: 12048), nucleoside derivatives (Haushalter KA et al., 199, Curr. Biol. 9: 174), and bovine-drug serum albumin conjugate (drug- BSA) (Tanaka et al., 1999, Mol.Pharmacol 55: 356) have been used in expression cloning. Another useful technique for closely associating ligand binding with DNA encoding the target is phage display. The presentation of phage, which has been predominantly used in the field of monoclonal antibody, peptide or Protein libraries are created on the viral surface and are projected for activity (Smith GP, 1985, Science 228: 1315). The phages are deposited for the target that is connected to a solid phase (Parmley SF et al., 1988, Gene 73: 305). One of the advantages of phage display is that the cDNA is in the phage and thus it is not required to separate the cloning step. A non-limiting example includes linkage reaction conditions wherein the ligand comprises a label such as biotin, fluorescein, digoxigenin, green fluorescent protein, radioisotope, histidine tag, a magnetic granule, an enzyme or combinations thereof. In one embodiment of the invention, the targets can be projected in a mechanism-based assay, as an assay to detect ligands that bind to the target. This may include a solid phase or fluid phase that binds the event with either the ligand or the protein or an indicator of any being detected. Alternatively, the gene encoding the previously undefined function protein can be transfected with a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein) into a cell and projected against the library preferably by a yield projection high or with individual members of the library. Other mechanism-based binding assays can be used, for example, biochemical assays that measure an effect on enzyme activity, cell-based assays in which the target and a reporter system (eg, luciferase or β-galactosidase) have been introduced into a cell, and binding assays that detect changes in free energy. The binding assays can be performed with the target fixed to a cavity, pellet or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound ligands can usually be detected using surface plasmon or colorimetric resonance or fluorescence. In certain embodiments, the present invention further contemplates methods of treating or preventing a disease (e.g., cancer) by modulating the function (e.g., activity or expression) of an objective (cellular component) that is identified in accordance with the invention. To illustrate, if a target is identified to promote tumor growth, a therapeutic agent can be used to modify or reduce the function (activity or expression) of the target. Alternatively, if a target is identified to inhibit tumor growth, a therapeutic agent may be used to increase the function (activity or expression) of the target. The therapeutic agent includes, but is not limited to, an antibody, a nucleic acid (e.g., an antisense oligonucleotide or a small inhibitory RNA for RNA interference), a protein, a small molecule (e.g., a compound of the invention). ) or a peptidomimetic.

Erastin Targets In certain embodiments, the present invention provides erastin and erastin analogue targets, which are generally referred to herein as erastin targets. The erastin targets can be linked directly or indirectly to erastin or an erastin analog as described above. Optionally, the erastin target can mediate the anti-tumor activity of a compound such as erastin or an erastin analog in a cell. Exemplary erastin targets include, but are not limited to, VDAC1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a, and Sec22b. Voltage-dependent anion channels (VDACs) are a family of proteins that form the pore encoded by different genes, with at least three protein products (VDAC1, VDAC2 and VDAC3) expressed in mammalian tissues. The main recognized functional role of VDAC is to allow almost free permeability of the outer mitochondrial membrane (ODF). See, for example, Shoshan-Barmatz et al., 2003, Cell Biochem Biophys 39: 279-92. VDAC2 and VDAC3 must have an alternative structural organization and different functions in the ODF than in the mitochondria (Hinsh et al., 2004, J Biol Chem. 279: 15281-8). Representative VDAC sequences from several species have been deposited in GenBank. For example, amino acid and nucleic acid sequences of human VDAC1 can be found in GenBank Access Numbers NP_003365 and NM_003374; amino acid and nucleic acid sequences of human VDAC2 can be found in GenBank Accession Numbers NP_003366 and NM_003375; and human VDAC3 nucleic acid and nucleic acid sequences can be found in GenBank Accession Numbers NP_005653 and NM_005662. Prohibitin is an evolutionarily conserved gene that is expressed ubiquitously. It is considered to be a negative regulator of cell proliferation and may be a tumor suppressor (eg, Fusaro et al., 2003, J. Biol. Chem. 278: 47853-47861; Fusaro et al., 2002, Oncogene 21 : 4539-4548). Representative prohibitin sequences from several species have been deposited in GenBank. For example, human prohibitin nucleic acid and amino acid sequences can be found in GenBank Accession Numbers NP_002625 and NM_002634. Riboforinas (for example, I and II) are proteins that seem to be involved in the ribosome binding. There are highly conserved, abundant glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum (eg, Fu et al., 2000, J. Biol. Chem. 275: 3984-3990; Crimaudo et al., 1987, EMBO J. 6 : 75-82). Riboforin sequences representative of several species have been deposited in GenBank. For example, human ribophorin I nucleic acid and nucleic acid sequences can found in GenBank Access Numbers NP_002941 and NM_002950; and human riboform II nucleic acid and nucleic acid sequences can be found in GenBank Accession Numbers NP_002942 and N M_002951. Sec61-alpha proteins are suggested to play a role in ?? insertion of secretory or membrane polypeptides in the endoplasmic reticulum (see, for example, Higy et al., 2004, BioJhemistry 43: 12716-22). Sequences of Sec61 alpha representative of several species have been deposited in GenBank. For example, amino acid sequences and nucleic acid of human Sec61-alpha-l can be found in GenBank Accession Numbers NP_037468 and NM_013336; and Sec61-human alpha-nucleic acid sequences can be found in GenBank Accession Numbers NP_060614 and NM_018144. Sec22-beta proteins are suggested to play a role in the ER-Golgi protein trafficking and the SNARE complex (eg, Parlati et al., 2000, Nature 407: 194-198, Mao et al., 1998, Proc. Nati, Acad. Sci. USA 95: 8175-8180). Sec61-beta sequences representative of several species have been deposited in GenBank. For example, human Sec61-beta amino acid and nucleic acid sequences can be found in GenBank Accession Numbers NP_004883 and NM_004892. In certain embodiments, the present invention relates to Methods to identify candidate anti-tumor therapeutic agents by using an erastin blank. In such methods, a test agent that binds to a erastin blank or increases or decreases the function (eg, activity or expression or interactions) of a erastin blank can be identified as a candidate anti-tumor therapeutic agent. The candidate anti-tumor therapeutic agent can also be tested in vivo or in vitro for its anti-tumor activity. Methods for identifying candidate anti-tumor agents can be carried out in a similar manner by screening methods as described above. Delivery Methods Certain embodiments of the invention utilize methods to deliver proteins (e.g., small T antigen, VDAC inhibitors, PP2A, etc.) or DNA encoding such proteins to a target cell, which can be performed by any standard molecular biology and techniques. of molecular medicine. The modalities illustrated below are but a bit of such techniques that can be used for such purposes. In one aspect of the invention, construction of expression of the subject proteins, or to generate antisense molecules, can be administered in any biologically effective carrier, for example any formulation or composition capable of effectively transfecting cells in vivo with a gene recombinant. Approaches include insertion of the subject gene into viral vectors that include recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-1, or recombinant bacterial or eukaryotic piasmids. Viral vectors can be used to transfect cells directly; Plasmid DNA can be delivered with the aid of, for example, cationic (eg lipofectin) or derivatized liposomes (eg, conjugated antibody), polylysine conjugates, gramacidin S, artificial viral layers or other such intracellular carriers, as well as also direct injection of the construct gene or CaP04 precipitation carried out in vivo. It will be appreciated that due to the transduction of appropriate target cells represent the first critical step in gene therapy, choice of the particular gene delivery system will depend on such factors as the target target phenotype and the route of administration, eg, locally or systemically. A preferred approach for the live introduction of nucleic acid encoding one of the subject proteins in a cell is by the use of a viral vector containing a nucleic acid, an oDNA encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of the target cells can receive the nucleic acid. Additionally, molecules modified within the viral vector, for example, by a cDNA contained in the vector viral, are efficiently expressed in cells that have nucleic acid uptake of the viral vector. Retroviral vectors and adeno-associated viral vectors are generally understood to be delivery systems of the recombinant gene of choice for the transfer of exogenous genes in vivo, particularly in humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A subset of the retrovirus family qualified as "lentivirus" for long duration of its latent phases after integration, are represented by the human immunodeficiency virus (HIV) and the feline immunodeficiency virus (FIV). Vector systems derived from both of those viruses have been used effectively in pre-clinical models and show greater commitment for therapeutic application (Humeau et al., Mol Ther 2004, 9 (6): 902-13; Curran et al. , Mol Ther, 2000, 1 (1): 31-8; Engel and Kohn, Front Biosci, 199, 4: e26-33). Most different viruses, HIV and FIV (and vectors derived from them) have the ability to transduce undivided cells (Humeau et al., Mol Ther 2004, 9 (6): 902-13; Curran et al., Mol Ther, 2000, 1 (1): 31-8). This property can be advantageous depending on the type of target cell. In addition, VIF can distinguish itself from other retroviruses by its increased capacity of the transgene transporter (Curran et al., Mol Ther. 2000, 1 (1): 31-8). A major prerequisite for the use of retroviruses is to ensure the safety device of their use, particularly with respect to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "encapsulation cells") that produces only defective retroviruses by replication have increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in transferring gene for gene therapy purposes ( for a review see Miller, AD, Blood 76: 271, 1990). In this way, recombinant retroviruses can be constructed in which part of the retroviral sequence encoding (gag, pol, env) has been replaced by nucleic acid encoding a subject polypeptide, which provides the defective retrovirus by replication. The replication-defective retrovirus is then packaged in virions that can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al., (eds), John Wiley & Sons, Inc., Greene Publishing Associates, (2001), Sections 9.9-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM that are well known to those skilled in the art. Examples of packaging virus lines Suitable for preparing both ecotropic and amphotropic retroviral systems include? p ?, v | / Cre,? 2 and ??? t ?. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo (see for example Eglitis et al., Science 230: 1395-1398, 1985, Danos and Mulligan, PNAS USA 85: 6460-6464, 1988, Wilson et al., PNAS USA 85: 3014-3018, 1988, Armentano et al., PNAS USA 87: 6141-6145, 1990; Huber et al., PNAS USA 88: 8039-8043, 1991; Ferry et al., PNAS USA 88: 8377-8391, 1991; Chowdhury et al., Science 254: 1802-1805, 1991; van Beusechem et al., PNAS USA 89: 7640-7644, 1992; Kay ef al., Human Gene Therapy 3: 641-647, 1992; Dai ef al., PNAS USA 89: 10892-10895, 1992; Hwu et al., J. Immunol., 150: 4104-4115, 1993; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and therefore of retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle (see, for example, PCT publications W093 / 25234). , WO94 / 06920, and W094 / 11524). For example, strategies for modifying Spectrum of infection of retroviral vectors include: specific binding antibodies for cell surface antigens to the viral env protein (Roux et al., PNAS USA 86: 9079-9083, 1989; Julan et al., J. Gen Virol 73: 3251 -3255, 1992; and Goud et al., Virology 163: 251-254, 1983); or cell surface ligands coupling to viral env proteins (Neda et al., J. Biol. Chem. 266: 14143-14146, 1991). The coupling may be in the form of chemical cross-linking with a protein or other variety (eg, lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (eg, antibody / env fusion proteins). single chain). This technique, while useful to limit or otherwise direct infection to certain types of tissues, and can also be used to convert an ecotropic vector to urT amphotropic vector. Another viral gene delivery system useful in the present invention utilizes adenovirus derived vectors. The genome of an adenovirus can be manipulated in such a way that it encodes a product of the gene of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., BioTechniques 6 : 616, 1988; Rosenfeld et al., Science 252: 431-434, 1991; and Rosenfeld er al., Cell 68: 143-155, 1992). Suitable adenoviral vectors derived from adenovirus strain type Ad 5 d1324 or other Adenovirus strains (eg, Ad2, Ad3, Ad7, etc.,) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting undivided cells and can be used to infect a wide variety of cell types, including the airway epithelium (Rosenfeld et al., (1992) supra) , endothelial cells (Lemarchand et al., PNAS USA 89: 6482-6486, 1992), hepatocytes (Herz and Gerard, PNAS USA 90: 2812-2816, 1993) and muscle cells (Quantin et al., PNAS USA 89: 2581-2584, 1992). In addition, the virus particle is relatively stable, receptive to purification and concentration, and as described above, can be modified to affect the spectrum of infectivity. Additionally, the introduced adenoviral DNA (and previous DNA contained in it) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that may occur as a result of insertional mutagenesis in situations where DNA is introduced which becomes integrated into the host genome (for example, retroviral DNA). In addition, the carrying capacity of the adenoviral genome for the anterior DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., Supra; Haj-Ahmand and Graham J., Virol. 57: 267, 1986). The majority of defective adenoviral replication vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral genes E1 and E3 but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al., Cell 16: 683, 1979; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, EJ Murray, Ed. (Humana, Clifton, NJ, 1991) vol 7. pp. 109-127). The expression of the inserted subject gene can be under the control of, for example, the E1A promoter, the major forward promoter (MLP) and associated guide sequences, the viral E3 promoter, or exogenously added promoter sequences. Yet another viral vector system useful for the delivery of the subject genes is the adeno-associated virus (AAV). The adeno-associated virus is a defective virus that occurs naturally that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. Immunol. (1992) 158: 97-129, 1992). It is also one of the few viruses that can integrate its DNA into undivided cells, and have a high frequency of stable integration (see, for example, Flotte et al., Am. J. Respir Cell. Mol. Biol. 7: 349 -356, 1992; Samulski ef al., J. Viroi 63: 3822-3828, 1989; and McLaughlin ef al., J. Virol. 62: 1963-1973, 1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can be integrated. The space for exogenous DNA is limited to approximately 4. 5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5: 3251-3260, 1985 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., PNAS USA 81: 6466-6470, 1984; Tratschin et al., Mol. Cell. Biol. 4: 2072 -2081, 1985, Wondisford et al., Mol.Endocrinol., 2: 32-39, 1988, Tratschin et al., J. Virol., 51: 611-619, 1984, and Flotte et al., J. Biol. Chem. 268: 3781-3790, 1993). Other viral vector systems that may have application in gene therapy have been derived from the herpes virus, vaccinia virus, and various RNA viruses. In particular, herpes virus vectors may provide a unique strategy for persistence of the subject recombinant gene in cells of the central nervous system and ocular tissue (Pepose er al., Invest Ophthalmol Vis Sci 35: 2662-2666, 1994). In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a subject protein in the tissue of an animal. Most non-viral methods of gene transfer depend on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, the gene delivery systems do not turn! of the present invention depend on endocytic sequences for the uptake of the gene subject by the target cell. Exemplary gene delivery systems of this type include liposome-derived systems, poly-lysine conjugates, and artificial viral layers. In a representative embodiment, a gene encoding a subject polypeptide can be trapped in liposomes that carry positive charges on its surface (e.g., lipofectins) and (optionally) that are labeled with antibodies against cell surface antigens of the target tissue (Mizuno et al. al., No Shinkei Geka 20: 547-551, 1992, PCT publication WO91 / 06309, Japanese patent application 1047381, and European patent publication EP-A-43075). For example, lipofection of neuroglioma cells can be carried out using liposomes labeled with monoclonal antibodies against antigen associated with glioma (Mizuno et al., Neurol, Med. Chir. 32: 873-876, 1992). In still another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand that is crosslinked with an agent that binds the gene as poly-lysine (see, for example, PCT publications WO93 / 04701, W092 / 22635 , WO92 / 20316, W092 / 19749 and WO92 / 06180). For example, the subject gene construct can be used to transfect specific cells in vivo using a soluble polynucleotide carrier comprising an antibody conjugated to a polycation, eg, poly-lysine (see U.S. Patent 5,166,320). It will also be appreciated that the effective delivery of the subject nucleic acid constructs via peptide-mediated endocytosis can be improved by using agents that enhance the escape of the gene from the endosomal structures. For example, all adenoviruses or fusogenic peptides of the influenza gene product HA can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., Science 260-926, 1993; Wagner et al. ., PNAS USA 89: 7934, 1992, and Christiano et al., PNAS USA 90: 2122, 1993). In clinical settings, the gene delivery systems can be introduced into a patient by any number of methods, each of which is familiar in the art. For example, a pharmaceutical preparation of the gene delivery system can be introduced systemically, for example, by intravenous injection, and specific transduction of the construct into target cells that occur predominantly from the transfection specificity provided by the delivery vehicle of the invention. gene, cell type or tissue type expression due to the transcriptional regulatory sequences that control the expression of the gene, or a combination thereof. In other embodiments, the initial supply of the recombinant gene is more limited with introduction into the animal that is completely localized. By example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al., PNAS USA 91: 3054-3057, 1994). In addition, the subject proteins can provide as a fusion polypeptide together with a second peptide that promotes "transcytosis", eg, uptake of the peptide by target cells. To illustrate, the subject protein can be provided as part of a fusion polypeptide with all or a fragment of the N-terminal domain of the HIV Tat protein, eg, Tat residues 1-72 or a smaller fragment thereof that can promote the expression of transcytosis. In another embodiment, the subject polypeptide can be provided as a fusion polypeptide with all or a portion of the Antennapedia III protein. Synthetic peptides have also been used effectively for transport proteins, peptides and small molecules through biological membranes that include blood brain barriers and therefore, may be applicable to this application (Rothbard et al., Nat Med. 2000, 6 (11): 1253-7; Rothbard et al., J Med Chem. 2002, 45 (17): 3612-8). While examples of synthetic protein transduction sequence are provided, they are characterized by a high density of arginine residues, other similar functionality but molecules or dissimilar sequences could be substituted structurally To further illustrate, the subject (or peptide mimetic) polypeptide can be provided as a chimeric peptide that includes a heterologous peptide sequence ("internalization peptide" or "internalization domain") that triggers the translocation of an extracellular form of a polypeptide attached through a cell membrane to facilitate intracellular localization of the subject polypeptide. In this regard, the subject therapeutic polypeptide is one that is intracellularly active. The internalization peptide, by itself, is capable of traversing a cell membrane by, for example, transcytosis, at a relatively high speed. The internalization peptide is conjugated, for example, as a fusion protein, to the subject polypeptide, optionally in a dissociable manner. The resulting chimeric peptide is transported in cells at a higher rate relative to the activating polypeptide alone, thus providing a means to improve its introduction into the cells to which it is applied, for example, to improve topical applications of the subject polypeptide. In addition to proteins and peptidomimetics, a drug agent can be coupled to a compound that improves delivery to a substance (e.g., receptor-mediated compounds such as Vitamin B12). In one embodiment, the internalization peptide is derived from the Antennapedia Drosophila protein, or homologues of the same The 60 amino acids along the homeodomain of the homeo-protein antennapedia has been shown to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled. See, for example, Derossi ef al. (1994) J Biol Chem 269: 10444-10450; and Pérez et al. (1992) J Cell Sci 102: 717-722. It has been shown that fragments as small as 16 amino acids along this protein are sufficient to trigger internalization. See Derossi ef al. (1996) J Biol Chem 271: 18188-18193. The present invention also provides a polypeptide (small t antigen or VDAC) or peptidomimetic sequence as described herein, and at least a portion of the Antennapedia protein (or homologue thereof) sufficient to increase the transmembrane transport of the protein chimeric, relative to the subject polypeptide or peptidomimetic, for a statically significant amount. Such a polypeptide or peptidomimetic thereof can be used in subject methods to assist in efficient and specific killing of cancer cells. Another example of an internalization peptide is the HIV transactivator protein (TAT). This protein appears to be divided into four domains (Kuppuswamy et al. (1989) Nucí Acids Res. 17: 3551-3561). The purified TAT protein is absorbed by cells in tissue cultures (Frankel and Pabo, (1989) Cell 55: 1189-1193), and peptides, such as the fragment corresponding to residues 37-62 of TAT, are rapidly absorbed by the cell in vitro (Green and Loewenstein, (1989) Cell 55: 1179-1188) . The highly basic region mediates the internalization and direction of the internalization portion to the nucleus (Rubén et al., (1989) J. Virol. 63: 1-8). Another exemplary transcellular polypeptide can be generated to include a sufficient portion of mastoparan (T. Higcshijima et al., (1990) J. Biol Chem. 265: 14176) to increase the transmembrane transport of the chimeric protein.

While not wishing to be bound by any particular theory, it is noted that the hydrophilic polypeptides can also be transported physiologically through the membrane barriers by coupling or conjugating the polypeptide to a transportable peptide that is capable of traversing the membrane by transcytosis mediated by the receiver. Suitable internationalization peptides of this type can be generated using all or a portion of, for example, a histone, insulin, transferrin, basic albumin, prolactin and growth factor I as insulin (IGF-I), growth factor II as insulin ( IGF-II) or other growth factors. For example, it has been found that an insulin fragment, which shows affinity for the insulin receptor in hair cells, and that is less effective than insulin in reducing blood sugar, is capable of transporting the insulin. transmembrane by transcytosis mediated by the receptor and can therefore serve as an internalization peptide for the subject transcellular peptides and peptidomimetics. Preferred growth factor-derived internalization peptides include peptides derived from EGF (epidermal growth factor), such as CMH I ESLDSYTC and CMYIEALDKYAC; peptides derived from TGF-beta (transforming beta growth factor); peptides derived from PDGF (platelet-derived growth factor) or PDGF-2; peptides derived from IGF-I (growth factor as insulin) or IGF-II; and peptides derived from FGF (fibroblast growth factor). Another class of translocation / internalization peptides has a pH-dependent membrane bond. For an internalization peptide that assumes a helical conformation at an acid pH, the internalization peptide acquires the property of amphiphilicity, for example, it has both hydrophobic and hydrophilic interfaces. More specifically, within a pH range of about 5.0-5.5, an internalization peptide forms an alpha-helical, amphiphilic structure that facilitates insertion of the portion into a target membrane. An environment of acidic pH that induces the alpha-helix can be found, for example, in the low pH environment present within cellular endosomes. Such internalization peptides can be used to facilitate transport of Subject polypeptide and peptidomimetics, are absorbed by an endocytic mechanism, from compartments endosomal to the cytoplasm. An internalization peptide that binds the preferred pH-dependent membrane includes a high percentage of residues that form the helix, such as glutamate, methionine, aianine and leucine. In addition, a preferred internalization peptide sequence includes residues having pKa's within the range of pH 5-7, such that a sufficient uncharged membrane binding domain will be present within the peptide at pH 5 to allow insertion into the membrane of white cell. A particularly preferred pH dependent membrane binding internalization peptide in this regard is aa1 -aa2-aa3-EAALA (EALA) 4-EALEALAA-amide, which represents a modification of the Subbarao peptide sequence ef al. (Biochemistry 26: 2964, 1987). Within this peptide sequence, the first amino acid residue (aa1) is preferably a single residue, such as cysteine or lysine, which facilitates chemical conjugation of the internalization peptide to a targeting protein conjugate. Residues of amino acids 2-3 can be selected to modulate the affinity of the internalization peptide for different membranes. For example, if both residues 2 and 3 are lys or arg, the internalization peptide will have the ability to bind to membranes or lipid patches that have a negative surface charge. If residues 2-3 are neutral amino acids, the internalization peptide will be inserted into neutral membranes. Still other preferred internalization peptides of apo-lipoprotein A-1 and B; peptide toxins, such as melittin, bombolitin, delta-hemolysin and the pardaxins; antibiotic peptides, such as alamethycin; peptide hormones, such as calcitonin, corticotrophin releasing factor, beta endorphin, glucagon, parathyroid hormone, pancreatic polypeptide; and peptides corresponding to signal sequences of numerous secreted proteins. In addition, exemplary internalization peptides can be modified through the binding of substituents that improve the alpha-helical character of the internalization peptide at acidic pH. Yet another class of internalization peptides suitable for use within the present invention includes hydrophobic domains that are "hidden" at physiological pH, but are exposed in the lower pH environment of the target cell endosome. Under pH-induced cleavage and exposure of the hydrophobic domain, the portion binds to lipid bilayers and effect translocation of the polypeptide covalently bound in the cell cytoplasm. Such internalization peptides can be modeled after the sequences identified in, for example, Pseudomonas exotoxin A, Clathrin, or Diphtheria toxin. Proteins or peptides that form the pore can also serve as internalization peptides in it. The proteins or peptides that form the pore can be obtained or derived from, for example, C9 complement protein, cytolytic T cell molecules or NK cell molecules. These portions are capable of forming membrane-like structures, thus allowing transport of the bound polypeptide through the membrane and into the interior of the cell. The pure membrane intercalation of an internalization peptide may be sufficient for translocation of the subject or peptidomimetic polypeptide, through the cell membranes. However, the translocation can be improved by binding to the internalization peptide a substrate for intracellular enzymes (ie, an "accessory peptide"). It is preferred that an accessory peptide be linked to a portion or portions of the internalization peptide that protrudes through the cell membrane to the cytoplasmic surface. The accessory peptide can be advantageously linked to a term of a translocation / internalization portion or anchor peptide. An accessory portion of the present invention may contain one or more amino acid residues. In one embodiment, an accessory portion can provide a substrate for cellular phosphorylation (for example, the accessory peptide can contain a tyrosine residue).

An exemplary accessory portion in this regard would be a peptide substrate for N-myristoyl transferase, such as GNAAAARR (Eubanks et al., In: Peptides, Chemistry and Biology, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp. 566-69). In this construct, an internalization peptide would be attached to the C-terminus of the accessory peptide, since the N-terminal glycine is critical for the activity of the accessory portion. This hybrid peptide, under binding to an E2 peptide or peptidomimetic in its C-term, is N-myristylated and further anchored to the target cell membrane, for example, serves to increase the local concentration of the peptide in the cell membrane. To further illustrate the use of an accessory peptide, a phosphorylatable accessory peptide is first covalently linked to a C-terminus of an internalization peptide and then incorporated into a fusion protein with a subject or peptidomimetic polypeptide. The peptide component of the fusion protein intercalates into the white cell plasma membrane and, as a result, the accessory peptide is translocated through the membrane and protrudes into the cytoplasm of the target cell. On the cytoplasmic side of the plasma membrane, the accessory peptide is phosphorylated by cellular kinases at neutral pH. Once phosphorylated, the accessory peptide acts to irreversibly anchor the fusion protein in the membrane. The location for the cell surface membrane can improve the translocation of the polypeptide in the cellular cytoplasm. Suitable accessory peptides include peptides that are kinase substrates, peptides that possess a single positive charge, and peptides that contain sequences that are glycosylated by membrane bound glycosyltransferases. Accessory peptides that are glycosylated by membrane bound glycosyltransferases can include the sequence x-NLT-x, where "x" can be for example another peptide, an amino acid, coupling agent or hydrophobic molecule. When this hydrophobic tripeptide is incubated with microsomal vesicles, through vesicular membranes, it is glycosylated on the luminal side, and is trapped inside the vesicles due to its hydrophilicity (C. Hirschberg et al., (1987) Ann. Rev. Biochem 56: 63-87). Accessory peptides containing the sequence x-NLT-x in this manner will improve retention of the target cell of the corresponding polypeptide. In another embodiment of this aspect of the invention, an accessory peptide can be used to enhance the interaction of a polypeptide or peptidomimetic with the target cell. Exemplary accessory peptides in this regard include peptides derived from cell adhesion proteins containing the sequence "RGD", or laminin-derived peptides containing the sequence CDPGYIGSRC. Extracellular matrix glycoproteins, such as fibronectin and laminin, come together to cell surfaces through processes measured by the receptor. A tripeptide sequence, RGD, has been identified as necessary to bind to cell surface receptors. This sequence is present in fibronectin, vitronectin, complement C3bi, von-Willebrand factor, EGF receptor, transforming beta growth factor, type I collagen, E. coli lambda receptor, fibrinogen and protein with Sindbis coat (E. Ruoslahti , Ann. Rev. Biochem. 57: 375-413, 1988). Cell surface receptors that recognize RGD sequences have been grouped into a superfamily of related proteins designated "integrins". The binding of "RGD peptides" to cell surface integrins will promote cell surface retention, and ultimately translocation of the polypeptide. As described above, the internalization and accessory peptides can each, independently, be added to the polypeptide or peptidomimetic by either chemical crosslinking or in the form of a fusion protein. In the case of fusion proteins, unstructured polypeptide linkers can be included between each of the peptide portions. In general, the internalization peptide will be sufficient for the direct export of the polypeptide. However, when an accessory peptide, such as an RGD sequence, is provided, it may be necessary to include a signal sequence of secretion to export directly from the fusion protein of your host cell. In preferred embodiments, the secretion signal sequence is located at the N-terminus, and is (optionally) flanked by a proteolytic site between the secretion signal and the rest of the fusion protein. In an exemplary embodiment, a polypeptide or peptidomimetic is genetically modified to include a nuclear localization signal of RGD / SV40 peptide that binds the integrin (see, for example Hart SL et al., 1994; J. Biol. Chem., 269: 12468-12474), as encoded by the nucleotide sequence provided in the Nde1-EcoR1 fragment: catatggutgactgccgtggcgatatgttcggttgcggtgctcctccaaaaaagaagaga aaggtagctggattc, which encodes the nucleotide sequence RGD / SV40: MGGCRGDMFGCGAPPKKKRKVAGF. In another embodiment, the protein can be genetically modified HIV-1 tat (1-72) polypeptide, for example by Nde1-EcoR1 fragment: catatggagccagtagatcctagactagagccctggaagcatccaggaagtcagccta aaactgcttgt-accaattgctattgtaaaaagtgttgctttcattgccaagtgtttcataacaaaagcccttgg catctcctatggcaggaagaagcgagacagcgacgaagacctcctcaaggcagtcag actcatcaagtttctctaagtaagcaaggattc, coding sequence of HIV-1 tat peptide ( 1-72): MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCF-HCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ. In yet another embodiment, the fusion protein includes HSV-1 VP22 polypeptide (Elliott G., O'Hare P (1997) Cell, 88: 223-233) provided by the Nde1-EcoR1 fragment. In yet another embodiment, the fusion protein includes the C-terminal domain of the VP22 protein from, for example, the nucleotide sequence (fragment of Nde1-EcoR1). In certain cases, it may also be desirable to include a nuclear localization signal as part of the subject polypeptide. In the generation of fusion polypeptides including a polypeptide, it may be necessary to include unstructured linkers to ensure proper folding of the various peptide domains. Many synthetic and natural linkers are known in the art and can be adapted for use in the present invention, including the linker (Gly3Ser) 4. Methods of Treatment In certain embodiments, the invention provides a method for treating or preventing cancer in an individual. The terms "cancer", "tumor" and "neoplasia" are used interchangeably herein. As used herein, a cancer (tumor or neoplasm) is characterized by one or more of the following properties: cell growth is not regulated by normal biochemistry and physical influences in the environment; anaplasia (for example, lacks normal coordinated cell differentiation); and in some cases, metastasis. Cancerous diseases include, for example, carcinoma anal, bladder carcinoma, breast carcinoma, cervical carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukemia, head and neck carcinoma, lung carcinoma (small cell), multiple myeloma, lymphoma no Hodgkin, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung carcinoma (non-small cell), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, and tissue sarcoma soft. Additional cancer disorders can be found in, for example, Isselbacher er al. (1994) Harrison's Principles of Internal Medicine 1814-1877, incorporated herein by reference. Typically, the cancers described above and which are treatable by the methods described herein exhibit deregulated VDAC expression. In one embodiment, the cancers described above contain a mutation in the Ras signaling sequence, resulting in high Ras signaling activity. For example, the mutation could be a constitutively active mutation in the Ras gene, such as Ras V12. In other embodiments, the cancer may contain loss of function mutations in PP2A, and / or activation mutations of MEK1 and / or ERK1. In certain additional embodiments, the cancer is characterized by cells that express the SV40 small oncoprotein t, or are phenotypically similar to cells expressing sT, and / or oncogenic HRAS. In certain preferred embodiments, the cells express substantially wild type level of Rb (eg, at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, or 150 %, etc.). In one embodiment, the invention relates to a method of treating or preventing cancer in an individual, comprising administering to the individual a therapeutically effective amount of a compound that is selectively toxic to a genetically modified human tumorigenic cell, or a genotype cancer cell. specific (or specifically altered genotype). In certain embodiments, the cancer is characterized by cells expressing SV40 small oncoprotein T, or having modulations of oncogenic sT and / or RAS targets. In a related embodiment, the invention contemplates practicing the method of the invention in conjunction with other anti-tumor therapies such as conventional chemotherapy directed against solid tumors and for controlling the establishment of metastases. The administration of the compounds of the invention can be conducted during or after chemotherapy. Such agents are typically formulated with a pharmaceutically acceptable carrier, and can be administered intravenously, orally, buccally, parenterally, by an inhalation atomizer, by topical application or transdermally. An agent can also administered by local administration. Preferably, one or more additional agents administered in conjunction with an anti-cancer chemotherapeutic agent (e.g., a compound of the invention) inhibits cancer cells in an additive or synergistic manner. A wide series of conventional compounds have been shown to have anti-tumor activities. These compounds have been used as pharmaceutical agents in chemotherapy to contract solid tumors, prevent metastasis and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-tumor compounds induce undesirable side effects. In many cases, when two or more different treatments are combined, the treatments can treat synergistically and allow the dosage reduction of each treatment, thus reducing the harmful side effects exerted by each compound at higher doses. In other cases, malignancies that are refractory to one treatment may respond to a combination therapy of two or more different treatments. Therefore, compounds and pharmaceutical compositions of the present invention can be co-administered with a conventional anti-tumor compound. The compounds conventional anti-tumor include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, luorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide , imatimib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate , pentostatin, plicamycin, porfimer, proca rbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. In other embodiments, compounds and pharmaceutical compositions of the present invention can be co-administered with a conventional anti-tumor compound selected from: a receptor antagonist of EGF, arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine, carminomycin, mechlorethamine hydrochloride, pentamethylmelamine, thiotepa, teniposide, cyclophosphamide, chlorambucil, demethoxy hypocrellin A, melphalan, ifosfamide, trofosfamide, treosulfan, podophyllotoxin, or podophyllotoxin derivatives, etoposide phosphate , teniposide, etoposide, leurosidine, leurosin, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, metopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, ion 1 -methyl-4- phenylpyridinium, semustine, staurosporine, streptozocin, phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin, deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin, epirubican, pir arubican, zorubicin, mitoxantrone, bleomycin sulfate, actinomycin D, safracins, saframycin, quinocarcin, discodermolyds, vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel, tamoxifen, raloxifene, thiazofuran, thioguanine, ribavirin, EICAR, estramustine, estramustine sodium phosphate , flutamide, bicalutamide, buserelin, leuprolide, pteridines, enediins, levamisole, aflacone, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, betamethasone, gemcitabine hydrochloride, verapamil, VP-16, altretamine, tapsigargin, oxaliplatin, iproplatin, tertraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin, docetaxel, deoxygenated paclitaxel, TL-139, 5'- nor-anhydrovinblastine (hereafter: 5'-nor-vinblastine), camptothecin, irinotecan (Camptosar, CPT-11), topotecan (Hycamptine), BAY 38-3441, 9-nitrocamptothecin (Oretecin, rubitecan), exatecan (DX) -8951), lurtotecan (G I-147211 C), gimatecan, diflomotecan homocamptothecin (BN-80915) and 9-aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), indolocarbazoles (for example, NB) -506), protoberberins, intoplicins, idenoisoquinolones, benzo-phenazines or NB-506. In another related embodiment, the invention contemplates the practice of the method in conjunction with other anti-tumor therapies such as radiation. As used herein, the term "radiation" is intended to include any treatment of a neoplastic cell or subject by photons, neutrons, electrons, or other type of ionizing radiation. Such radiations include, but are not limited to, X-rays, gamma radiation, or heavy ion particles, such as alpha or beta particles. Additionally, the radiation can be radioactive. Means for irradiating neoplastic cells in a subject are well known in the art and include, for example, external beam therapy, and brachytherapy.

Methods for determining whether a cancer (tumor or neoplasm) has been treated are well known to those skilled in the art and include, for example, a decrease in the number of tumor cells (e.g., a decrease in cell proliferation or a decrease in the size of the tumor). It is recognized that the treatment of the present invention may be a constant and complete response or may encompass a partial or transient response. See, for example, Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference. Assays to test sensitization or increased death of tumor cells are well known in the art, including, for example, standard dose response assays that assess cell viability; agarose gel electrophoresis of DNA extractions or flow cytometry to determine DNA fragmentation, a characteristic of cell death; assays that measure the activity of polypeptides involved in apoptosis; and assay for morphological signs of cell death. The details regarding such tests are described elsewhere herein. Other assays include chromatin assays (eg, counting the condensed nuclear chromatin frequency) or drug resistance assays as described in, for example, Lowe et al. (1993) Cell 74:95 7-697, incorporated herein by reference. See also U.S. Patent No. 5,821,072, also incorporated herein by reference. Pharmaceutical Compositions Prospective therapeutic agents can be profiled to determine their adaptability for inclusion in a pharmaceutical composition. A common measurement for such agents is the therapeutic index, which is the ratio of the therapeutic dose to a toxic dose. Thresholds for therapeutic doses (efficacy) and toxic doses can be adjusted as appropriate (eg, the need for a therapeutic response or the need to minimize a toxic response). For example, a therapeutic dose may be the therapeutically effective amount of an agent (relative to the treatment of one or more conditions) and a toxic dose may be a dose that causes death (eg, an LD50) or causes an undesirable effect on a proportion of the population treated. Preferably, the therapeutic index of an agent is at least 3, more preferably at least 5, and even more preferably at least 10. Profiling a therapeutic agent may also include the measurement of the pharmacokinetics of the agent, to determine its bioavailability and / or absorption when administered in various formulations and / or via different routes. A compound of the present invention, such as erastin or a tubulin inhibitor, can be administered to an individual in need thereof. In certain embodiments, the individual is a mammal such as a human being, or a non-human mammal. When administered to an individual, the compound of the invention can be administered as a pharmaceutical composition containing, for example, the compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or carriers such as glycols, glycerol, oils such as olive oil or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, the aqueous solution is free of pyrogens, or substantially free of pyrogens. The excipients may be chosen, for example, to effect delayed release of an agent or for target selectivity of one or more cells, tissues or organs.

A pharmaceutically acceptable carrier may contain physiologically acceptable agents that act, for example, to stabilize or to increase the absorption of a compound such as erastin or a tubulin inhibitor. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including an agent Physiologically acceptable, depends, for example, on the route of administration of the composition. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, which may be incorporated herein, for example, a compound of the invention. Liposomes, for example, consisting of phospholipids or other lipids, are non-toxic, physiologically acceptable and metabolizable carriers that are relatively simple to process and administer. A pharmaceutical composition (preparation) containing a compound of the invention can be administered to a subject by any of a variety of administration routes including, for example, orally; intramuscularly; intravenously anally; vaginally parenterally; nasally; intraperitoneally; subcutaneously; and topically. The composition can be administered by injection or by incubation. In certain embodiments, the compound (e.g., erastin) of the present invention can be used alone or in conjunction with another type of anti-tumor therapeutic agent. As used herein, the phrase "co-administration" refers to any form of administration in combination of two or more different therapeutic compounds such that the second compound is administered while the therapeutic compound previously therapeutic is still effective in the body (for example, the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In this way, an individual who receives such treatment may benefit from a combined effect or different therapeutic compounds. It is contemplated that the compound (e.g., erastin) of the present invention will be administered to a subject (e.g., a mammal, preferably a human) in a therapeutically effective amount (dose). By "therapeutically effective amount" is meant the concentration of a compound that is sufficient to produce the desired therapeutic effect (eg, treatment of a condition, death of a neoplastic cell). It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors that influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent that is administered with the compound of the invention. Typically, for a human subject, an effective amount will vary from about 0. 001 mg / kg of body weight to approximately 50 mg / kg of body weight. A larger total dose can be supplied by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996) Harrlson's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference. EXAMPLIFICATION The invention which is in general now described will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. EXAMPLE 1 Identification of Compounds with Power or Augmented Activity in the Presence of Specific Cancer-Related Alleles The work described here is carried out to identify compounds with potency or increased activity in the presence of hTERT, LT, ST, E6, E7 or RASV12. Although the work described here makes use of hTERT, LT, ST, E6, E7 and RASV12 as transforming genes, future studies can make use of a wide variety of alleles associated with cancer using this methodology to define signaling networks that involve many oncogenes and suppressors smokers. Genetically modified cell lines with these genetic elements were used to project 23,550 compounds, including 20,000 compounds from a combinatorial library, 1990 compounds from the National Cancer Institute's diversity collection, and 1,540 known biologically active compounds that were selected and purchased by the Applicant and formatted in a projected collection. The primary projection tested (in quadruplicate) the effect to treat tumorigenic cells genetically modified of BJ-TERT / LT / ST / RASV Z tumorigenic with each compound during 48 hours at a concentration of 4 pg / mL, corresponding to 10 μ? for a compound with a molecular weight of 400, which is the approximate average molecular weight of the libraries. Cell viability was measured using calcein acetoxymethylester dye (AM calcein) (Wang et al., 1993, Hum Immunol 37, 264-270), which is a non-fluorescent compound that diffuses freely in cells. In live cells, calcein AM is dissociated by intracellular esterases, forming the fluorescent anionic derivative calcein, which can not be diffused out of living cells. Here, living cells show a green fluorescence when incubated with calcein AM, while dead cells do not. Compounds presenting 50% or greater inhibition of staining with calcein AM dye viability in J-TERT / LT / ST / RASV12 B cells were subsequently tested in a double dilution series in BJ and BJ-TERT / LT / ST / RASV12 cells to identify compounds that exhibit sufficient lethality, which is lethality in tumbrigenic cells but not in isogenic primary cells. The IC 50 value (concentration required to inhibit 50% of the calcein AM signal) was calculated for each compound in each cell line (Table 1). This results in the identification of nine compounds (Figure 2) that were at least four times more potent in tumorigenic BJ-TERT / LT / ST / RASV12 cells relative to primary BJ cells (compounds for which at least one concentration four times higher was required in primary BJ cells to obtain the same 50% inhibition of the calcein AM signal). Then there is a more detailed analysis of these nine compounds. Three of these compounds (doxorubicin, daunorubicin and mitoxantrone) are in current clinics used as anti-cancer drugs, one (camptothecin) is a natural analog product of clinically used anticancer drugs (topotecan and irinotecan), and one (equinomycin) was recently tested in phase II clinical trials. All nine compounds were subsequently tested to replicate in multiple doses and each panel of genetically modified cells to confirm that the observed selectivities were seen in multiple independently derived cell lines (Figure 1 and Table 1).

The Applicants developed a metric selectivity that measures the change in IC50 (concentration required for 50% inhibition of viability signal) of a compound in two different cell lines. To calculate this selectivity result between two cell lines, the IC50 value for a compound in a cell line was divided by the IC50 value for the same compound in a second cell line. Thus, a compound to be used at a four-fold higher concentration in a cell line relative to a second cell line would have a selectivity result of 4. The "tumor selectivity result" was calculated for each compound, dividing the IC5o value for the compound in primary, parental BJ cells by the IC50 value for the compound in genetically modified BJ-TERT / LT / ST / RASV12 cells, containing all four genetic elements required to create tumorigenic cells ( Table 1) · These genetically modified tumorigenic cells make use of dominantly acting viral oncoproteins such as LT, ST, E6 and E7. These viral proteins are possibly involved in cell transformation in specific forms of cancer, namely malignant mesothelioma induced by simian virus 40 (Testa and Giordano, 2001, Semin Gancer Biol 11, 31-8) and cervical carcinoma induced by the virus. human papilloma (Bosch et al., 2002, J. Clin Pathol 55, 244- 65), and have been used to interrupt p53 and pRB function to transform cells in vitro and in vivo (Elenbaas et al., 2001, Genes Dev 15, 50-65, Jorcyk et al., 1998, Prostate 34, 10- 22, Perez-Stable et al., 1997, Cancer Res 57, 900-6, Rich et al., 2001, Cancer Res 61, 3556-60, Sandmoller et al., 1995, Cell Growth Differ 6, 97-103). . Applicants make use of these two different methods to inactivate cellular proteins (the effects of both LT-based and E6 / E7 inactivation of pRB and p53 have been tested) for control of the kinosyncratic effects that must be observed with a specific viral protein. . The selectivity of these compounds was also confirmed in a cell line expressing dominant negative inhibitors of p53 and pRB that are not derived from viral elements. This cell line expresses (i) a truncated form of p53 (p53DD) that interrupts the tetramerization of endogenous p53, (ii) a mutant CDK4R24C resistant to inhibition by p16IN 4A and p15lNK B (the main negative regulators of CDK4) and (iii) Cyclin D1. The effects of the nine selective genotype compounds were tested at a range of concentrations in those cells, which are referred to as BJ-TERT / p53DD / CDK4R24C / D1 / ST / RASV12 cells (Table 1). The results show that there is a modest overall reduction in activity for all of the compounds when tested in these cells. However, the total results of the analyzes were not changed by the use of non-viral proteins in this cell line (Table 1). EXAMPLE 2 Determination of the genetic basis of the selectivity of the compounds The Applicants determine the genetic basis of selectivity for each compound. That is, for each compound, an attempt is made to define the gene or combination of genes responsible for providing cells sensitive to the compound (Table 1). The results show that these nine compounds could be categorized into three groups, namely (i) compounds that do not exhibit simple genetic selectivity, (ii) compounds that exhibit selectivity for cells harboring TERT and inactive RB, and (iii) compounds that require I ?? presence of both RAS and ST oncogenes to present lethality. The compounds in group (i), sangivamycin, bouvardine, NSC146109 and equinomycin, have no clear genetic basis for their tumorigenic cell selectivity. For example, equinomycin becomes somewhat more active as each genetic element is introduced (Figure 3a). The Applicants have observed that the rate of cell proliferation increases when each of these genetic elements is introduced. In this way, it is likely that the compounds in group (ii) are simply selective for rapidly dividing cells. Supporting this interpretation is the fact that all of these compounds are reported to act by inhibiting DNA or protein synthesis, the need for which is greater in rapidly dividing cells. For example, equinomycin is reported to function as a bis-DNA intercalator (Van Dyke and Dervan, 1984, Science 225, 1122-7; Waring and Wakelin, 1974, Nature 252, 653-7), bouvardina is reported to work as an inhibitor of protein synthesis (Zlacain er al., 1982, FEBS Lett 148, 95-7), sangivamycin is a nucleotide analogue (Rao, 1968, J Med Chem 11, 939-41), and NSC146109 structurally resembles a DNA intercalator (Figure 2). It should be noted that sangivamycin has been reported to function as an inhibitor of PKC (Loomis and Bell, 1988, J Biol Chem 263, 1682-92), although this activity seems unlikely to be relevant in this context because other PKC inhibitors they show no selectivity in this system. The Applicants were able to identify compounds that are simply more active in rapidly dividing cells, such as this group (i) compounds, because they do not show clear genetic basis for selectivity. Without additional work these compounds were made. In this way, they were able to focus mechanistic studies on the compounds in groups (ii) and (iii), which exhibit selectivity. The compounds in group (ii), mitoxantrone, doxorubicin and daunorubicin, are topoisomerase II poisons, which bind to topoisomerase II and DNA and prevent religation of double-stranded DNA breaks introduced by topoisomerase II. These compounds, and anthracyclines in general, have also been reported to induce the formation of reactive oxygen species (ROS) in some cell types (Laurent and Jaffrezou, 2001, Blood 98, 913-24, Muller et al., 1998 , Int J Mol Med 1, 491-4; Richard et al., 2002, Leuk Res 26, 927-31), although the Applicants do not observe ROS formation in these genetically modified cells in the presence of these three compounds. It was found that these compounds become more potent (active at a lower concentration) when hTERT is introduced and again when RB is inactivated by introduction of LT or HPV E7. In cells, E7 was introduced after E6, so it is possible that the increased potency of these compounds in the cells harboring E7 also depends on the presence of E6, yet E6 itself does not confer increased potency to these compounds. The introduction of hTERT and inactivation of RB causes an increase in topoisomerase Ia expression (Figure 5A) and only a very modest increase in topoisomerase Ia expression. The introduction of oncogenic RAS causes a further increase in topoisomerase Ia expression, although the Applicants do not observe additional sensitization to topoisomerase II poisons in the presence of oncogenic RAS (Figure 5A). The compounds in group (III) are camptothecin (CPT) and a novel compound of a combinatorial library, whose Applicants have named erastin, for eradicating cells expressing RAS and S_J_ (Figure 2). Cell death induced by erastin and induced by efficient CPT requires the presence of both ST and RASV12 (Figures 3 and 4 and Table 1). Although CPT and erastin have a similar genetic basis of selectivity, they have different mechanisms of action. CPT is partially active in cells lacking RB function (via E7 expression), while erastin is not, and CPT requires two days to cause death in BJ-TERT / LT / ST / RASV12 cells, while the erastina is 100% effective within 18 hours (Figures 3 and 4). The okadaic acid phosphatase inhibitor was able to otherwise sensitize the primary BJ cells resistant to CPT (Figure 5E), possibly because the okadaic acid stimulates TOP1 (Figure 5F). Okadaic acid does not provide BJ or BJ-TERT cells sensitive to erastin, consistent with a model in which CPT and erastin act via different mechanisms. In addition, the Applicants found that the lethal compound of podophyllotoxin, a tubulin inhibitor, does not sensitize BJ or BJ-TERT cells to CPT, confirming that the sensitization of BJ cells to CPT by okadaic acid is specific and not the result of two weak cell death stimuli that have an additive, but a functionally irrelevant effect. In attempts to understand the molecular basis for increased sensitivity to CPT of cells expressing RAS and ST, the Applicants determined the level of expression in the genetically modified cells of topoisomerase I (TOP1), the putative target of CPT (Adoh et al., 1987, Proc Nati Acad Sci USA 84 , 5565-9; Bjornsti et al., 1989, Cancer Res 49, 6318-23; Champoux, 2000, Ann NY Acad Sci 922, 56-64; D'Arpa et al., 1990, Cancer Res 50, 6919-24 Eng et al., 1988, Mol Pharmacol 34, 755-60, Hsiang et al., 1989, Cancer Res 49, 5077-82, Hsiang and Liu, 1988, Cancer Res 48, 1722-6, Liu et al., 2000, Ann NY Acad Sci 922, 1-10, Madden and Champoux, 1992, Cancer Res 52, 525-32; Tsao er al., 1993, Cancer Res 53, 5908-14). It was found that cells expressing both RASV12 and ST stimulate TOP1 (Figure 5B). As putative mechanisms of CPT of action in other cell types implies a gain of function, namely introduction of double-strand DNA breaks in a TOP1-dependent manner (Liu et al., 2000, Ann NY Acad Sci 922, 1- 10), the stimulation of TOP1 could explain the increased sensitivity of cells expressing RASV12 and ST to CPT. In support of this interpretation, it was found that genetic inactivation of TOP1 with a small interfering RNA (siRNA) in J-TERT / LT / ST / RASV12 B cells confers partial resistance to CPT (Figure 5C, D). The Applicants additionally tested other erastin analogues for activity and selectivity in tumor cells versus normal cells. Another analogous compound was identified as active and selective, but less potent than erastin. This compound was named erastin B (see Figure 8). BJELR cells are BJ-TERT / LT / ST / RASV12 cells, and BJEH are BJ-TERT cells. Other compounds tested in both BJELR and BJEH cells are as follows: EXAMPLE 3 Characterization of cell death Requesters should characterize the type of cell death induced by CPT and erastin in tumorigenic cells BJ-TERT / LT / ST / RASV12. In other contexts, CPT has been found to induce apoptotic cell death (Tráganos et al., 1996, Ann NY Acad Sci 803, 101-10), which is characterized by alterations in nuclear morphology including piknosis, kariorhexis and / or chromatin marginalization. (Majno and Joris, 1995, Am J Pathol 146, 3-15). To determine if erastin or CPT induces apoptosis in their system, the Requesters verified the nuclear morphology of tumorigenic cells treated with CPT and erastin using fluorescence microscopy. Although kariorhexis and chromatin marginalization were clearly visible in cells treated with CPT, without such morphological alternation it was visible in cells treated with erastin (Figure 7A). Since the nuclear morphological change is required of apoptotic cells, the Applicants concluded that the cell death induced by erastin is not apoptotic. Support Additional to this conclusion were observations that the CPT, but o erastin, induces DNA fragmentation (which is the formation of a DNA ladder), that a pan-caspase inhibitor (50 μ? Boc-Asp (Ome) -fluoromethyl) -cetone, Sigma # B2682 (Chan et al., 2001, Neuroreport 12, 541-545)), partially blocked cell death induced by CPT, but not by erastin, and that CPT, but erastin, causes an increase in staining of Annexin V (Figure 7B) and the appearance of active, dissociated caspase 3 (Figure 7C). Additionally, the nuclei remain intact in tumor cells treated with erastin (Figure 9). The ability of erastin to induce non-apoptotic cell death is selective for cells expressing ST and RASV12. Larger treatments and higher concentrations of erastin have little effect on the viability of cells lacking RASV12 or ST, confirming the qualitative nature of erastin selectivity (Figure 6A, C). As the cells treated with erastin do not undergo apoptosis, Applicants must confirm that erastin genuinely induces cell death, rather than disinterest. Cell viability is quantified in the presence of erastin using Blue Alamar (Ahmed et al., 1994, J. Immunol.Modes 170, 211-224), a viability dye that measures the intracellular reductive potential. Erastin shows selective lethality in tumorigenic cells BJ-TERT / LT / ST / RASV12 in relation to cells BJ-TERT in this homogeneous Blue Alamar viability assay (Figure 6B). B cells J-TERT / LT / ST / RASV12 treated with erastin for 18 hours rounded up and separated (Figure 6C), failed to exclude the vital dye Tripane Blue, exhibits a loss of mitochondrial membrane potential as assayed by the dye potentiometric JC-1, and had a small cell size characteristic of dead cells. The Applicants determined that the loss of viability induced by erastin is irreversible once completed, in which BJ-TERT / LT / ST / RASV12 cells treated with erastin for 24 hours rounded up, separated and were unable to recover when repositioned in a erastina free medium. In this way, erastin induces non-apoptotic, irreversible (12-24 hours) rapid cell death in a ST-dependent and RASV12-dependent manner. Studies were also conducted that show that erastin induces the formation of reactive oxygen species (see Figure 10). Projections were made for suppressors (inhibitors) of erastin activity. All anti-oxidants that suppress erastin activity were identified, one of which was the anti-oxidant, α-tocopherol. The following methods and materials were used in the examples described herein.

Constructs and retroviruses The expression constructs for hTERT, LT, ST, SV40 Early Region, and HRASV12 were used as previously described (Hahn et al., 1999, supra; Hahn et al., 2002, supra)). The hTERT-pWZL-Blaste, E6-pWZL-zeoe, and E6E7-pWZL-Zeos were previously described (Lessnick et al., 2002, supra). The E6 and LT cDNAs were cloned into the retroviral vector pWZL-Hygroe (a species present of J. Morgenstern, Millenium Pharmaceuticals). Pseudo-reverted GV-glycoprotein retroviruses of vesicular stomatitis were prepared, and the infections were carried out as previously described (Lessnick et al., 2002, supra). Cell Lines TIP5 primary fibroblasts (Lessnick ef al., 2002, supra) were prepared from the neonatal foreskin discharged and immortalized by infection with hTERT-pWZL-blasts or hTERT-pBabe-hygro retroviruses and selection with either blasticidin or hygromycin , respectively. The BJ cells were a present of Jim Smith. Fibroblasts immortalized with hTERT were infected with the retroviruses indicated and selected for the appropriate markers. All BJ derivatives were cultured in a 1: 1 mixture of DMEM and M199 supplemented with 15% inactivated fetal bovine serum, penicillin and streptomycin (pen / strep). TIP5 cells were developed in DMEM containing 10% FBS and pen / estrep. All cell cultures were incubated at 37 ° C in a humidified incubator containing 5% C02. Composite libraries An annotated composite library (ACL) comprising 1, 540 compounds, a NCI diversity set of 1,990 compounds obtained from the National Cancer Institute and a combinatorial library (Comgenex International, Inc.) containing 20,000 compounds were used in the tumor-selective synthetic lethal projections. All the composite libraries were prepared as 4 mg / ml solutions in DM-SO in 384 well polypropylene plates (columns 3-22) and stored at -20 ° C. Camptothecin were obtained (cat # C9911, MW 348.4), doxorubicin (cat # D1515 MW 580.0), daunorubicin (cat # D8809, MW 564.0), mitoxantrone (cat # M6545, MW 517.4), okadaic acid (cat # 04511, MW 805.0 ), equinomycin (cat # E4392, MW 1101), sangivamycin (cat # S5895, MW 309.3) from Sigma-Aldrich Co. Bouvardina (MW 772.84) and NSC146109 (MW 280.39) were obtained from the Therapeutic Development Program of the National Cancer Institute. Erastin (MW 545.07) was obtained from Comgenex International, Inc. Calcein Acetoxymethylester AM (AM) Feasibility Assay is a non-fluorescent, cell membrane permeable compound that is dissociated by intracellular esterases to form the calcein Fluorescent compound, cell-impermeable, anionic. Viable cells are stained by calcein due to the presence of intracellular esterases and because the intact plasma membrane prevents fluorescein calcein from cell loss (Wang et al., 1993, supra). Cells were seeded in 384-well plates using a Zymark Sciclone ALH, treated with each compound in triplicate at 4 pg / mL in the primary projection for two days, washed with phosphate-buffered saline in a Packard Minitrak equipment with a 384 wells and incubated for four hours with 0.7 g / mL calcein (Molecular Probes). The total fluorescence intensity in each well was recorded in a Packard Fusion plate reader, and converted to a percent inhibition of signal by subtraction of the antecedent instrument and dividing by the average signal obtained when the cells were not treated with any compound. Feasibility Study of Blue Alamar The Alamar Blue was reduced by the activity of the mitochondrial enzyme in viable cells, causing both chlorimetric and fluorescent changes (Nociari et al., 1998, J. Immunol.Methods 13, 157-167). Cells were seeded at a density of 6000 cells (50 μ?) Per well in a 384 well, black bottom plate using a volume distribution syringe (Zymark). 10 μ? of a serially diluted erastin plate twice (final concentrations 6X) using a fixed cannula head 384, which makes the final concentration of 20 pg / ml in the well with the highest concentration. The plates were incubated for 24 hours. Blue Alamar (Biosource International) was added to each well by diluting 1:10 and incubated for 16 hours at 37 ° C. The fluorescence intensity was determined using a Packard Fusion plate reader with a excitation filter centered at 535 mm and a emission filter centered at 590 mm. The percentage of average inhibition in each concentration was calculated. The bar error indicates a standard deviation. The Alamar Blue test does not imply that it cleans the cells. Projection Descendant replica plates were prepared with a Zymark Sciclone ALH and integrated Twister II by diluting deposit plates 50 times in a medium lacking serum and pen / strep to obtain a compound concentration in descending plates of 80 pg / ml with 2 % of DMSO. Test plates were prepared by seeding cells in 384 well, clear bottom, black plates in columns 1-23 (6000 cells / well in 57 μ?) Using a volume distribution syringe. Columns 3-22 were treated with compounds from a downstream library plate by transferring 3 μ? of the descending library plate using fixed cannula array at position 384. The final compound concentrations in test plates were thus 4 pg / ml. The test plates are incubated for 48 hours at 37 ° C in a humidified incubator containing 5% C02. The plaque processing for the calcein AM viability assay was performed using an integrated robotic system Minitrak / Sidetrak from Packard Bioscience (Perkin Elmer). Test plates were washed with phosphate buffered saline, and 20 μ? Calcein AM (0.7 g / ml) per well. Plates were incubated at room temperature for 24 hours. The fluorescence intensity was determined using a Fusion plate reader with filters centered on an excitation of 485 nm and an emission of 535 nm. Retest or retest of compounds in a dilution series Compounds that are new assays were purchased from the manufacturers. Stock solutions were prepared in DMSO at a concentration of 1 mg / ml in polypropylene plates of 384 wells with a twice-diluted, 16-point dilution curve of each compound in a column, in duplicates. Column 1-2 and 23-24 were left empty for controls. Probe rejection plates were prepared from a tank retest plate by diluting 66.6 times in DMEM in 384 well deep-deep well plates (4.5 μ? Transfer in 300 μ?). The cells were seeded at a density of 6000 per well in 40 μ ?, and 20 μ? they were added from a descendant retest plate. The plates were incubated for two days at 37 ° C with 5% C02. Data Analysis A means of RFU (relative fluorescence units) was calculated for untreated cells by averaging columns 1, 2 and 23 (wells with cells but lacking compounds). The calcein background was calculated by averaging column 24 (wells with calcein, but lacking cells). The percent inhibition of each well was calculated as [1 - (RFU - calcein control) / (untreated cell - calcein control) * 100]. Compounds causing at least 50% inhibition of calcein staining in the primary projection were tested for selectivity to BJ-TERT / LT / ST / RASV12 genetically modified tumor cells when tested in BJ and BJ-TER ~ i7LT / ST / primary cells. RASV12 at a range of concentrations. New assays of selective compounds were made on all genetically modified cell lines. Nuclear Morphology Assay 200,000 tumorigenic BJ-TERT / LT / ST / RASV12 cells were seeded in 2 ml_ of one glass cover slip in each well of a six-well plate, treated with nothing (NT), 9 μm erastin. or 1.1 μ? of camptothecin (CPT) in a growth medium for 18 hours while it was incubated at 37 ° C with 5% C02. The nuclei were stained with 25 μg / mL of Hoechst 33342 (Molecular Probes) and checked using an oily 100X objective immersion in a microscope. fluorescence. Measurements of cell size 200,000 BJ-TERT / LT / ST / RASV12 cells were seeded in six-well dishes in 2 mL of a growth medium only (No treatment), with 9 μ? of erastine or with 1.1 μ? of camptothecin (CPT). After 24 hours, the cells were released with trypsin / EDTA, diluted with 10 mL in a growth medium, and the cell size distribution of each sample was determined in a Coulter Counter. Cell count assay for camptothecin activity J-TERT / LT / ST / RASV12 B cells were seeded in 6-well dishes (200,000 cells / well, 2 ml per well) and transfected in an antibiotic-free medium and in serum using Oligofectamine (Life Technologies), with 100 nM of siRNA per well in a total volume of one milliliter. 500 μ? of a medium containing 30% FBS was added 4 hours after transfection. The cells were treated with the indicated concentrations of camptothecin 30 hours after transfection. 500 μ? of a 5X solution of the desired camptothecin concentration was added to each well. The cells were removed with trypsin / EDTA and counted using 75 hours after transfection. The control experiments indicate that the transfection efficiency was approximately 10%.

Caspase-3 Western Blot Analysis BJ-TERT / LT / ST / RASV12 cells were seeded prior to the experiment in 5X105 cells in 60 mm dishes. The cells were treated with 5 pg / ml of erastin (9 μ?) For 2, 4, 6, 8 or 10 hours. One dish was maintained for treatment of camptothecin (0.4 pg / ml for 24 hours) as a positive control. The cells were used after each time point in the tisis buffer (50 mM HEPES KOH pH 7.4, 40 nM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3V0, 50 mM NaF, 10 mM of sodium pyrophosphate, 10 mM of sodium beta-glycerophosphate and one tablet of the protease inhibitor (Roche)). The protein content was quantified using a Biorad protein assay reagent. Equal amounts of protein were resolved in 16% SDS-polyacrylamide gel. The electrophoresed proteins were transtined in a PVDF membrane, blocked with 5% milk and incubated with a polyclonal anti-active caspase-3 antibody (BD Pharmingen) at 1: 1500 dilution overnight at 4 ° C. The membrane was then incubated in an anti-rabbit HRP (Santa Cruz Biotechnology) at 1: 3000 dilution for 1 hour and developed with an improved chemiluminescence mixture (NEN Ufe science, Renaissance). To test the equivalent load in each path, the stains are stabilized, blocked, and tested with an anti-elF-4E antibody (BD Transduction Laboratories) at 1: 1000 dilution. Topoisomerase The cells were seeded BJ, BJ-TERT, B J-TERT / LT / ST, BJ-TERT / LT / ST / RASV12, BJ-TERT / LT / RASV12 and BJ-TERT / LT / RAS 12 / ST in 1X106 cells per plate in 60 mm dishes. After overnight incubation of the cells at 37 ° C with 5% C02, the cells were used as described above and the proteins were resolved in a 10% polyacrylamide gel. The membrane was incubated with a monoclonal anti-human Na p170 topoisomerase antibody (TopoGEN) at a dilution of 1: 1000 overnight at 4 ° C and then with anti-mouse HRP (Santa Cruz Biotechnology). Topoisomerase 1 (TOP1) A double-stranded siRNA of 21 nucleotides directed against TOP1 (nuceotides 2233-2255, which number from the initial codon, Genbank access J03250) was synthesized (Dharmacon, purified and desalted / deprotected) and transfected (100 nM) in BJ-TERT / LT / ST / RASV12 cells in six-well dishes with oligofectamine (Life Technologies). After 75 hours, the cells were used and the expression level of TOP1 was determined by Western blot analysis (Topogen, Cat # 2012-2, dilution 1: 1000). The level of protein loading was determined by washing and polluting the same stain with an antibody against F-4E (BD Biosciences, Cat # 610270, dilution 1: 500). Alternatively, cells were seeded 1x10 in 60 mm dishes and grown overnight at 37 ° C with 5% C02, then used with 150 μ? of lysis buffer The cells were removed with a scraper and transferred to microcentrifuge tubes and incubated on ice for 30 minutes. The protein contents in the lysates were quantified using a Biorad protein estimation assay reagent. Equal amounts of protein were loaded on 10% gradient polyacrylamide-SDS gel. The electrophoresed proteins were transtined in a PVDF membrane. After blocking with 5% dry milk, the membrane was incubated with anti-human mouse topoisomerase I antibody (Pharmingen) overnight at 4 ° C, then with anti-mouse peroxidase conjugate antibody (Santa Cruz Biotechnology) . Annexin V-FITC Apoptosis Assay J-TERT / LT / ST / RASV12 B cells were seeded in 1X106 cells per dish in 100 mm dishes and allowed to grow overnight. The cells were treated with erastin (5 or 10 pg / ml) for 6, 8 or 11 hours. A control treated with camptothecin (0.4 pg / ml) was maintained, treated at the time of sowing for 20 hours. After the treatment, the cells were harvested with trypsin / EDTA and washed once with fresh medium containing serum and then twice with phosphate buffered saline. The cells were suspended in 1X-binding buffer (BD Pharmingen) at a concentration of 1X10 cells / ml. 100 μ? (1X105 cells) with 5 μ? of Annexin V-FITC (BD Pharmingen) and propidium iodide (BD Pharmingen) for 15 minutes in the dark at room temperature. Then 400 μ? of the 1X linker buffer and the cells were analyzed by flow cytometry (Becton-Dickinson). Data were acquired and analyzed using Cellquest software. Only viable cells that were not stained with propidium iodide for Annexin V-FITC staining using the FL1 channel were analyzed. ROS analysis: flow cytometric analysis using H2DCF-DA The 2 ', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) is a non-fluorescent cell permeable compound. The endogenous esterase enzyme within the cell dissociates the diacetate part, and may not spend more time outside the cell. In this way it accumulates in the cell. The H2DCF then reacts with ROS to form fluorescent dichlorofluorescene (DCF) which can be measured by flow cytometry in I channel FL1. 1. Sow cells at 3X105 cells per plate in plates 60 mm and allowed to grow overnight. 2. Treat with the test compound for a different period of time (1-10 hours). 3. Maintain a dish for untreated cells, a cell dish treated with compound and positive control (treated with hydrogen peroxide) for each time point. 4. Incubate the cells with 10 μ? of H2DCF-DA for 10 minutes at 37 ° C. 5. For positive control cells, after 5 minutes loading of H2DCF-DA, add 500 μ? of hydrogen peroxide and incubate for an additional 5 minutes. 6. Harvest the cell by trypsinization. 7. Wash with cold PBS twice. 8. Resuspend the pellet or granule in 100 μ? of PBS and transfer into a 5 ml FACS tube. 9. Add 5 μ? of propidium iodide (50 pg / ml) and incubate for 10 minutes on ice in the dark. 10. Add 400 μ? of PBS and analyze by flow cytometry (Becton-Dickinson). 11. Acquire the data and analyze using a software program CelIQuest. 12. Take only propidium iodide negative cells (viable cells) for the DCF staining analysis using the FL1 channel, Pl in the FL3 channel, plot a quadrant graph. ACL library projections for compounds that can suppress erastin activity in BJELR cells. Method: ACL library comprises 1,540 compounds and all compounds were prepared in DMSO at 4 pg / ml in plates 384 well polypropylene and stored at -20 ° C. Replica replica plates were prepared for each library plate using Zymark Scilone ALH. The descending plates were diluted 50 times in DMEM and the concentration of the compound in the descending plate is 80 pg / ml with 2% DMSO. In the test plate compound of the descending plate was diluted 20 times with cell suspension, in this way the final concentration of each compound is 4 pg / ml. BJELR cells were seeded in 6000 cells / well (57 pl) (for co-treatment projection) and 5000 cells / well (57 μ?) (for pretreatment projection) on 384-well clear, black background plates using a volume distribution syringe. For co-treatment suppressive projection, cells were treated with 3 μ? of composite of the ACL library descending plates (final concentration in assay plate at 4 g / ml) and at the same time treated with 5 g / ml of erastin. The transfer of the compound was done using a fixed cannula head 384. The plates were incubated for 48 hours at 37 ° C in an incubator with 5% C02. For the pretreatment projections, cells were pre-incubated with the ACL down-library library compound overnight and then treated with 5 pg / ml erastin for an additional 48 hours. The plates were processed by Calcein assay using a robotic system MiniTrak / SideTrak from Packard BioScience. Test plates were washed with PBS and incubated with Calcein AM (0.7 pg / ml) for 4 hours at room temperature. The fluorescence intensity was determined using a Fusion plate reader with filters centered on an excitation of 485 nm and an emission of 535 nm. BJELR cells are BJ-TERT / LT / ST / RASV12 cells. Table 1 shows the potencies of tumor selective compounds in genetically modified cell lines. Nine tumor selective compounds were retested at point 16, double dilution dose curves in all genetically modified cell lines. The table lists the concentration (in pg / mL) required to achieve 50% inhibition of calcein staining AM (IC50) for each compound in each cell line. The IC50 value in primary BJ cells was divided by the IC50 value in tumorigenic B cells J-TERT / LT / ST / RASV12 to obtain a tumor selectivity ratio for each compound. The selectivity of the compound for each genetic element is determined by calculating the selectivity ratio for each subsequent pair of cell lines in a series. Selective compounds of small T oncoprotein were considered to be selective for PP2A (the target of small T oncoprotein), whereas selective compounds of E6 were considered to be selective for loss of selective compounds of p53 and E7 They considered that they are selective for loss of RB.

Table 2 shows the potencies of tumor selective compounds in genetically modified cell lines. The list lists the inhibition (values of negative%) or improvement (values of% positive) of calcein staining AM (IC50) for each compound and each cell line. Table 2% Average% Average inhibition / improvement inhibition / improvement BJELR cell molecule of BJEH cells ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? EXAMPLE 4 Identification and Characterization of Erastin Binding Molecules or Partners Decrease assays using immobilized erastin and cell lysates were used in an initial attempt to identify erastin binding partners within a cell. Initial decrease experiments were performed with total cell lysates HEK293, BJEH and BJELR. In those experiments, a derivative of eraylin methyl-amino (ERA-A6) was immobilized to Affigel 10 and incubated with lysate under standard depletion conditions. The spheres were washed and eluted with either 100 μ? of erastine or 0.8% of N-lauroyl sarcosine (sarcosyl). The eluates were subjected to mass spectrometry analysis. The analysis of the erastin decrease assay is inverted up to a high speed of mitochondrial proteins or ER membrane, identified in this first set of decrease experiments with HEK293 or BJEH and BJELR lysates. This suggests that membrane pores may be a target of erastin or its analogues. However, this result does not include the possibility that the erastin must have additional and / or different targets that have not been identified in this set of decrease experiments. Several proteins were observed repeatedly in the erastin or erastin analogue decrease experiments. These proteins, including VDAC1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a and Sec22b, are probably Erastin targets. Since the total cell lysate mixtures were still quite complex, those proteins were only detected in sarcosyl elutions from the decrease experiments. This potentially complicates mass spectrometry analysis. In this way, to simplify the mixing, several separation methods were used to isolate or pre-concentrate some of the proteins identified from the total cell lysate decrease experiments. Since Prohibitin and VDAC isoforms are all mitochondrial proteins, Requesters enrich the potential erastin targets by isolating the mitochondria from cellular ones. The isolated mitochondrial extracts were then used in erastin decrease experiments. In those experiments of decreasing erastin with mitochondrial extracts, Prohibitin, VDAC and Riboforin were also identified by Western Blot analysis. Figure 14 shows the Western Blot analysis of a decrease in where a mitochondrial extract is contacted with active Erastin (A6) and inactive (B1) derivatives immobilized in spheres. The decreases were made with 0.25 mg of total protein of the mitochondrial extract. The spheres were incubated with the extracts for 1.5 hours at 4 ° C and then washed several times. times. The proteins bound to the immobilized Erastin derivatives were eluted with 50 μl of 0.8% N-lauroyl sarcosine solution. Proteins were identified by Western blot analysis with a mixture of anti-Riboforin, -Sec6, -Prohibitin and anti-VDAC antibodies. The proteins were also identified by MS analysis. Riboforin and Prohibitin are rather acidic proteins with a calculated PI of 5.57 (Prohibitin) and 5.96 (Riboforin I). The Applicants separated those two proteins from the most basic proteins (VDAC isoforms, Sec22 and Sec61a) by ion exchange chromatography on a MonoQ column at a pH of 6.8. The fractions were then tested for their contents of Prohibitin or Riboforin using antibodies. The fractions were also tested by binding to a BIACORE ™ surface containing immobilized ERA-A6 and ERA-B1 compounds. Prohibitin and Riboforin were found in the fractions that show binding in the BIACORE ™ experiments. Interestingly, an unknown 45 kDa protein that is not reacted with any of the antibodies used was observed to bind the spheres of ERA-A6 or ERA-B1 on a silver stained SDS-PAGE gel. To confirm those stuck, the decrease experiments with ERA-A6 and ERA-B1 were performed from the fractions of the MonoQ elutions. Again, the Prohibitin and Riboforin were identified as links to the Erastin spheres from several fractions. These experiments clearly support the notion that the VDAC isoforms, as well as the binding of Prohibitin and Riboforin to erastin and the erastin analogues. In this way these proteins are all potential targets / partners of erastin binding in vivo. EXAMPLE 5 Expression Levels of Various VDAC Isoforms Successful decreases with the Erastin ERA-A6 analogue consistently produces higher EM-based identification results for VDAC3 when decreases of the Used BJELR are made, compared to the Used derived from the BJEH cells. . The superior results for that isoform of VDAC are consistent with a higher abundance of that isoform in the Used BJELR in relation to the Used BJEH, giving a comparable amount of total protein. These different levels of white protein should have an impact on the selectivity of Erastin. To address this point, the Applicants tested the relative expression levels of the various VDAC isoforms using quantitative PCR (Q-PCR). Other possible methods include Western blot analysis and mass spectrometry. Quantitative PCR experiments (Q-PCR) were performed to determine the relative amounts of mRNA (as a substitute marker for gene expression) for a variety of genes in the "normal" BJEH cell line, and the BJELR tumorigenic line. For each of the VDAC isoforms (VDAC1, 2 and 3), two regions of the mRNA were targeted for amplification. These regions were referred to as 1 and 1-2, 2-1 and 2-2, and 3-1 and 3-2, respectively. The Q-PCR signal for amplification of the mRNA fragment for each gene of interest was compared to a series of internal standards, and classified relative to the signal derived from GAPDH mRNA in the target cells. The results depicted in Figure 11 indicate that the expression of VDAC3 is significantly elevated in BJELR cells relative to that in BJEH cells. This finding is in contrast to the results observed for several different genes, which were suppressed in BJELR cells in relation to those observed in BJEH cells. Figure 12 was generated using the same Q-PCR data as in Figure 11, but Figure 12 focuses exclusively on the relative expression levels of the VDAC isoforms in the target cells. The Q-PCR signal for each amplified mRNA fragment was compared to a series of internal standards, and expressed relative to the VDAC1 mRNA-derived signal in the target cells, which is defined as 100%. As in Figure 11, the two amplified regions of the mRNA for each of the isoforms of VDAC (VDAC1, 2 and 3), are referred to as 1, 1-2, 2-1, 2-2, 3-1 and 3-2, respectively. The results indicate that the expression of VDAC3 is expressed at a level 2 to 2.5 times higher in BJELR cells than in BJEH cells. Taken together, these findings suggest a possible mechanism to explain the differential sensitivity of BJELR cells with erastin treatment. EXAMPLE 6 Functional Evaluation of Erastin in Various VDAC Isoforms Functional assays help to validate proteins identified as functional targets for erastin. In certain modalities, the isolated mitochondria should be used to see if the erastin has any functional or phenotypic effects on mitochondrial function. For example, phenotypic effects could be observed by microscopy, while detection of changes in mitochondrial membrane potential, or the release of oxidative species with erastin treatment could be observed by using certain dyes, known in the art to detect species of reactive oxygen (ROS). In certain different embodiments, validation experiments should include photo-affinity tag of the target protein with azido-erastin derivatives, or erastin analogs or derivatives coupled to a crosslinker labeled with bidentate affinity (such as SBED), or a dissociable crosslinker .

In still other modalities, recombinant and overexpressed proteins must be used in certain in vitro assays to assess any possible effects that erastin must have on its functions. Such in vitro assays could include, but are not limited to: direct link (in vitro or BIACORE ™), or flow or emanation assays that could determine the channel properties of the VDAC isoforms. In still other embodiments, agonistic mutants (cells or organisms) of those target proteins can be used. Compared with wild types, these mutants could become either resistant or hypertensive to erastin. Those agénic cell lines could also be used in high-throughput (HTS) projections to determine and / or evaluate the specificity of erastin or its analogues. In still other modalities, RNAi experiments for VDAC, Prohibitin and Riboforin can also be used to assess any phenotypes with erastin treatment (eg, resistance to erastine or hypersensitivity). According to this embodiment of the invention, SMARTPOOL® steering siRNAs VDAC 1, VDAC2 and VDAC3, respectively, can be purchased from Dharmacon (Lafayette, CO). Transfection conditions are then optimized, for example, using FUGENE ™ and oligofectamine in 384-well bales, and a fluorescently labeled siRNA duplex. Such a procedure resulted in ~ 90% transfection efficiency. The cells ELR tumors can then be transfected with siRNAs against VDAC1, VDAC2 or VDAC3, and the response to the dose of erastin can be measured. EXAMPLE 7 Inhibition of Cell Growth The ability of a compound to inhibit the growth of BJELR and BJEH cells is measured. The compounds are tested by primary projection of Sytox, a phenotypic assay that monitors alterations in the proliferation of cell survival as a result of treatment with the compound. It is invented as a high-throughput method to identify compounds that specifically alter the growth potential of the cells harboring the causative mutations found in cancer patients while not affecting the growth of normal cells. The assay depends on a reliable and simple, inexpensive output reading of a fluorescent membrane-impermeable dye (Sytox, from Molecular Probes) that binds to the nucleic acid. In healthy cells, no signal is detected because the cell membrane is intact and the dye will not enter. However, if a cell membrane is compromised as a result of apoptosis or necrosis, a fluorescent signal proportional to the number of cells similarly affected will be detected. Using a two-stage output reading (final reading in the presence of a detergent to allow labeling of all cells), the assay can identify compounds that they produce cytostasis, cytotoxicity and / or mitogenesis. The first "dead cell" reading or reading provides an estimate of the toxicity of a given compound by indicating the number of dead or stained cells in the culture at the time of the test. The second reading or reading of "total cell" captures both the cumulative effects of cytoxicity in reducing the cell population size as well as any anti-proliferative or cytostatic effects a test compound can exert on cells in the test population in the absence of toxicity. For the purpose of projection, the previously described BJ-TERT line is defined as the "normal" reference cell line and the J-TE RT / LT / ST / RASV12 B cells are the tumorigenic cell line. Cells were seeded overnight in 96 well plates at densities that without treatment could allow 95% confluence in the wells 72 hours later. The next day, the cells are exposed to test compounds in a dilution series for a period of 48 hours. After this incubation period, the Sytox reagent is added to the cultures at the manufacturer's recommended concentration and fluorescence reading is taken from the dead cell. After completion of this measurement, e! Saponin detergent to each well of the cultures to permeabilize the membranes that allow the Sytox reagent to enter each cell, thus facilitating the measurement of the total number of cells that remain in the culture. EXAMPLE 8 Synthesis of 3- (2-ethoxy in i I) -2- (pi perazi n-1-ylmethyl) quinazolin-4 (3 H) -one (Compound 5) Compound 1 Compound 2 compound 5 compound 3 compound 4 Step 1: Preparation of 2- (chloromethyl) -4H-benzordl-1, 31-oxazin-4-one (Compound 2) Method 1: Under a nitrogen atmosphere, the anthranilic acid (compound 1, 15.3 g) was dissolved in 300 mL of dichloromethane (CH2Cl2). Triethylamine (TEA, 1.1 equiv.) Was then added and the mixture was cooled in a water-ice bath. A solution of chloroacetyl chloride (1.1 equiv.) In dichloromethane (150 mL) was added dropwise and the mixture was allowed to stir for two hours with heating at room temperature. (The bath with wire can be removed at the end of the addition or the mixture can be allowed to warm to room temperature for two hours.) The solids were isolated by filtration and washed with cold water (2x) followed by 5% diethyl ether (Et20). ) in hexane and dried with air to yield compound 2 as a white powdery solid (22.5 g, quantitative yield). The final product was characterized by LC / MS m / z MH + 196.13; > 95% pure; 1 H NMR. Method 2 - Use of Dimethylformamide (DMF) as a reaction solvent: grams of anthranilic acid were dissolved in 300 mL of DMF. TEA (1.5 equiv.) Was added and the mixture was cooled in an ice / water bath. A solution of chloroacetyl chloride (1.3 equiv.) In DMF (100 mL) was added dropwise to the cooled reaction mixture. The ice / water bath was removed, and the reaction mixture was allowed to stir for 2 hours. The reaction mixture was poured into ice water (200-300 mL) and extracted with ethyl acetate (EtOAc, 3x extraction). The organic layers were combined, washed with water and brine and dried over sodium sulfate (Na 2 SO 4). The concentration produced a solid which was triturated with 100 mL of 10% Et20 / hexane to obtain compound 2 as a white powder (12 g, 85% yield). The final product was characterized by LC / MS. Step 2j Preparation of 2- (chloromethy-3- (2-ethoxyphenyl) quinazolin-4 (3H) -one (Compound 3) Method 1 - using trichlorophosphine (PCI3): Under a nitrogen atmosphere, compound 2 (8.8 g) was dissolved in 440 mL of acetonitrile (CH3CN) and He added 2-ethoxybenzenamine (1.5 equiv.) to it and stirred. To this well stirred reaction mixture, PCI3 drops (2 equiv.) Were added. The resulting suspension was heated at 50 ° C for 6-12 hours. The reaction mixture was poured into a saturated Na 2 CO 3 / ice mixture, stirred for 30 minutes, and extracted with EtOAc (3x300 mL). The combined organic layers were washed with (a minimum amount of) water and brine and dried over Na2SO4. The solution was concentrated, and the crude solid / oil mixture was triturated with 3% Et20 / Hexane (2x100 mL) to remove most of the unreacted phenethidine. The resulting solids were isolated by filtration and further purified by passing through a column of silica gel (20% EtOAc / hexane). Compound 3 was isolated as a white powder (11 g, 75-80%). The final product was characterized by LC / MS m / z MH + 315/317; > 98% pure. Method 2 - using phosphoryl trichloride (POCI3): Under a nitrogen atmosphere, compound 2 (1.2 g, 6.0 mmol, 1.0 equiv.) And 2-ethoxybenzenamine (1.2 mL, 9.0 mmol, 1.5 equiv.) Were dissolved in 30 mL of CH3CH. To this solution was added POCI3 drops (1.1 mL, 12 mmol, 2.0 equiv.) And the mixture was heated to reflux for a period of 3 hours. The reaction was cooled to room temperature, poured into an ice / saturated NaHC03 suspension, and extracted with EtOAc (3x200 mL). The combined organic layers were washed with water and brine and dried over Na2SO4. Analysis of the reaction mixture by LC / MS confirmed the presence of the desired product compound 3 (97%) together with a small amount of the composite byproduct 3c (2-3%). The MH + m / z (333/334/335) in LC / MS is consistent with the diamine structure of compound 3c. The desired product was typically isolated after chromatography on silica gel (as described above in Method 1 of Step 2) to yield compound 3 as a white powder (1.3 g, 80-85% yield). Step 3: Preparation of 3- (2-ethoxyphenyl -2- (piperazin-1-methylmethyl) quinazolin-4 (3H) -one (Compound 5) compound 3 compound S Method 1: Under a nitrogen atmosphere, Compound 3 (5 g) was dissolved in CH3CN (0.08 - 0.2 M concentration of compound) and potassium carbonate (K2C03, 1.2 equiv., commercial powder) was added thereto. , piperazine (2 equiv.) and tetrabutylammonium iodide (0.2 equiv.) in that order. The mixture heated at 60 ° C (warm bath) for 8-10 hours. The reaction was carried out in any of the following two ways: (a) 80% of the solvent was evaporated, water (20 mL) was added and the mixture was extracted with EtOAc (4x60 mL); or (b) the reaction mixture was diluted with 400 mL of EtOAc and washed with water (3x20 mL). The combined organic layers were washed with brine and concentrated to yield a light yellow oil. Compound 5 was obtained as a white powder (80-85% yield) after purification by means of pressure chromatography (CombiFlash®) on silica (10-25% MeOH / dichloromethane). The product was characterized by 1 H NMR and LC-MS MH + 365. Step 4: Preparation of 4 - ((3- (2-ethoxyphenyl) -4-oxo-3,4-dihydroquinazolin-2-yl) methyl) piperazine-1-carboxylate of tert-butyl (Compound 4) Compound 3 Compound 4 Method 1: Under a nitrogen atmosphere, Compound 3 (4.0 g, 12.7 mmol, 1.0 equiv.) Was dissolved in 60 mL of CH3CN, and solid samples were added to this solution.

K2C03 (2.1 g, 15 mmol, 1.2 equiv.), Boc-piperazine (4.73 g, 25 mmol, 2.0 equiv.) And sodium iodide (Nal, 570 mg, 3 mmol, 0.3 equiv.) In that order. The mixture (a suspension) was heated at 80 ° C for 3-6 hours. Resulting in a white solution. Examination of the reaction using TLC and LC / MS showed complete conversion of Compound 3 to Compound 4. Approximately 30 ml_ of CH 3 CN were removed by distillation under reduced pressure, and to the resulting suspension was added 60 ml_ of water and the mixture was extracted with EtOAc (4x60 mL). The combined organic layers were washed with water, a saturated suspension of ammonium chloride (to remove unreacted Boc-piperazine), a saturated solution of NaHCO 3 and brine was dried over Na 2 SO 4. Concentration by filtration produced solids which were triturated with hexane to yield compound 4 as a white solid (5.6 g, quantitative yield). This material was used in the subsequent deprotection step without further purification. The final compound was characterized by H NMR, LC / MS. Step 5: Preparation of 3- (2-ethoxyphenyl) -2- (piperazin-1-methylmethyl) quinazolin-4 (3H) -one (Compound 5) compound 4 compound 5 Method 1: Compound 4 (2.7 g, 5.8 mmol, 1.0 equiv.) Was suspended in 15 mL of anhydrous dioxane at room temperature. A solution of 4N HCI / dioxane (17 mL, 12 equiv.) Was added dropwise at room temperature; After 30 minutes of reaction, an additional 17 mL of 4N HCI / dioxane was added and the progress of the reaction was monitored by LC / MS. (This was an exothermic reaction and gas evolution was observed, the reaction system must be opened to release pressure while maintaining anhydrous conditions). If any amount of the unreacted compound 4 remains, an additional 8-10 mL of HCI / 4N dioxane can be added to drive the reaction to completion. At the end of the reaction, 40 mL each of water and CH2Cl2 were added to the reaction and the mixture was made basic by adding a sufficient amount of saturated aqueous Na2CO3 solution until a pH of 8-9 was reached. The layers were separated and the aqueous layer was extracted with CH2Cl2. (3x60 mL). The organic layers were combined, washed with water (4 x 10 mL, until a pH of aqueous extract is around neutral) and brine and dried over Na2SO4. The concentration produced a slightly yellow oil which was purified by means of pressure chromatography (CombiFlash®) on silica (10-25% MeOH / dichloromethane) to yield compound 5 as a white powder (85-95% yield) after trituration with hexane containing 5-10% diethyl ether. Compound 5 was characterized by 1 H NMR and LC / MS. General: Analytical Method The following CL conditions were used to analyze Compound 5: Column: XTerra® for MS; C18, 3.5 pm Dimension: 2.1 x 1.50 mm Gradient: 75% CH3CN (containing 0.08% formic acid) / 10% water (containing 0.1% formic acid). All compounds were analyzed by LC / MS under the following column and mobile phase conditions: Column: XTerra® for MS; C18, 3.5 pm Dimension: 2.1x150 mm Gradient: aaua time / 0.1% FA CH3CN / 0.08% 0:00 95 5 7:00 5 95 8:00 5 95 9:00 95 5 13:00 95 5 EXAMPLE 9 Sytox Primary Projection Sytox primary projection is a test phenotypic that monitors alterations in proliferation of cell survival as a result of the treatment of the compound. It was invented as a high-throughput method to identify compounds that specifically alter the growth potential of cells harboring the causative mutations found in cancer patients as long as they do not affect the growth of normal cells. The assay depends on a reliable and simple, inexpensive output reading of a fluorescent membrane-impermeable dye (Sytox, from Molecular Probes) that binds to the nucleic acid. In healthy cells, no signal is detected because the cell membrane is intact and the dye will not enter. However, if a cell membrane is compromised as a result of apoptosis or necrosis, a fluorescent signal proportional to the number of cells similarly affected will be detected. Using a two-stage output reading (final reading in the presence of a detergent for allow labeling of all cells), the assay can identify compounds that produce cytostasis, cytotoxicity and / or mitogenesis. The first reading or reading of "dead cell" provides an estimate of the toxicity of a given compound by indicating the number of dead or stained cells in the culture at the time of the test. The second reading or reading of "total cell" captures both the cumulative effects of cytoxicity in reducing the cell population size as well as any anti-proliferative or cytostatic effects a test compound can exert on cells in the test population in the absence of toxicity. For the purpose of projection, the BJ-TERT line previously described is defined as the "normal" reference cell line and the BJ-TERT / LT / ST / RASV12 cells are the tumorigenic cell line. Cells were seeded overnight in 96 well plates at densities that without treatment could allow 95% confluence in the wells 72 hours later. The next day, the cells are exposed to test compounds in a dilution series over a period of 4.8 hours. After this incubation period, the Sytox reagent is added to the cultures at the manufacturer's recommended concentration and fluorescence reading is taken from the dead cell. After the completion of this measurement, the Saponin detergent is added to each well of the crops for permeabilize the membranes that allow the Sytox reagent to enter each cell, thus facilitating the measurement of the total number of cells that remain in the culture. For evaluation data, no distinction is made between compounds that have cytotoxic or cytostatic effects. To be considered for additional testing, the compounds have met two convincing criteria: i. produce a change in the signal of at least 2 magnitudes of standard deviation over the tumor cell lines in either dead cell or total cell readings (or both) ii. produce a change in the signal of less than one magnitude of standard deviation over the "normal" control cells. See Tables 3 and 4 and Figures 15-17 for in vitro data corresponding to the compounds of the invention.

Table 3 EXAMPLE 10 Tumor Treatment Study HT-1080: Evaluation of the Antitumor Activity of Compound 6 PRLX and Compound 5 PRLX Mouse strain: Balb / c nude (strain Nu / Nu, Charles River Laboratories), female, 5-6 weeks old (~ 20 g average body weight). Formulation of Test Items: Both compounds were formulated in an identical manner. For the dose level of 100 mg / kg, each compound was supplied at a concentration of 10.0 mg / ml, in an injection volume of 0.2 ml. For the dose level of 50 mg / kg, each compound was supplied at a concentration of 5.0 mg / ml, in an injection volume of 0.2 ml. For both dose levels, the vehicle contained 0.025% Tween-80, 0.01% benzyl alcohol, 35 mM acetic acid (HOAc), 100 mM potassium phosphate buffer, and 32 mM sucrose, pH = 6.5. Study Groups: A: compound 6 PRLX @ 100 mg / Kg, QD x 5 days, IP, n = 8 B: compound 6 PRLX @ 50 mg / Kg, QD x 5 days, IP, n = 8 C: compound 5 PRLX @ 100 mg / Kg, QD x 5 days, IP, n = 8 D: compound 5 PRLX @ 50 mg / Kg, QD x 5 days, IP, n = 8?. Vehicle Control, QD x 5 days, IP, n = 8 F: Control not treated, n = 8 Treatment Table: Beginning when the average tumor volume reaches ~200 mm3, and continue through day 5, each day, a simple IP injection of one of the above treatments was administered to each animal, for a total of 5 doses . Tumor Implants and Period: Each of the 70 mice was implanted with 1 x 107 HT-1080 cells per SC injection of 0.1 ce inoculum on the right posterior side. A needle size of 25 G x 5/8"was used.The tumor cell inoculum was prepared using HT-1080 cells (ATCC isolation, 6th refrigerator storage passage) which has been cultured in DMEM [Gibco, No 10569-010] + 10% FCS [Gibco, No. F-2442] At the time of collecting cells, the cells had a confluence growth of 95-100% .The HT-1080 inoculum was prepared in a medium Sterile DMEM + 10% FCS at a density of 1.0 x 108 cells / ml In the post-tumor implant of day + '9, the animals were balanced by group in treatment and control groups, with each group consisting of 8 A total of 22 absentees were excluded from the study due to tumors that were either quite small or quite large.This was considered in the Day 1 study, and treatment was started on this day. prepared the following solutions for injection recently in each of the 5 days of administration of the compound: Dose of 100 mg / kg (groups A and C) First, a stock solution of 100 mg / ml was prepared for each compound by dissolving 35 mg of compound 6 PRLX or compound 5 PRLX in 0.35 ml of solvent (0.25% Tween-80, 0.1% benzyl alcohol, and 350 mM acetic acid). The final solutions for injection were then prepared by diluting the resulting stock solutions 1:10, by mixing each with 3.15 ml of diluent (100 mM potassium phosphate buffer and 32 mM sucrose, pH 6.8). The solutions were then sterilized by filter (0.45 μ? T? Membrane). The resulting solutions contained compound 6 PRLX or compound 5 PRLX at a final concentration of 10.0 mg / ml, pH = 6.5. Dosage of 50 mg / Kg (groups B and D). For each of the two compounds, 1.0 ml of the 10 mg / ml injection solutions (described above) were diluted 1: 2 by the addition of 1.0 ml of diluent (100 mM potassium phosphate buffer and 32 mM sucrose, pH 6.8). The resulting solutions would contain a final concentration of compound PRLX or compound 5 PRLX of 5.0 mg / ml at pH = 6.5. Vehicle Control (group E). Vehicle Control was prepared by diluting the solvent (0.25% Tween-80, 0.1% benzyl alcohol, and 350 mM acetic acid) 1:10 using the diluent (100 mM potassium phosphate buffer and 32 mM sucrose, pH 6.8). The final composition of the Vehicle Control contained 0.025% Tween-80, 0.01% benzyl alcohol, and 35 mM HOAc, in 100 mM potassium phosphate and 32 mM sucrose with a final pH = 6.8. Dosage Summary: Tumor Measurement: Starting on Day 1, all animals were weighed, and the tumor dimensions (length (L) and width (W)) were measured every other day. Tumor measurements were then converted to tumor volume (mm3) using the following formula: Tumor Volume = L x W x W / 2. The resulting tumor volume values were averaged for each study group for each time point, and were then plotted against time. The difference as standard mean error (± SEM). The results of these experiments are shown in Figures 18-20. EXAMPLE 11 Study of Tumor Treatment PANC-1: Evaluation of the Antitumor Activity of Compound 6 PRLX and Compound 5 PRLX Preparation and Implantation of Xenograft PANC-1 The experimental plan for the study of PANC-1 was essentially identical to that of the HT study -1080 described above in Example 10 with the following exceptions: about 30-40 mg of passive PANC-1 tumor tissue fragment was implanted subcutaneously on the right side of an immunodeficient nude mouse. Tumor growth is monitored daily and when tumors reached approximately 100 mm3, animals that harbor similarly sized tumors were balanced in groups and compound dosing was initiated. The administration of compound 5 occurs once a day for five consecutive days in the doses listed below. In the PANC-1 xenografts, gemcitabine, administered at the maximum tolerated dose for the model, was used as a control. The gemcitabine regimen was 180 mg / kg three times a day on every third day for a period of 9 days. The results of these experiments are shown in Figures 21-22.

INCORPORATION BY REFERENCE All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will be controlled. EQUIVALENTS While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art with review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, together with its full scope of equivalents, and the specification, together with such variations.

Claims (97)

1. R E I V I N D I C A C I O N S 1. A method for treating a condition in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt thereof, 00 characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from Ci-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, C1-4 alkyl, C1-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci-8 alkyl, aryl, C -4 aralkyl, and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C 2-6 alkenyl, and alkynyl wherein the condition is characterized by cells with enhanced Ras signaling activity and altered activity of a cellular target protein of the SV40 small t antigen.
2. 2. The method according to claim 1, characterized in that the condition is further characterized by the substantially wild type level of the Rb activity.
3. 3. The method according to claim 1, characterized in that R1 is selected from Z-Q-Z, -alkyl of Ci. 8-N (R2) (R4), -C1-8 -OR3 alkyl, aryl, heteroaryl, and C1-4 aralkyl.
4. The method according to claim 1, characterized in that aryl is optionally substituted with a group selected from Ci-6 alkyl, CF3, hydroxyl, Ci-4 alkoxy, aryl, aryloxy, halogen, NR2R4, nitro, carboxylic acid , carboxylic ester, and sulfonyl.
5. 5. A method for treating a condition in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt thereof, (D characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from Ci.8-N (R2) (R4), -alkyl from Ci-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, C1-4 alkyl, C1.4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, provided that when both of R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci-8 alkyl, aryl, Ci-4 aralkyl, and heteroaryl; R3 is selected from H, C1-alkyl, Ci-4 aralkyl, aryl, and heteroaryl; Q is selected from O and NR2; and Z is independently selected from Ci-6 alkyl, C 2-6 alkenyl, and alkynyl C2-6 where the condition is characterized by cells with enhanced Ras signaling activity and altered activity of a small white SV40 t cell antigen.
6. 6. The method according to claim 5, characterized in that the condition is further characterized by the substantially wild type level of the Rb activity.
7. 7. The method according to claim 5, characterized in that R1 is selected from Z-Q-Z, -alkyl of d. 8-N (R2) (R4), -alkyl of Ci.8-OR3, aryl, heteroaryl, and aralkyl
8. 8. The method according to claim 7, characterized in that R4 is selected from Ci-4 aralkyl and acyl.
9. 9. The method according to claim 8, characterized in that R4 is acyl.
10. 10. The method according to claim 9, characterized in that R 4 is -C (0) -C 1-3 alkyl-Y, and Y is selected from H, alkyl, alkoxy, aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl.
11. The method according to claim 9, characterized in that R 4 is -C (0) -C 1-3 alkyl-Y, and Y is selected from aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl.
12. 12. The method according to claim 9, characterized in that R4 is -C (0) -alkyl of Ci-3-Y, and Y is selected from aryloxy and heteroaryloxy.
13. 13. The method according to any of claims 1-12, characterized in that the compound kills cells in the mammal by a non-apoptotic mechanism.
14. 14. The method according to claim 13, characterized in that the cells have improved Ras signaling activity.
15. 15. The method according to claim 13, characterized in that the cells overexpress the SV40 small t antigen.
16. 16. The method according to claim 13, characterized in that the cells have substantially reduced phosphatase PP2A activity.
17. 17. The method according to claim 13, characterized in that the cells over-express VDAC.
18. 18. The method of compliance with any of the claims 1-12, characterized in that the condition is cancer.
19. 19. The method according to claim 13, characterized in that the cells are induced to express the SV40 small t antigen.
20. The method according to claim 19, characterized in that the cells are induced to express the SV40 small t antigen by infecting the cells with a viral vector which over-expresses the SV40 small t antigen.
21. 21. The method according to claim 20, characterized in that the viral vector is a retroviral vector or an adenoviral vector.
22. 22. The method according to any of claims 1-12, further characterized by comprising administering to the mammal together an agent that kills cells through an apoptotic mechanism.
23. 23. The method according to claim 22, characterized in that the agent is a chemotherapeutic agent.
24. 24. The method according to claim 23, characterized in that the chemotherapeutic agent is selected from: an EGF receptor antagonist, arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine, carminomycin, mechlorethamine chloride, pentamethylmelamine, thiotepa, teniposide, cyclophosphamide, chlorambucil , demethoxy hypocrelin A, melphalan, ifosfamide, trofosfamide, Treosulfan, podophyllotoxin or derivatives of podophyllotoxin, etoposide phosphate, teniposide, etoposide, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, metopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin , 1-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin, phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine (ara C), porfiromycin, 5-fluorouracil, 6 -mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenolic acid, daunorubicin, deferoxamine, floxuridine, doxifluridine, raltitrexed, idarubicin, epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate, actinomycin D, safracin, saframycin, quinocarcin, discodermolide, vincristine, vinblastine , vinorelbine tartrate, vertoporphine, paclitaxel, tamoxifen, raloxifene, thiazofuran, thioguanine, ribavirin, EICAR , estramustine, estramustine sodium phosphate, flutamide, bicalutamide, buserelin, leuprolide, pteridines, enedines, levamisole, aflacone, interferon, interleukins, aldesleucine, filgrastim, sargramostim, rituximab, BCG, tretinoin, betamethasone, gemcitabine hydrochloride, verapamil, VP- 16, altretamine, tapsigargin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, satraplatin, docetaxel, deoxygenated paclitaxel, TL-139, 5'-without-anhydrovinblastine (from here on forward: 5'-without-vinblastine), camphexine, irinotecan, (Camptosar, CPT-11), topotecan (Hycamptine), BAY 38-3441, 9-nitrocamptothecin (Oretecin, rubitecan), exatecan (DX-8951), lurtotecan ( GI-147211 C), gimatecan, diflomotecan of homocanptothecins (BN-80915) and 9-aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), indolocarbazoles (for example, NB-506), protoberberines, intoplicins, denoisoquinolones, benzo-phenazines or NB-506.
25. 25. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell: (1) an effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt of the same, characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from C1-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently each present selected from H, C1-4 alkyl, C1- aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are on the same N atom and not both on H , are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci-β alkyl, aryl, Ci aralkyl -4, and heteroaryl; R3 is selected from H, Ci-4 alkyl, Ci-4 aralkyl, aritG, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is each independently selected from C 1-6 alkyl, C 2-6 alkenyl. and alkynyl (2) an agent that increases the abundance of VDAC in the cell.
26. 26. The method according to claim 25, characterized in that R1 is selected from Z-Q-Z, -alkyl of C ^ 8-N (R2) (R4), -alkyl of C1-8-OR3, aryl, heteroaryl, and aralkyl
27. 27. The method according to claim 25, characterized in that aryl is optionally substituted with a group selected from C 1-6 alkyl, CF 3, hydroxyl, C 1-4 alkoxy, aryl, aryloxy, halogen, NR 2 R 4, nitro, carboxylic acid, carboxylic ester, and sulfonyl.
28. 28. The method according to claim 25, characterized in that the VDAC is VDAC 1, VDAC2 or VDAC3.
29. 29. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell: (1) an effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt of the same, 00 characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from C1-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl , heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, C1- aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, provided that when both of R2 and R4 are in the same N atom and not both in H, are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C-alkyl. -8, aryl, C1-4 aralkyl, and heteroaryl; R 3 is selected from H, Ci. 4 alkyl) Ci-4 aralkyl, aryl, and heteroaryl; Q is selected from O and NR2; and Z is independently each one that is selected from Ci-6 alkyl, C2-6 alkenyl. and C2-6 alkynyl; and (2) an agent that increases the abundance of VDAC in the cell.
30. 30. The method according to claim 29, characterized in that R is selected from Z-Q-Z, -alkyl Ci. 8-N (R2) (R4), -alkyl of Ci-8-OR3, aryl, heteroaryl, and aralkyl of C i -.
31. 31. The method according to claim 30, characterized in that R4 is selected from Ci aralkyl. and acyl.
32. 32. The method according to claim 31, characterized in that R4 is acyl.
33. 33. The method according to claim 32, characterized in that R 4 is -C (0) -C 1-3 alkyl-Y, and Y is selected from H, alkyl, alkoxy, aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl.
34. 34. The method according to claim 32, characterized in that R4 is -C (0) -Ci-3-Y alkyl, and Y is selected from aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl.
35. 35. The method according to claim 32, characterized in that R4 is -C (0) -C 1-3 alkyl-Y, and Y is selected from aryloxy and heteroaryloxy.
36. 36. The method according to claim 29, characterized in that the VDAC is VDAC 1, VDAC2 or VDAC3.
37. 37. The method according to any of claims 25-35, characterized in that the cell is a cancer cell.
38. 38. The method according to any of claims 25-35, characterized in that the agent comprises a polynucleotide encoding VDAC.
39. 39. The method according to any of claims 25-35, characterized in that the agent is a VDAC protein adapted to be transported in the cell.
40. 40. The method according to claim 39, characterized in that the agent is a VDAC protein merged with a heterologous internalization domain.
41. 41. The method according to claim 39, characterized in that the agent is a liposome preparation comprising a VDAC protein.
42. 42. The method according to any of claims 25-35, characterized in that the agent improves or inhibits the expression of endogenous VDAC.
43. 43. The method according to claim 42, characterized in that the agent stimulates the expression of VDAC.
44. 44. The method according to claim 42, characterized in that the agent inhibits the function of a VDAC inhibitor.
45. 45. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell: (1) an effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt of the same, (D characterized because - R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from d-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, C4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C1-8 alkyl, aryl, C1- aralkyl, and heteroaryl; R3 is selected from H, Ci-4 alkyl, C-i- aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently each one selected from C 1-6 alkyl, C 2-6 alkenyl > and C2-6 alkynyl; and (2) an agent that decreases the abundance of VDAC in the cell.
46. 46. A compound having the structure of Formula I or a pharmaceutically acceptable salt thereof, (D characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from Ci-8-N (R2) (R4), -alkyl from Ci-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, C4 alkyl, Ci.4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same atom of N and not both in H, are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, alkyl of C -8, aryl, aralkyl of Ci-4, and heteroaryl; R 3 is selected from H, C 1-4 alkyl, C 1-4 aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently selected from C 1-6 alkyl, C 2-6 alkenyl, and alkynyl
47. 47. The method according to claim 46, characterized in that R1 is selected from Z-Q-Z, -alkyl of d. 8-N (R2) (R4), -alkyl of d.8-OR3, aryl, heteroaryl, and aralkyl
48. 48. The method according to the rei indication 46, characterized in that aryl is optionally substituted with a group selected from C 1-6 alkyl, CF 3, hydroxyl, C 1-4 alkoxy, aryl, aryloxy, halogen, NR 2 R 4, nitro, carboxylic acid, carboxylic ester, and sulfonyl.
49. 49. A compound having the structure of Formula I or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from H, -ZQ-Z, -alkyl from C1-8-N (R2) (R4), -alkyl from Ci-8-OR3, carbocyclic or heterocyclic from 3 to 8 members, aryl, heteroaryl, and aralkyl of Ci-4; R2 and R4 are each independently selected from H, C1-4 alkyl, C1.4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and not both in H, they are different, and when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci-8 alkyl, aryl, Ci-4 aralkyl, and heteroaryl; R 3 is selected from H, C 1-4 alkyl) Ci-4 aralkyl, aryl, and heteroaryl; Q is selected from O and NR2; and Z is each independently selected from C 1-6 alkyl, C 2-6 alkenyl. and alkynyl C2-6-
50. 50. The method according to claim 49, characterized in that R1 is selected from Z-Q-Z, -alkyl of C-i. 8-N (R2) (R4), -C1-8 -OR3 alkyl, aryl, heteroaryl, and aralkyl
51. 51. The method according to claim 50, characterized in that R 4 is selected from C 4 aralkyl and acyl.
52. 52. The method according to claim 51, characterized in that R4 is acyl.
53. 53. The method according to claim 52, characterized in that R4 is -C (0) -alkyl of C -3-Y, and Y is selected from H, alkyl, alkoxy, aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl .
54. 54. The method according to claim 52, characterized in that R4 is -C (0) -alkyl of Ci-3-Y, and Y is selected from aryloxy, aryl, heteroaryl, heteroaryloxy, and cycloalkyl.
55. 55. The method according to claim 52, characterized in that R4 is -C (0) -alkyl of C -3-Y, and Y is selected from aryloxy and heteroaryloxy.
56. 56. A method for identifying a candidate anti-tumor agent, characterized in that it comprises: a) contacting a cell with a sufficient amount of a test agent under suitable conditions; and b) determining whether the test agent improves or inhibits the level of a component of a erastin protein that binds the complex or a nucleic acid encoding a component of a complex that binds the erastin protein.
57. 57. The method according to claim 56, characterized in that the protein that binds erastin is VDAC1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a, or Sec22b.
58. 58. The method according to claim 56, characterized in that it further comprises: a) contacting the test agent with a tumor cell; and b) determining whether the test agent inhibits the growth of the tumor cell.
59. 59. A method for identifying a candidate anti-tumor agent, characterized in that it comprises: a) contacting a protein that binds erastin or a cell that expresses a protein that binds erastin to a test agent; and b) determining whether the test agent binds the protein that binds the erastin.
60. 60. The method according to claim 59, characterized in that the protein that binds the erastin is VDAC1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a, or Sec22b.
61. 61. The method according to claim 59, further characterized in that it comprises: a) contacting the test agent with a tumor cell; and b) determining whether the test agent inhibits the growth of the tumor cell.
62. 62. The method according to claim 56 or 59, characterized in that the test agent is a small organic molecule.
63. 63. The method according to claim 56 or 59, characterized in that the test agent is a peptide, protein, carbohydrate or nucleic acid.
64. 64. The method according to claim 56 or 59, characterized in that the method is repeated for a library of different test agents.
65. 65. The method according to claim 59, characterized in that the test agent or the protein that binds erastin is labeled with a detectable marker.
66. 66. The method according to claim 65, characterized in that the detectable label is biotin, fluorescein, digoxigenin, green fluorescent protein (GFP), isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST).
67. 67. A method for increasing the sensitivity of a tumor cell to a chemotherapeutic agent, characterized in that it comprises contacting a tumor cell with a tumor cell. compound that increases or decreases the abundance of a protein that binds erastin.
68. 68. The method according to claim 67, characterized in that the protein that binds the erastin is VDAC 1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a, or Sec22b.
69. 69. The method according to claim 67, characterized in that the chemotherapeutic agent is the compound of claim 46.
70. 70. A method for reducing the sensitivity of a normal cell to a chemotherapeutic agent, characterized in that it comprises a normal cell with a compound that decreases or increases the abundance of a protein that binds erastin.
71. 71. The method according to claim 70, characterized in that the protein that binds the erastin is VDAC 1, VDAC2, VDAC3, Prohibitin, Riboforin, Sec61a, or Sec22b.
72. 72. The method according to claim 70, characterized in that the chemotherapeutic agent is the compound of claim 46.
73. 73. A method for identifying a candidate therapeutic agent for inhibiting unwanted cell proliferation, characterized in that it comprises: a) mixing a test agent and a VDAC protein or a protein complex comprising at least one VDAC protein and optionally one or more other proteins; b) determining whether the test agent binds to the VDAC protein; and c) if the test agent binds to the VDAC protein, contact the test agent with a cell and determine whether the test agent alters the proliferation of the cell.
74. 74. The method according to claim 73, characterized in that the test agent is a small organic molecule, a peptide, a protein, a peptidomimetic, a nucleic acid, or an antibody.
75. 75. The method according to claim 73 or 74, characterized in that the binding of the VDAC protein to the test agent is detected by a physical binding assay.
76. 76. A method for reducing the growth rate of a tumor, comprising administering an amount of a therapeutic agent sufficient to reduce the rate of tumor growth, characterized in that the therapeutic agent is: (a) an agent that increases or inhibits the growth of the tumor. level of a VDAC protein; (b) an agent that increases or inhibits the activity of a VDAC protein; (c) an agent that binds to a VDAC protein; (d) an agent that binds, modulates, or binds and modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (e) an agent comprising a VDAC polypeptide or functional variants thereof; or (f) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof.
77. 77. A method for treating a patient suffering or suffering from a cancer, characterized in that it comprises administering to the patient a therapeutic agent selected from: (a) an agent that increases or inhibits the level of a VDAC protein; (b) an agent that increases or inhibits the activity of a VDAC protein; (c) an agent that binds to a VDAC protein; (d) an agent that binds, modulates, or binds and modulates a protein complex comprising at least one VDAC and optionally one or more other proteins; (e) an agent comprising a VDAC polypeptide or functional variants thereof; and (f) an agent comprising a nucleic acid encoding a VDAC polypeptide or functional variants thereof.
78. 78. The method according to claim 76 or 77, characterized in that the VDAC protein is VDAC1, VDAC2, or VDAC3.
79. 79. The method according to claim 76 or 77, characterized in that the agent is a small organic molecule, a peptide, a protein, a peptidomimetic, a nucleic acid, or an antibody.
80. 80. The method according to claim 76 or 77, characterized in that the agent is formulated with a pharmaceutically acceptable carrier.
81. 81. The method according to claim 76 or 77, characterized in that the agent is administered intravenously, orally, buccally, parenterally, by an inhalation atomizer, by topical or transdermal application.
82. 82. The method according to claim 76 or 77, characterized in that the agent is administered via local administration.
83. 83. The method according to claim 76 or 77, characterized in that it further includes administering at least one additional anti-cancer chemotherapeutic agent that inhibits cancer cells in an additive or synergistic manner with the therapeutic agent.
84. 84. A compound having a structure of formula II or a pharmaceutically acceptable salt thereof, (?) characterized in that: Ar is a substituted phenyl; R is selected from H, Ci-8 alkyl, -ZQ-Z, -C 8 alkyl -N (R2) (R4), -C 1-8 alkyloxy, OR3, carbocyclic or 3- to 8-membered heterocyclic. , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, C1-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci alkyl. e, aryl, C1.4 aralkyl, aryl, and heteroaryl; R3 is selected from H, Ci-4 alkyl, C -4 aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; W is "Q is selected from O and NR2, and Z is independently selected as selected from Ci.6 alkyl, C2-6 alkenyl and alkynyl C2-6-
85. 85. A compound that has a structure of the formula III or a pharmaceutically acceptable salt thereof, (m) characterized in that Ar is a substituted phenyl; R1 is selected from H, C -8 alkyl, -ZQ-Z, -C 1-8 alkyl-N (R2) (R4), -C 1-8 alkyl-OR3, carbocyclic or 3- to 8-membered heterocyclic , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, C1-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, Ci alkyl. 8, aryl, Ci-4 aralkyl, and heteroaryl; R3 is selected from H, Ci-4 alkyl, C -4 aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2; and Z is independently selected from Ci.6 alkyl, C2-e alkenyl, and alkynyl
86. 86. A compound having a structure of formula IV or a pharmaceutically acceptable salt thereof, (IV) characterized in that Ar is a substituted phenyl; R1 is C8 alkyl; R2 and R4 are each independently selected from H and C1-8 alkyl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2.
87. 87. A compound having a structure of the formula a pharmaceutically acceptable salt thereof, (V) characterized in that R1 is selected from H and C1-8 alkyl; R2 is selected from H and C1-8 alkyl; R3 is selected from halogen, alkoxy and Ci-8 alkyl; R 4 is selected from H, halogen, alkoxy and C 1-8 alkyl; R5 is selected from H, halogen and nitro; and n is 1 or 2.
88. 88. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell an effective amount of a compound having a structure of Formula I or a pharmaceutically salt. acceptable of it, 00 characterized in that: R1 is selected from H, -Z-Q-Z, -alkyl from C1-8-N (R2) (R4), - C -8-OR3 alkyl, carbocyclic or heterocyclic of 3 to 8 members, aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently each occurring selected from H, Ci-4 alkyl, C -4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the condition that when both of R2 and R4 are in the same N atom and not both in H, they are different, and that when both of R2 and R4 are on the same N and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C8 alkyl, aryl, C1-4 aralkyl, and heteroaryl; R3 is selected from H, C1-4 alkyl, Ci-4 aralkyl, aryl, and heteroaryl; W is selected from Q is selected from O and NR2; and Z is independently each selected from Ci-6 alkyl, C2- alkenyl
89. 89. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell an effective amount of a compound having a structure of Formula II or a pharmaceutically acceptable salt thereof, (?) characterized in that: Ar is a substituted phenyl; R 1 is selected from H, C 1-8 alkyl, -ZQ-Z, -C 1-8 alkyl-N (R2) (R4), -C 1-8 alkyl-OR3, carbocyclic or 3- to 8-membered heterocyclic , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, Ci-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, C- alkyl ,. 8, aryl, Ci-4 aralkyl, aryl, and heteroaryl; R3 is selected from H, d-4 alkyl, C1-4 aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; Q is selected from O and NR2; and Z is independently selected from C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl.
90. 90. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, which comprises administering to the cell an effective amount of a compound having a structure of Formula III or a pharmaceutically acceptable salt thereof, characterized in that Ar is a substituted phenyl; R1 is selected from H, C1-8 alkyl, -ZQ-Z, -Ci-8-Nalkyl (R2) (R4), -C 8 alkyl-OR3, carbocyclic or 3- to 8-membered heterocyclic , aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, Ci-4 aralkyl) aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, provided that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, β-alkyl, aryl, Ci-4 aralkyl, and heteroaryl; R3 is selected from H, C1- alkyl, C1- aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are United; W is selected from Q is selected from O and NR2; Z is each independently selected from C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; and wherein Ar does not have an ethoxy substituent in an ortho position for the bond to the nitrogen of the quinazolinone ring.
91. 91. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, comprising administering to the cell an effective amount of a compound having a structure of Formula IV or a pharmaceutically acceptable salt thereof, (IV) characterized in that Ar is a substituted phenyl; R1 is Ci-8 alkyl; R2 and R4 are each independently selected from H and C1-8 alkyl; R5 represents 0-4 substituents on the ring to which they are attached; W is selected from Q is selected from O and NR2.
92. 92. A method to kill a cell, which promotes cell death or inhibits cell proliferation, which comprises administering to the cell an effective amount of a compound having a structure of Formula I or a pharmaceutically acceptable salt thereof, characterized in that R1 is selected from H and Ci.8 alkyl; R2 is selected from H and C1-8 alkyl; R3 is selected from halogen, alkoxy and Ci-8 alkyl; R4 is selected from H, halogen, alkoxy and C-i-e 'alkyl, R5 is selected from H, halogen and nitro; and n is 1 or 2.
93. 93. A method for increasing sensitivity of a tumor cell or a normal cell to a chemotherapeutic agent, characterized in that it comprises contacting the cells with a compound according to claims 46, 84, 85, 86 or 87.
94. 94. A method for treating cancer in a mammal, characterized in that it comprises administering to the mammal a therapeutically effective amount of a compound of according to claims 46, 84, 85, 86 or 87 or a pharmaceutically acceptable salt thereof.
95. 95. A method for killing a cell, which promotes cell death or inhibits cell proliferation, characterized in that it comprises administering to the cell: (1) an effective amount of a compound according to claims 84, 85, 86 or 87 or a pharmaceutically acceptable salt thereof, and (2) an agent that increases the abundance of VDAC in the cell.
96. 96. A method for killing a cell, which promotes cell death or which inhibits cell proliferation, characterized in that it comprises administering to the cell: (1) an effective amount of a compound according to claims 84, 85, 86 or 87 or a pharmaceutically acceptable salt thereof, and (2) an agent that decreases the abundance of VDAC in the cell.
97. 97. A method for preparing a compound represented by the formula F, characterized in that it comprises reacting a compound D, D, with an amine HNR2 wherein Ar is a substituted phenyl R is selected from H, C 1-8 alkyl, -Z-Q-Z, -alkyl C -8-N (R2) (R4), -C1-8-OR3 alkyl, carbocyclic or 3- to 8-membered heterocyclic, aryl, heteroaryl, and C1-4 aralkyl; R2 and R4 are each independently selected from H, C1-4 alkyl, C1-4 aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl, and arylsulfonyl, with the proviso that when both of R2 and R4 are in the same N atom and either R2 or R4 is acyl, alkylsulfonyl, or arylsulfonyl, then the other is selected from H, alkyl of d. 8, aryl, Ci-4 aralkyl, and heteroaryl; R3 is selected from H, C1- alkyl, C1- aralkyl, aryl, and heteroaryl; R5 represents 0-4 substituents on the ring to which they are attached; W is selected from Q is selected from O and NR2; Z is independently each or selected from C 1-6 alkyl, C 2- alkenyl