CN101218211A

CN101218211A - Quinoxaline derivatives as antitumor agents

Info

Publication number: CN101218211A
Application number: CNA2006800097335A
Authority: CN
Inventors: 罗伯特·R·贝克林; 辛迪·卢·彻帕诺斯克; 约翰·M·佩尔特; 齐龙武; 保罗·B·罗宾斯; 苏迪尔·R·萨哈斯拉布德; 罗伯特·塞利亚; 基思·西门斯; 布伦特·R·斯托克韦尔; 拉杰·葛佩尔·文卡特; 莫里茨·冯雷兴贝格; 尤金·阵
Original assignee: Columbia University in the City of New York; Whitehead Institute for Biomedical Research; Prolexys Pharmaceuticals Inc
Current assignee: Columbia University in the City of New York; Whitehead Institute for Biomedical Research; Prolexys Pharmaceuticals Inc
Priority date: 2005-01-25
Filing date: 2006-01-25
Publication date: 2008-07-09
Also published as: TWI389892B; TW200637830A

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

Quinoxaline derivatives as antineoplastic agent

Government-funded

This paper work, all or part of, be under the subsidy of the state-run cancer research fund 1R01CA97061-01 of institute, to finish.United States Federal Government is enjoyed certain right in the present invention.

Background of invention

Many medicines that are used for the treatment of disease are as target with the general difference between disease cell and normal cell.For example, be used for the treatment of ovarian cancer and mammary cancer and suppress the taxol of microtubule function, be considered to show specificity (Miller and Ojima, Chem.Rec.1:195-211,2002) tumour cell based on the higher proliferation rate of the relative normal cell of tumour cell.Yet, although this is consistent viewpoint, very big (the people such as Weinstein of the external activity of taxol difference between different tumor cell lines, Science 275:343-349,1997), show gene can regulate tumor cell to the susceptibility of taxol, and the reactivity of tumour cell is not simply to be decided by proliferation rate.

A kind of promising novel method people such as (, Cancer Cell 1:117-23,2002) Shawver of cancer therapy drug exploitation has been represented in molecular targeted treatment.Utilize this method, the design small molecules is used for directly suppressing the sudden change of specific tumors cell type or crosses the cancer protein of expressing.By the specific molecular defective that target is found in tumour cell, this method finally can produce the method that is suitable for the various tumour genetic compositions of particular treatment.Recently the example of two molecular targeted anticancer therapeutic agents of success is Gleevec (imatinib mesylate imatinib mesylate), the inhibitor of a kind of breaking point of in Philadelphia chromosome positive chronic myelogenous leukemia, finding bunch Ji Qu-abelsen kinases (BCR-ABL) cancer protein (people such as Capdeville, Nat Rev DrugDiscov 1:493-502,2002), and Herceptin (trastuzumab trastuzumab), monoclonal antibody (Mokbel and the Hassanally of the HER2/NEU cancer protein that a kind of target is found in metastatic breast cancer, Curr Med Res Opin 17:51-9,2001).

A kind of complementary strategy relates to seeks optionally anti-tumor agent comprising salmosin of genotype, described reagent only particular cancer albumen exist or the condition of specific tumors inhibition disappearance under tumour cell is only lethality.This genotype alternative cpd is the target cancer protein directly, perhaps they can target other participate in the link key protein of signal network of cancer proteins.The compound of the demonstration synthetic lethal of having reported comprises (i) forms of rapamycin analogs CCI-779 (people such as Shi in lacking the myeloma cell of PTEN, Cancer Res 62:5027-34,2002), (ii) Gleevec (people such as Druker in the BCR-ABL-transformant, Nat Med 2:561-6,1996) and (iii) a large amount of compound that fully characterizes (people such as Stockwell, Chem Biol 6:71-83,1999; People such as Torrance, Nat Biotechnol 19:940-5,2001).

Although above-mentioned research is arranged, still be starved of exploitation and/or identify can selectivity target tumor cell compound.

The invention summary

Utilize a kind of synthetic lethal sieve method, especially synthetic lethal high flux screening method, this method can effectively be identified reagent or the medicine that is used for the treatment of or prevents illness or disease, described illness or disease for example tumour or other are the existence or the development of the illness (for example leukemia) of feature with hyperplasia, the applicant has identified a large amount of compound/reagent/medicine and has been used in individuality treatment or preventing cancer (for example tumour or leukemia), and described individuality for example needs the people that treats or prevent.The present invention also provides and the direct or indirect bonded cell protein of some compound/reagent that therapeutic value is arranged that identify.It is the additive method of the disease or the illness of feature with hyperplasia (for example leukemia) that this cell protein provides treatment.

The term of Shi Yonging " reagent " and " medicine " can exchange use herein; They can be compound or molecule.

In one embodiment, the present invention points to compound disclosed herein, comprises its salt.

In another embodiment, the present invention points to pharmaceutical composition, comprises pharmaceutically acceptable carrier and compound disclosed herein.

Still in another embodiment, the present invention is a kind of method that promotes necrocytosis, comprises the compound disclosed herein of pair cell effective dosage.

On the one hand, the present invention is a kind of method of the candidate's of evaluation anti-tumor agent comprising salmosin, may further comprise the steps:

A) under conditions suitable, use capacity test agent exposing cell;

B) detect the level whether test agent strengthens or suppress protein-bonded level of erastin or the protein-bonded nucleic acid of coding erastin.

This method can also may further comprise the steps:

A) with tumour cell contact measured reagent (body is interior or external);

B) detect the growth whether test agent suppresses tumour cell.

On the other hand, the present invention is a kind of method of the candidate's of evaluation anti-tumor agent comprising salmosin, comprising:

A) conjugated protein or express the protein-bonded cell of erastin with test agent contact erastin, wherein conjugated protein the or test agent of erastin can be selected the detectable mark for use;

B) whether the detection test agent is conjugated protein in conjunction with erastin.

This method can also comprise:

A) with tumour cell contact measured reagent (body is interior or external);

B) detect the growth whether test agent suppresses tumour cell.

Use different test agent libraries, can repeat above-mentioned two kinds of methods.

On the other hand, the present invention relates to the screening method of authenticating compound, described compound kills and wounds or suppresses the growth of tumorigenic cell, through engineering approaches tumorigenic cell for example, but be not the homology normal cell of their correspondences.This method has been used to identify the known and new genotype-select active compound of having, and comprises i.e. herein the erastin of compound known Zorubicin, daunorubicin, mitoxantrone, camptothecine, sangivamycin, Quinomycin A, bouvardin, NSC 146109 and new compound.These compounds have the activity of increase under the situation that one or more following materials exist: hTERT cancer protein, the big T cancer protein of SV40 (LT), little T cancer protein (ST), human papillomavirus Class1 6 (HPV) E6 cancer protein, HPV E7 cancer protein and carcinogenic HRAS, NRAS and KRAS.The applicant detects crossing of hTERT and E7 or LT and expresses the expression that increases topoisomerase 2a, and crosses the RAS that expresses in the cell of expressing hTERT ^V12Increase the expression of topoisomerase 1 and make cell sensitization with ST to the initial non-apoptotic cell death step of erastin.

The present invention relates to the method for a kind of indentifying substance (for example medicine), described reagent has selection toxicity (for example kill and wound or suppress and grow) to tumorigenic cell, for example the through engineering approaches tumorigenic cell comprises people's tumorigenic cell (for example through engineering approaches tumorigenic cell and/or tumour cell).In one embodiment, the reagent (for example medicine) that the present invention relates to identify selective killing or suppress through engineering approaches people tumorigenic cell growth (deleterious) comprises with the through engineering approaches people tumorigenic cell of candidate agent contact as cell to be measured; Detect the cell activity to be measured that contacts with candidate agent; The activity of cell activity to be measured and appropriate control is compared.In all embodiments, come the evaluation vigor by detection reagent (for example medicine) killer cell or cell growth inhibiting/propagation and ability that both have concurrently.If the vigor of cell to be measured is lower than control cells, then identify the reagent (for example medicine) that through engineering approaches people tumorigenic cell is had selection toxicity (kill and wound or suppress and grow).Suitable contrast is the cell with cell same type to be measured, except control cells is not engineered to tumorigenesis.For example, control cells may be the parent's primary cell that derives cell to be measured.Control cells contacts candidate agent under the condition identical with cell to be measured.Suitable contrast can be carried out simultaneously, perhaps establishes (for example, standard of establishing in advance or reference) in advance.

In one embodiment, evaluation has tumorigenic cell selects toxic compositions and methods also to comprise the toxicity of evaluate reagent, described reagent through evaluation be through engineering approaches people's tumorigenic cell of appropriate animal model or other based on cell or results of screening based on acellular system or in testing.For example, reagent of so identifying or medicine can be evaluated it to such as tumour cell that obtains from individuality or leukemia cell's toxicity or to the toxicity of (one or more) cancer (tumour) clone.For example, present method can also be included in and evaluate the selection toxicity of reagent (perhaps medicine) to tumorigenic cell in suitable mouse model or the non-human primate.The invention still further relates to the method for a kind of production, for example through engineering approaches people tumorigenic cell is had the toxic reagent (for example medicine) of selection by the reagent (for example medicine) of the inventive method evaluation.Utilize currently known methods synthetic to tumorigenic cell demonstration selection toxic reagent (for example medicine).

The present invention also relates to the method for evaluation to the virose reagent of through engineering approaches tumorigenic cell (for example medicine), for example through engineering approaches people tumorigenic cell in addition.In one embodiment, the present invention relates to identify the compositions and methods of the growth (toxic) that kills and wounds or suppress through engineering approaches people tumorigenic cell, comprise cell to be measured is contacted with candidate agent people's tumorigenic cell that described cell to be measured is a through engineering approaches it; Detect the cell activity to be measured that contacts with candidate agent; The activity of cell activity to be measured and appropriate control is compared.If the vigor of cell to be measured is lower than control cells, then identify the reagent (for example medicine) that through engineering approaches people tumorigenic cell is had toxicity (kill and wound or suppress and grow).Here, suitable contrast is the cell (for example through engineering approaches people tumorigenic cell) with cell same type to be measured, except control cells does not contact with candidate agent.Suitable contrast can be carried out simultaneously, perhaps establishes (for example, standard of establishing in advance or reference) in advance.For example, reagent of so identifying or medicine can be evaluated it to such as tumour cell that obtains from individuality or leukemia cell's toxicity or to the toxicity of (one or more) cancer (tumour) clone.

In one embodiment, evaluation also comprises the toxicity of evaluating reagent to the virose compositions and methods of through engineering approaches tumorigenic cell, and described reagent is to identify in the results of screening based on cell or in based on the through engineering approaches people's tumorigenic cell in acellular system or the test of appropriate animal model or other.For example, present method also is included in and evaluates the toxicity of reagent (for example medicine) to tumorigenic cell in suitable mouse model or the non-human primate.The invention still further relates to a kind of method of reagent (for example medicine) of production the inventive method evaluation, for example through engineering approaches people tumorigenic cell is had toxic reagent (for example medicine).Utilize currently known methods to synthesize tumorigenic cell is shown toxic reagent (for example medicine).

In another embodiment, the present invention is a kind of method that reduces the tumor growth rate, comprises that administration enough reduces the treatment reagent of the amount of tumor growth rate, wherein treats reagent to be:

(a) reagent of enhancing or inhibition VDAC protein level;

(b) reagent of enhancing or inhibition VDAC protein-active;

(c) in conjunction with the proteic reagent of VDAC;

(d) combination and/or adjusting comprise the reagent of at least one VDAC and optional one or more other proteic albumen compositions;

(e) comprise the reagent of VDAC polypeptide or its function varient;

(f) comprise the reagent of the nucleic acid of coding VDAC polypeptide or its function varient;

Suitable reagent is in existing form or have described activity after the metabolism wholly or in part.

On the one hand, the present invention is a kind of cancer patients's of treatment a method, comprise patient's administration is selected from following treatment reagent:

(a) reagent of enhancing or inhibition VDAC protein level;

(b) reagent of enhancing or inhibition VDAC protein-active;

(c) in conjunction with the proteic reagent of VDAC;

(d) in conjunction with and/or regulate the reagent comprise at least a VDAC and optional one or more other proteic albumen composition;

(e) comprise the reagent of VDAC polypeptide or its function varient;

The suitable agent that is suitable for reducing the tumor growth rate and treating the cancer patients includes but not limited to little organic molecule, peptide, protein, simulating peptide, nucleic acid, antibody and combination thereof.This reagent is usually made preparation with acceptable carrier, can intravenously, oral, cheek, parenteral, spray by sucking, by topical application or through percutaneous drug delivery.Reagent can also pass through the administration of topical mode.Reagent can also with at least a other anti-cancer chemotherapeutic agents couplings, described chemotherapeutics is to add up or the cooperative mode anticancer.

On the other hand, the present invention is a kind of method that increases tumour cell to chemotherapeutics susceptibility, and wherein tumour cell contacts with the compound of increase or the conjugated protein abundance of minimizing erastin.In related fields, the present invention is a kind of method that reduces normal cell to chemotherapeutics susceptibility, and wherein normal cell contacts with the compound of minimizing or the conjugated protein abundance of increase erastin.

In certain embodiments of the invention, comprise that by screening (annotated) library of compounds, combinatorial library or other libraries of the unknown or known compound (for example reagent, medicine) or both marks identify candidate agent.

In certain embodiments, the present invention is a kind of method of identifying the candidate therapeutic agent that suppresses unwanted cells propagation, comprising:

A) with test agent and VDAC albumen or comprise at least a VDAC and optional one or more other proteic albumen composition mixes;

B) whether detect test agent in conjunction with VDAC albumen;

C) if test agent in conjunction with VDAC albumen, with test agent and cells contacting (in the body or external) and detect the propagation whether test agent changes cell.

VDAC albumen is can be for example with combining of test agent, by the physical bond test, detect in conjunction with test, the two assorted tests of yeast, fluorescence polarization test, superficial cell plasmagene group (plasmon) resonance or fluorescence resonance energy transmission (FRET) test such as immunity.

In certain embodiments, the present invention relates to a Verbindung rastin and a class erastin related compound (for example, compound of the present invention).

In other embodiments, the present invention relates to compound, erastin B and its related compound.

In other embodiments, the present invention relates to compound, erastin A and its related compound.

In other embodiments of the present invention, the present invention relates to the analogue of (deleterious) erastin of selective killing or the growth of inhibition through engineering approaches people tumorigenic cell to it.Optional, these compounds of the present invention are prepared into pharmaceutical composition with pharmaceutically acceptable carrier.

The invention still further relates to the method that participates in tumorigenic cellular component of identifying.Cellular component comprises, for example, and albumen (for example enzyme, acceptor), nucleic acid (for example thymus nucleic acid, Yeast Nucleic Acid), and fat (for example phosphatide).In one embodiment, the present invention relates to the method that a kind of evaluation participates in tumorigenic (one or more) cellular component, wherein (a) cell contacts with erastin such as through engineering approaches people tumorigenic cell; (b) cellular component direct or indirect and erastin effect is differentiated.The cellular component of being differentiated is to participate in tumorigenic cellular component.In other embodiments, the present invention relates to the method for (one or more) cellular component of a kind of evaluation and erastin effect, (a) cell wherein, such as through engineering approaches people tumorigenic cell, tissue, organ, organism or one of above-mentioned split product or extract, contact with erastin; (b) cellular component direct or indirect and erastin effect is differentiated.The cellular component of being differentiated is a cellular component direct or indirect and erastin effect.

The invention still further relates to the method for treatment or preventing cancer.In one embodiment, the present invention relates to the method for a kind of treatment or preventing cancer, wherein treat the compound of significant quantity, such as, for example erastin or its analogue, perhaps below the compound of formula I-V, administration needs the individuality of cancer therapy.In certain embodiments, the characteristics of cancer are that the RAS approach in the cell is activated.In certain other embodiments, the characteristics of cancer are the little T cancer protein of cell expressing SV40, and are perhaps similar to the cell phenotype of expressing ST, and/or carcinogenic HRAS.In certain preferred aspects, cell is expressed the Rb (for example, about at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% or 150% or the like) of wild-type level basically.

The invention still further relates to the method for the reagent (for example medicine) of evaluation and one or more cellular component effects, described cellular component direct or indirect and erastin effect.In one embodiment, the present invention relates to the compositions and methods of a kind of evaluation and cellular component effect, described cellular component and erastin effect comprise that (a) is with erastin exposing cell, tissue, organ, organism or one of above-mentioned split product or extract; (b) differentiate the cellular component that acts on erastin (direct or indirect); (c) with candidate agent exposing cell, tissue, organ, organism or one of above-mentioned split product or extract, described reagent is reagent or the medicine for the treatment of the ability of assessed and cellular component effect, described cellular component and erastin effect; (d) detection reagent whether with (b) in cellular component (directly or indirectly) effect.If reagent with (b) in the cellular component effect, it is exactly reagent, described cellular component and erastin effect with the cellular component effect.

In related fields, the invention still further relates to the method for the reagent (for example medicine) of evaluation and one or more cellular component effects, described cellular component is known and erastin direct or indirect action, this method comprises: (a) with candidate agent exposing cell, tissue, organ, organism or one of above-mentioned split product or extract, described reagent is reagent or the medicine for the treatment of the ability of assessed and cellular component effect, the known and erastin effect of described cellular component; (b) detection reagent whether with (a) in cellular component (directly or indirectly) effect.If reagent with (a) in the cellular component effect, it is exactly reagent, described cellular component and erastin effect with the cellular component effect.

In certain embodiments, cell is a kind of through engineering approaches people's tumorigenic cell.In other embodiments, the present invention relates to and the direct or indirect interactional compound of (one or more) cellular component, described cellular component and erastin interact.In certain embodiments, participating in tumour with the interactional cellular component of erastin takes place.Use synthetic demonstration of currently known methods and the interactional reagent of cellular component (for example medicine), described cellular component and erastin interact.

The invention still further relates to the method for a kind of indentifying substance (for example medicine), described reagent is induced death in tumour cell, for example by apoptosis or acellular apoptosis mechanism.In one embodiment, a kind of evaluation in tumour cell induces dead reagent to comprise that (a) is used in the candidate agent contact measured cell of inducing death in the tumour cell by acellular apoptosis mechanism, and described cell to be measured is tumour cell (organ or the tissue that perhaps comprise tumour cell); (b) whether evaluation reagent is apoptosis-induced in cell to be measured; (c) with the middle apoptosis induced of suitable comparing (b).If apoptosis is induced in control cells rather than cell to be measured, then identify in tumour cell and induce dead reagent (for example medicine) by acellular apoptosis mechanism.Suitable contrast is the cell with cell same type to be measured, except control cells contacts with known reagent apoptosis-induced in cell.Suitable contrast can be carried out simultaneously, perhaps can set up (for example, setting up standard or reference in advance) in advance.In certain embodiments, cell to be measured is a through engineering approaches people tumorigenic cell.

" one " of Shi Yonging and " one " are meant the thing of one or more indications herein.

In some aspects, the invention provides drug development active method.In one embodiment, the present invention relates to a kind of drug development active method of carrying out, comprising: (a) identifying has the toxic reagent (for example medicine) of selection to through engineering approaches people tumorigenic cell; (b) reagent or effect and the toxicity of its analogue in animal of evaluation evaluation in (a); (c) preparation comprises the pharmaceutical preparation of the reagent that one or more are evaluated in (b).The effect of evaluation can be the ability of reagent selective induction necrocytosis in the tumorigenic cell of animal.In another embodiment, carry out drug development active method and comprise that foundation is used to distribute the distribution system of pharmaceutical preparation for sale.Optional, set up the marketing that selling group is used for pharmaceutical preparation.In other embodiments, the present invention relates to a kind of protein science active method of carrying out, comprise that evaluation has through engineering approaches people tumorigenic cell to select toxic reagent (for example medicine) and will further develop through engineering approaches people tumorigenic cell had and select the right of toxic reagent to permit to the third party.

In another embodiment, the present invention relates to a kind of drug development active method of carrying out, comprising: (a) identify through engineering approaches people tumorigenic cell virose (one or more) reagent (for example medicine); (b) reagent or effect and the toxicity of its analogue in animal of evaluation evaluation in (a); (c) preparation comprises the pharmaceutical preparation of the reagent that one or more are evaluated in (b).For example, the reagent of evaluation is erastin.The effect of evaluation can be in the tumorigenic cell of animal, the ability that the growth of reagent selectivity inducing cell, toxicity or necrocytosis change.In another embodiment, carry out drug development active method and comprise that foundation is used to distribute the distribution system of pharmaceutical preparation for sale.Optional, set up the marketing that selling group is used for pharmaceutical preparation.In other embodiments, the present invention relates to a kind of protein science active method of carrying out, comprise that evaluation has through engineering approaches people tumorigenic cell to select toxic reagent (for example medicine) and will further develop through engineering approaches people tumorigenic cell had and select the right of toxic reagent to permit to the third party.

In another embodiment, the present invention relates to a kind of drug development active method of carrying out, comprising: identify and cellular component interactional (one or more) reagent (for example medicine) that (a) described cellular component and erastin interact; (b) reagent or effect and the toxicity of its analogue in animal of evaluation evaluation in (a); (c) preparation comprises the pharmaceutical preparation of the reagent that one or more are evaluated in (b).The reagent effect of evaluation can be the ability of its selective induction necrocytosis in the tumorigenic cell of animal.In another embodiment, carry out drug development active method and comprise that foundation is used to distribute the distribution system of pharmaceutical preparation for sale.Optional, set up the marketing that selling group is used for pharmaceutical preparation.In other embodiments, the present invention relates to a kind of protein science active method of carrying out, comprise and identifying and the interactional reagent of cellular component (for example medicine), described cellular component and erastin interact, permit to the third party that with will further developing described cellular component and erastin interact with the right of the interactional reagent of cellular component.

In another embodiment, the present invention is a kind of pharmacy active method of carrying out, and comprising:

(a) identify a kind of candidate therapeutic agent that suppresses cell proliferation, wherein candidate therapeutic agent is

(i) reagent of enhancing or inhibition VDAC protein level;

(ii) strengthen or suppress the reagent of VDAC protein-active;

(iii) with the protein bound reagent of VDAC;

(iv) in conjunction with and/or regulate and to comprise at least a VDAC and choose any one kind of them or multiple other the reagent of proteic protein complex;

(the reagent that v) comprises VDAC polypeptide or its functional variant;

(the reagent that vi) comprises the nucleic acid of coding VDAC polypeptide or its functional variant; Perhaps

(compound vii) disclosed herein,

(b) carry out in step (a) effect and the toxic treatment evaluation of candidate therapeutic agent in animal identified; With

(c) useful in preparing drug formulations, it comprises the candidate therapeutic agent with acceptable treatment evaluation that one or more are identified in step (b).

A step of replacing (b) and a step or two (c) go on foot or except step (b) with (c) the step or two steps, described method can comprise permits the right of the further exploitation of candidate therapeutic agent to the third party.In another embodiment, carry out drug development active method and comprise that foundation is used to distribute the distribution system of pharmaceutical preparation for sale.Optional, set up the marketing that selling group is used for pharmaceutical preparation.

(a) identify the candidate therapeutic agent that suppresses cell proliferation, wherein candidate therapeutic agent is

(i) enhancing or the proteic reagent of inhibition VDAC; Perhaps

(ii) strengthen or suppress between VDAC albumen and the second kind of albumen interactional reagent and

(b) right that will further develop candidate therapeutic agent is permitted to the third party.

In another embodiment, carry out drug development active method and comprise that foundation is used to distribute the distribution system of pharmaceutical preparation for sale.Optional, set up the marketing that selling group is used for pharmaceutical preparation.

Another aspect of the present invention is to carry out pharmacy active method, comprises following one or more: the market development, production, market development right permitted to third party and the right that will produce test kit permit that to the third party, wherein test kit comprises:

(a) detect in biological sample that erastin is conjugated protein, the protein-bonded activity of erastin or above both one or more reagent; With

(b) specification sheets of explanation test-results.

Usually, specification sheets can point out whether the protein-bonded level of erastin and/or activity be normal, its desired level and/or activity are to increase or reduce relatively, like this but whether detection level and/or activity should change, and perhaps prediction (partly) depends on whether the treatment of level and/or activity (for example cancer chemotherapy) can be successful.

In certain embodiments, specification sheets comprises conjugated protein one or more normal, the level or the active guidances that reduce and raise about erastin.

In certain embodiments,, erastin protein-bonded activity conjugated protein or both levels based on erastin, specification sheets comprises about the guidance with following one or more successive treatments:

(i) reagent of enhancing or inhibition VDAC protein level;

(ii) strengthen or suppress the reagent of VDAC protein-active;

(iii) in conjunction with the proteic reagent of VDAC;

(iv) comprise at least a VDAC and choose any one kind of them or multiple other the reagent of proteic protein complex in conjunction with, adjusting or combination and adjusting;

(the reagent that v) comprises VDAC polypeptide or its functional variant;

(the nucleic acid reagent that vi) comprises coding VDAC polypeptide or its functional variant; With

(compound vii) disclosed herein.

In certain embodiments,, erastin protein-bonded activity conjugated protein or both levels based on erastin, whether successful specification sheets comprise about following one or more treatments guidance:

(i) reagent of enhancing or inhibition VDAC protein level;

(ii) strengthen or suppress the reagent of VDAC protein-active;

(iii) in conjunction with the proteic reagent of VDAC;

(the reagent that v) comprises VDAC polypeptide or its functional variant;

(the reagent that vi) comprises the nucleic acid of coding VDAC polypeptide or its functional variant; With

(compound vii) disclosed herein.

In certain embodiments,, erastin protein-bonded activity conjugated protein or both levels based on erastin, specification sheets comprises the guidance about the cancer therapy probability of success.

Identify to increase of the hereditary change of people's cell, finally may allow from the mechanism carcinogenic signal network of analysis and customization embolic chemotherapy at the specific tumors type to specific compound susceptibility.The applicant has developed the micromolecular systematics method of a kind of discovery, and described small molecules has the activity of increase in the cell that has specific heredity variation.Use this system, they detect the anti-tumor agent comprising salmosin of several clinical uses, have more effectiveness and have more activity under the situation that specific genetic elements exists.In addition, they identify the compound of the selective killing cell that makes new advances, T cancer protein that described cell expressing is little and carcinogenic RAS.These genetic targets to small molecules can be used as the exploitation have good therapeutic index cancer therapy drug guide thing.

The present invention also provides the medicine of packing.In one embodiment, the medicine of packing comprises: (i) the treatment significant quantity has the toxic reagent of selection to through engineering approaches people tumorigenic cell; The specification sheets and/or the label that (ii) are used for the treatment of cancer patients's reagent administration.In a particular, reagent is erastin.In another embodiment, the medicine of packing comprises: (i) the treatment significant quantity has the toxic reagent of selection to through engineering approaches people tumorigenic cell; The specification sheets and/or the label that (ii) are used for the treatment of cancer patients's reagent administration.In another relevant embodiment, the medicine of packing comprises: (i) the treatment significant quantity with the interactional reagent of cellular component, described cellular component and erastin interaction; The specification sheets and/or the label that (ii) are used for the treatment of cancer patients's reagent administration.

Specification sheets or label can be stored at electronic media, for example CD, DVD, floppy disk, storage card or the like, and these can read by computer.

The present invention also provides any agent of using the present invention to identify to prepare the cancer therapy medicament, for example, utilizes erastin or its analogue to prepare the cancer therapy medicament.

In certain embodiments, method of the present invention also comprises one or more reagent of Combined Preparation, for example passes through the chemotherapeutics of Apoptosis Mechanism killer cell usually.The suitable agent that is suitable for reducing the tumor growth rate and treating the cancer patients includes but not limited to little organic molecule, peptide, protein, simulating peptide, nucleic acid, antibody and combination thereof.Can be contemplated that all embodiments of the present invention can make up with one or more other embodiments.

In yet another aspect, the present invention relates to be used for the sieve method of authenticating compound, the cytotoxicity of described compound arrestin in through engineering approaches cell rather than its isogenic normal cell resemblance.These methods have been used to identify the known and new compound with genotype selective active.Optional, there be active increasing under the situation in these compounds at mutain.

() method for example, medicine, described reagent selectivity in the through engineering approaches cell suppresses cytotoxicity to the present invention relates to a kind of indentifying substance.In one embodiment, the present invention relates to a kind of indentifying substance () method for example, medicine, described reagent is the proteic cytotoxicity of mutation inhibiting in the through engineering approaches cell, comprise with candidate agent contact measured cell (for example, expressing the through engineering approaches cell of mutain); Detect the vigor of the cell to be measured that contacts with candidate agent; With the vigor of cell to be measured and the vigor of appropriate control are made comparisons.If the selectivity that the vigor of cell to be measured, then identifies greater than the vigor of control cells suppresses Cytotoxic reagent (for example, medicine).Suitable contrast is the cell identical with cell type to be measured, does not carry out through engineering approaches except control cells and causes toxic albumen with expression.For example, control cells can be the parent's primary cell that derives cell to be measured.Control cells contacts candidate agent under the condition identical with cell to be measured.Suitable contrast can be carried out simultaneously, perhaps sets up (for example, setting up standard or reference in advance) in advance.

In some aspects, the invention provides sanatory method in Mammals, comprise the erastin analogue of administration Mammals treatment significant quantity, for example, the compound of general formula I representative:

Wherein the characteristics of illness are that cell has the proteic activity of the antigenic cell-targeting of the little t of SV40 of enhanced Ras signal activity and change (for example, reduce or increase); With the optional Rb activity of wild-type level basically; With

Wherein:

R ¹Be selected from H ,-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and C _1-4Aralkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴At identical nitrogen and R ²Or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

With

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl.When Z is thiazolinyl or alkynyl group, two keys or triple bond are preferred not at the end of group (thereby getting rid of for example, enol ether, alkynol ether (alkyno1 ether), enamine and/or ynamine).

In some embodiments, W is selected from Or

In some this embodiment, R ¹Be selected from-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl, and C _1-4Aralkyl;

In certain embodiments, W is

In some this embodiment, R ¹Be selected from-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl, and C _1-4Aralkyl.

In certain embodiments, R ¹Be selected from-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl, and C _1-4Aralkyl.

In certain embodiments, R ⁴Be selected from C _1-4Aralkyl and acyl group.In some this embodiment, R ⁴It is acyl group.

In certain embodiments, R ⁴Be acyl group, R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from H, alkyl, alkoxyl group, aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.In certain embodiments, Y is selected from aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.In preferred this embodiment, Y is selected from aryloxy and heteroaryloxy.In preferred this embodiment, C _1-3Alkyl-Y is-CH ₂The O-phenyl, wherein phenyl is optional replaces preferred chlorine with halogen.At some Y be-CH ₂In the preferred embodiment of O-phenyl, select remaining value from embodiment, to get rid of erastin.

In certain embodiments, aryl is optional with being selected from C _1-6Alkyl, CF ₃, hydroxyl, C _1-4Alkoxyl group, aryl, aryloxy, halogen ,-NR ²R ⁴, nitro, carboxylic acid, carboxylicesters and sulphonyl group replace.

Suitable reagent can have described activity after existing form or metabolism completely or partially.

In certain embodiments, the characteristics of illness are that cell has the Rb activity of wild-type level basically.In some this embodiment, cell characteristic also is the activity of enhanced Ras signal activity and/or the proteic change of the antigenic cell-targeting of the little t of SV40 (for example, reduce or increase).

In certain embodiments, compound is by acellular apoptosis mechanism killer cell.

In certain embodiments, compound is by the outer mechanism killer cell of acellular apoptosis mechanism.

In certain embodiments, cell has enhanced Ras pathway activities (for example, RasV12), cross the little t antigen of expression SV40, have the Phosphoric acid esterase PP2A activity that reduces in essence, and/or (for example regulate, strengthen or inhibition) VDAC level or activity, such as VDAC2 or VDAC3.

In certain embodiments, illness is a cancer.

In certain embodiments, cell for example, was used the described cell of the antigenic viral vector infection of the little t of expression SV40, for example retroviral vector or adenovirus carrier by the little t antigen of abduction delivering SV40.

In certain embodiments, virus vector is retroviral vector or adenovirus carrier.

In certain embodiments, method of the present invention also comprises a kind of reagent of the described Mammals of Combined Preparation, for example passes through the chemotherapeutics of apoptosis mechanism killer cell usually.In certain embodiments, the reagent of Combined Preparation is selected from: epidermal growth factor receptor antagonists, arsenic sulfide, Zorubicin, cis-platinum, carboplatin, Cimitidine Type A/AB, carminomycin, mustargen hydrochloric acid, pentamethylmelamine, plug is for group, Vumon, endoxan, Chlorambucil, de-methoxy Hypocrellin A A, alkeran, ifosfamide, trofosfamide, Treosulfan, podophyllinic acid lactone (prodophyllotoxin) or podophyllinic acid lactone derivative, Zuyeyidal phosphoric acid salt, Vumon, Zuyeyidal, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, methotrexate, ametycin, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, the 1-methyl-4-phenylpyridinium ion, semustine, staurosporine (staurosporin), U-9889, phthalocyanine, Dacarbazine, aminopterin, methotrexate, Trimetrexate, Tioguanine, purinethol, fludarabine, pentastatin, 2-chlorodeoxyadenosine (cladribin), cytosine arabinoside (ara C), methylmitomycin, 5 FU 5 fluorouracil, Ismipur, the Zorubicin hydrochloride, formyl tetrahydrofolic acid, mycophenolic acid, daunorubicin, desferrioxamine, floxuridine, doxifluridine, Raltitrexed, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, bleomycin vitriol, dactinomycin, Safranin, saframycins, quinocarcins, discodermolides, vincristine(VCR), vincaleucoblastine, preparing vinorelbine tartrate, vertoporfm, taxol, tamoxifen, raloxifene, tiazofurine, Tioguanine, virazole, EICAR, Emcyt, estramustine phosphate sodium, Drogenil, bicalutamide, buserelin, Leuprolide, pteridine, enediyne, L-tetramisole, aflacon, Interferon, rabbit, interleukin, rIL-2 (aldesleukin), filgrastim, sargramostim, Rituximab, bacille Calmette-Guerin vaccine, vitamin A acid, Betamethasone Valerate, the gemcitabine hydrochloride, verapamil, VP-16, hexamethyl melamine, thapsigargin (thapsigargin), oxaliplatin, iproplatin, four platinum, Lip river platinum, DCP, PLD-147, JM118, JM216, JM335, husky platinum, the Japanese yew terpene, deoxy taxol, TL-139,5 '-fall-the dehydrogenation vinealeucoblastine(VLB) (hereinafter: 5 '-vinealeucoblastine(VLB) falls), camptothecine, Rinotecan (Camptosar, CPT-11), Hycamtin (Hycamptin), BAY38-3441,9-nitrocamptothecin (Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C), gimatecan, homocamptothecins diflomotecan (BN-80915) and 9-aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), benzazole and carbazole (for example NB-506), protoberberine, intoplicine, idenoisoquinolones, benzo-azophenlyene or NB-506.

Another aspect of the present invention provides a kind of killer cell, promotes the method for necrocytosis or inhibition cell proliferation, comprises the administration cell: the compound of (1) significant quantity, represent by general formula I:

Wherein:

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R ²Or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from Or

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl.(when Z is thiazolinyl or alkynyl group, two keys or triple bond are preferred not at the end of group); With

(2) a kind of VDAC (for example, VDAC2, VDAC3) reagent of abundance of in cell, increasing.

Another aspect of the present invention provides a kind of method of killer cell, comprises the administration cell: the compound of (1) significant quantity, represent by general formula I:

Wherein:

R ¹Be selected from H ,-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and aralkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R2 or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

Or

Q is selected from O and NR ²With

(2) a kind of VDAC (for example, VDAC2, VDAC3) reagent of abundance of in cell, reducing.

In another embodiment, the present invention is a kind of method that promotes necrocytosis, comprises the compound of the formula (I) of pair cell effective dosage.

In certain embodiments, compound as mentioned above.

In certain embodiments, cell is a cancer cells.

In certain embodiments, reagent comprises the polynucleotide of coding VDAC such as VDAC3.

In certain embodiments, reagent be fit to transportation advance the VDAC albumen of cell (for example, VDAC3), for example, the albumen that merges with allos internalization structural domain.

In certain embodiments, reagent is to comprise VDAC albumen (for example, Liposomal formulation VDAC3).

In certain embodiments, reagent strengthens or suppresses endogenous VDAC and (for example, VDAC3) express, stimulate or suppress VDAC and (for example, VDAC3) express or strengthen or suppress VDAC (for example, the VDAC33) function of inhibitor.

In some aspects, this method also relates to VDAC in a kind of increase cell of administration (for example, VDAC2, VDAC3) reagent of abundance.In some aspects, this method also relates to VDAC in a kind of reduction cell of administration (for example, VDAC2, VDAC3) reagent of abundance.

In yet another aspect, the present invention be a kind of tumour cell that increases to the method for chemotherapeutics susceptibility (for example, that add up or collaborative), wherein tumour cell contacts with disclosed compound herein.In related fields, the present invention is a kind of method that reduces normal cell to chemotherapeutics susceptibility, and wherein normal cell contacts with disclosed compound herein.

In one embodiment, the present invention is a kind of evaluation has the patient of reaction to the treatment that utilizes a compound of the present invention method.Use standard known in the art to characterize authentication method, be defined as having the patient that tumour forms and show what one or more following signs will be considered to respond: unusual Ras signal pathway activity, the activation that it is characterized in that one or more approach member (for example, phosphorylation Erk1/2, phosphorylation MEK or the like), and/or the expression of VDAC albumen (1,2 or 3), and/or in external or body, be exposed to the susceptibility of the clone of the similar of compound of the present invention or homologous genes type.

Another aspect of the present invention provides a kind of compound of general formula I representative, perhaps its pharmacologically acceptable salts:

Wherein:

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they be different (except in certain embodiments, R wherein ²And R ⁴Be hydrogen), and work as R ²And R ⁴On identical nitrogen and R ²Or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

With

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl.When Z is thiazolinyl or alkynyl group, two keys or triple bond are preferred not at the end of group.

Another aspect of the present invention provides a kind of compound of general formula I I representative:

Wherein

Ar is the phenyl that replaces;

R ¹Be selected from H ,-C _1-8Alkyl ,-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and C _1-4Aralkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are worked as R ²And R ⁴At identical nitrogen and R ²Or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, aryl and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

R ⁵The 0-4 substituting group of representative on its bonded ring.

W is

Perhaps

Q is selected from O and NR ²With

Another aspect of the present invention provides a kind of compound of general formula III representative:

Wherein

Ar is that replace or unsubstituted phenyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are worked as R ²And R ⁴At identical nitrogen and R ²Or R ⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

R ⁵The 0-4 substituting group of representative on its bonded ring.

W is selected from

, perhaps

Q is selected from O and NR ²With

Another aspect of the present invention provides a kind of compound of general formula I V representative:

Wherein

Ar is that replace or unsubstituted phenyl;

R ¹Be-C _1-8Alkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H and C _1-8Alkyl;

R ⁵The 0-4 substituting group of representative on its bonded ring.

W is selected from , perhaps

Q is selected from O and NR ²

Another aspect of the present invention provides a kind of compound of general formula V representative:

Wherein

R ¹Be selected from H and C _1-8Alkyl;

R ²Be selected from H and C _1-8Alkyl;

R ³Be selected from halogen, C _1-8Alkoxyl group and C _1-8Alkyl;

R ⁴Be selected from H, halogen, C _1-8Alkoxyl group and C _1-8Alkyl;

R ⁵Be selected from H, halogen and nitro; With

N is 1 or 2.

Expect that the compound of above-mentioned general formula I-V representative can be used for following method 1) treat mammiferous illness, the described compound that comprises administration Mammals treatment significant quantity, 2) killer cell, comprise a) the described compound of significant quantity of administration cell, and b) (for example increases in the cell VDAC, VDAC2, the VDAC3) reagent of abundance, perhaps 3) killer cell, comprise a) the described compound of significant quantity of administration cell, and b) reduces VDAC (for example, VDAC2, VDAC3) reagent of abundance in the cell.

Expect whole embodiment of the present invention can with one or more other embodiment couplings, even they are described at different aspect of the present invention.In certain embodiments of the invention, compound or reagent are not the compounds that is disclosed in the table 2.

The accompanying drawing summary

Fig. 1 is the synoptic diagram that shows people's iuntercellular relation of experiment conversion.The BJ cell is former generation human foreskin fibroblast.BJ-TERT is cell-derived in the BJ cell, and expresses hTERT, the catalytic subunit of Telomere terminal transferase.The BJ-TRET/LT/ST cell by the genome structure body of introducing coding simian virus 40 big (LT) and little T (ST) cancer protein derived from the BJ-TRET cell.BJ-TERT/LT/ST/RAS ^V12Tumour cell is by introducing HRAS (RAS ^V12) carcinogenic allelotrope and derived from the BJ-TERT/LT/ST cell (people such as Hahn, 1999, Nat Med5,1164-70).BJ-TERT/LT/RAS ^V12Origin of cell is in the BJ cell, by introducing coding TERT, LT, RAS ^V12Construction and the cDNA of control vector (people such as Hahn, 2002, NatRev Cancer 2,331-41).BJ-TERT/LT/RAS ^V12/ ST cell originates from BJ-TERT/LT/RAS by the cDNA that introduces coding ST ^V12Cell (people such as Hahn, 2002, NatRev Cancer 2,331-41).The TIP5 cell is former generation human foreskin fibroblast.TIP5-deutero-clone passes through to introduce coding hTERT, LT, and ST, RAS or papillomavirus E6 or the proteic carrier of E7 prepare, as shown in the figure.E6 and E7 can replace jointly LT (people such as Lessnick, 2002, Cancer Cell 1,393-401).

Fig. 2 shows the chemical structure of nine genotype alternative cpds.

Fig. 3 shows Quinomycin A and the camptothecine effect diagram to the through engineering approaches cell.The cell of mark is used Quinomycin A (A) or camptothecine at 384 orifice plates, and (B C) handled 48 hours.The inhibition per-cent that shows the cell viability that uses fluorexon AM detection.Error line shows a standard deviation.(A) BJ, BJ-TERT, BJ-TERT/LT/ST and the BJ-TERT/LT/ST/RAS of Quinomycin A processing ^V12Cell; (B) BJ, BJ-TERT, BJ-TERT/LT/ST and the BJ-TERT/LT/ST/RAS of camptothecine processing ^V12Cell; (C) BJ-TERT/LT/RAS of camptothecine processing ^V12, BJ-TERT/LT/RAS ^V12/ ST and BJ-TERT/LT/ST/RAS ^V12Cell.

Fig. 4 shows the effect diagram of erastin to the through engineering approaches cell.The cell of mark was handled 48 hours with erastin at 384 orifice plates.The inhibition per-cent that shows the cell viability that uses fluorexon AM detection.Error line shows a standard deviation.(A) BJ, BJ-TERT, BJ-TERT/LT/ST and the BJ-TERT/LT/ST/RAS of erastin processing ^V12Cell; (B) BJ of erastin processing, BJ-TERT, BJ-TERT/LT/RAS ^V12Cell (lacking ST), BJ-TERT/LT/RAS ^V12/ ST (tumorigenic cell) and TERT/LT/ST/RAS ^V12(tumorigenic cell); (C) independent deutero-TIP5/TERT, TIP5/TERT/E6, TIP5/TERT/LT, TIP5/TERT/LT/ST and TIP5/TERTLT/ST/RAS ^V12Cell.

Fig. 5 shows that the protein targets of tumor-selective compound is marked in the through engineering approaches tumorigenic cell and is raised.(A-C) from BJ, BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS ^V12, BJ-TERT/LT/RAS ^V12And BJ-TERT/LT/RAS ^V12The Westernblot of/ST cell lysate uses anti-topoisomerase II (A) or TOPI (B, antibody C).On (C) hurdle, cell is used at TOPI, contrast double-stranded DNA duplex (TOPI dsDNA) transfection of the siRNA of lamic A/C or equal length.In all cases, each point with the antibody test of anti-eIF-4E to determine the difference of albumen applied sample amount.Relative quantity to every band is carried out quantitative assay.(D) TOPI siRNA prevents the necrocytosis that camptothecine causes in the through engineering approaches tumour cell.After using siRNA transfection and the processing of prescribed concentration camptothecine, detect cell number at TOPI.(E) okadaic acid, the inhibitor of PP2A and other cell Phosphoric acid esterases makes former generation people cell to the camptothecine sensitivity.The BJ primary cell is handled simultaneously with the camptothecine and the okadaic acid of prescribed concentration, measures the effect to fluorexon AM active coloring.Though okadaic acid kills and wounds the BJ cell at the maximum concentration of test, it self is invalid at 3.4nM, and it causes cell to the camptothecine sensitivity.(F) okadaic acid promotes the expression of TOPl.The BJ primary cell is handled with the okadaic acid of prescribed concentration, and the expression level of TOPI detects with western blot.Relative quantity to every band is carried out quantitative assay.

Fig. 6 shows that erastin is with ST/RAS ^V12The dependence pattern is induced quick necrocytosis.(A) erastin is to BJ-TERT and BJ-TERT/LT/ST/RAS ^V12The time dependent effect of cell.Under the situation that contains prescribed concentration erastin, cell seeding is in 384 orifice plates.Use fluorexon AM24, detecting the inhibition of cell viability after 48 and 72 hours.(B) at BJ-TERT (red) and BJ-TERT/LT/ST/RAS ^V12Erastin is to the effect of Alamar Blue active coloring in (indigo plant) cell.(C) BJ-TERT/LT/ST/RAS that handles with erastin ^V12Photo with BJ primary cell cell.Cell was handled 24 hours with 9 μ M erastin then in conjunction with spending the night, and took pictures then.

Fig. 7 shows camptothecine rather than the apoptosis-induced feature of erastin.(A) camptothecine is handled, rather than the erastin processing, BJ-TERT/LT/ST/RAS ^V12Cell shows broken nuclear (10-20%, redness and the blue arrow of total nuclear) as shown in the figure.(B) camptothecine is handled, rather than the erastin processing, BJ-TERT/LT/ST/RAS ^V12Cell shows Annexin V dyeing.Be untreated, erastin handle (9 μ M) and camptothecine handle (1 μ M) accordingly the cell per-cent in specified M1 zone be 6%, 6% and 38%.(C) camptothecine is handled, rather than the erastin processing, BJ-TERT/LT/ST/RAS ^V12Cell has activatory caspase3.Camptothecine and erastin handle the split product utilization of sample and analyze by western blot at the antibody of the caspase3 that activity/shear-form is arranged.Spot detects once more with the antibody of anti-eIF4E, to control the sample level.

Fig. 8 shows the chemical structure of erastin and erastin B.

The neoplastic cell nuclei that Fig. 9 is presented at the erastin processing is kept perfectly.

Figure 10 shows the formation of erastin induced activity oxygen.

Figure 11 shows the chemical structure of erastin A.

Figure 12 points out that with respect to the expression in the non-tumorigenesis BJEH cell, the expression of VDAC3 raises significantly in the tumorigenesis BJELR cell.

Figure 13 demonstration is made as 100% with the VDAC-1 level, the relative expression level of VDAC isoform in target cell.

Figure 14 shows the albumen that utilizes plastosome extract and immobilized activity (A6) and non-activity (B1) Erastin derivative to identify by Western blot and SDS-PAGE from the pull-down test.

Figure 15 is presented at (a) HCT116 cell, (b) DLD-I cell, (c) OVCAR-3 cell and (d)

compound

12,13 and 5 in the MCL test of BT549 cell.

Figure 16 is presented at (a) MiaPaca2 cell, (b) DU145 cell, (c) SK-MeI28 cell and (d)

compound

12,13 and 5 in the MCL test of Malm3M cell.

Figure 17 is presented at (a) BT549 cell, (b) MCF-7 cell, (c) HOP-92 cell and (d)

compound

12,13 and 5 of the MCL test of HOP-62 cell.

Figure 18 shows compound 6 induced tumor growth-inhibiting in the HT-1080 heterograft.

Figure 19 shows that compound 5 induces tangible tumor regression in the HT-1080 heterograft.

Figure 20 is presented at the body weight trend of using compound 5 in the HT-1080 heterograft.

Figure 21 shows compound 6 induced tumor growth-inhibiting in the PANC-1 heterograft.

Figure 22 shows that compound 5 induced tumor in the PANC-1 heterograft disappears.

Detailed Description Of The Invention

The genotype alternative cpd is as the ability of the molecular probe prerequisite based on chemical genetics, and namely little molecule can be used for identifying albumen and approach (Schreiber, 1998, Bioorg.Med.Chem.6, the 1127-1152 that exercises biology effect; Stockwell, 2000, Nat Rev Genet 1,116-25; Stockwell, 2000, Trends Biotechnol 18,449-55). For example, observe the natural products rapamycin and stop Growth of Cells, make the target of finding mammiferous rapamycin (mTOR) as the albumen of regulating Growth of Cells become possible (people such as Brown, 1994, Nature 369,756-758; The people such as Sabatini, 1994, Cell 78,35-43). The applicant is in conjunction with these two kinds of methods, and chemistry and molecular genetics are used for finding to be subjected to for example related approach that suddenlys change and affect of cancer of human diseases.

The applicant has processed a series of tumour cells with definite genetic elements, and for the identification of those critical paths, the destruction of these approach will cause tumorigenic phenotype (people such as Hahn, 1999, Nat Med 5,1164-70; The people such as Hahn, 2002, Nat Rev Cancer 2,331-41; The people such as Lessnick, 2002, Cancer Cell 1,393-401). The applicant infers cells that these experiments transform and makes that identified gene type selective reagent becomes possibility from known and novel compound source, and described reagent shows synthetic lethal in the situation that the relevant allele of particular cancers exists. Compound with genotype selection lethal can be used as the molecular probe of signal network in the tumour cell, and has the primer of the clinical active drug of good efficacy index as subsequent development, and/or as active drug.

Make in this way, the applicant carries out high flux screening research to natural and synthetic compound library, and has identified the compound that kills and wounds by force the cancer activity. Erastin and erastin analog such as erastin B and erastin A just in these compounds. However, much detect reagent or compound and can be used to screening study described herein (for example, identifying the method for antitumor material standed for). This detection reagent includes but not limited to, little organic molecule, peptide, simulating peptide, albumen (comprising antibody), nucleic acid, carbohydrate.

Therefore, the invention provides the compound of general formula I, described compound kills and wounds cancer cell, especially the special cancer cell of genotype, active such as those Ras signals with rising, the little t antigen of the SV40 of change target activity, and/or the cancer cell of complete Rb activity basically.

The applicant has also identified several direct or indirect cell proteins in conjunction with erastin and/or its analog. These albumen comprise: voltage dependence anion channel (VDAC1, VDAC2, and VDAC3), Prohibitin, ribophorin (ribophorin), Sec61a and Sec22b. Quantitative RT-PCR also shows the VDAC3 expression rising (for example, high 2-6 times, common high 2-2.5 doubly) that erastin is killed and wounded responsive cell. But do not wish to be fettered by any particular theory, these experiments show high-caliber VDAC, and especially VDAC3 and VDAC2 express the cell killing that strengthens erastin (with its analog) mediation, this in addition may be that effect is needed.

Therefore, one aspect of the present invention provides a kind of method of selective killing cancer cell, especially those Ras with rising are active, the little t antigen of the SV40 target activity that changes, active with preferred basically complete Rb and/or p53, described method comprises the compound by the general formula I representative to the mammalian subject drug treatment effective dose of needs treatment:

Wherein:

R ¹Be selected from H ,-Z-Q-Z ,-C_1-8Alkyl-N (R²)(R ⁴)、-C _1-8Alkyl-OR³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and aralkyl;

Each R that occurs²And R⁴All be independently to be selected from H, C_1-4Alkyl, C_1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R²And R⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R²And R⁴At identical nitrogen and R²Or R⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C_1-8Alkyl, aryl, C_1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C_1-4Alkyl, C_1-4Aralkyl, aryl and heteroaryl;

W is selected fromOr

Q is selected from O and NR² With

Each Z that occurs is independently selected from C_1-6Alkyl, C_2-6Thiazolinyl and C_2-6Alkynyl. When Z is thiazolinyl or alkynyl group, two keys or triple bond are preferred not at the end of group.

In certain embodiments, the compound of general formula I does not comprise erastin or erastin A.

As known in the art, the activation of the composition of Ras is the key factor of human cancer cell malignancy. In 20% to 30% human tumor, find RAS proto-oncogene (H-RAS, N-RAS, K-RAS) sudden change is the heredity distortion that takes place frequently, although the incidence in different tumor types alters a great deal (Bos, Cancer Res.49:4682-4689,1989). The highest RAS mutation rate is at pancreas adenocarcinoma (90%), finds in adenocarcinoma of colon (50%) and the adenocarcinoma of lung (30%). In thyroid folliculus and undifferentiated carcinoma, the generation of RAS sudden change also is quite high (50%). The most normal RAS that observes mutates the critical sites that present Ras regulates--and be

codon

12,13 and 61. Each of these sudden changes all causes the elimination of the normal GTPase activity of Ras. Ras activation is also for example observed in myelomatosis and the Huppert's disease through the hematologic malignancies of being everlasting. In myelodysplastic syndrome about 1/3rd (MDS) and the acute myelogenous leukemia (AML), the activation that suddenlyd change of RAS gene. The RAS sudden change is present in about 40% multiple myeloma patients of newly making a definite diagnosis, and frequency increases with course advancement.

On the other hand, polyomavirus infects multiple vertebrate (now known 12 members). The Muridae polyomavirus separated during by the leukaemia of Ludwig Gross at Study Mouse in nineteen fifty-three, and name is because it causes solid tumor in a plurality of sites like this. Second member of this family, Sweet and Hilleman have separated ape vacuolating virus 40 (SV40) (Hilleman in nineteen sixty at the former generation MK cells culture that is used for growth Sabin OPV, Dev Biol Stand 94:183-190,1998). Two kinds of human polyoma viruses, cBK virus (BKV) and JC virus (JCV), separated in 1971.

Three kinds of albumen that participate in cell transformation of polyomavirus coding are called large tumour antigen (LT), middle T antigen (mT) and little tumour antigen (sT). These three kinds of albumen are that the different montages by the early region transcription product cause, and comprise homologous sequence. The large T antigen of polyoma and tumor suppressor protein pRb interact, and can make former generation fibroblast immortalization in cultivation. Be positioned at the Dna J domain of N end, the HPDKYG sequence of especially between residue 42 and 47, finding, very crucial to the functional inactivation of Rb family, also be like this to the SV40 large T antigen. The expression of LT be not enough to produce fully transform cell phenotype--this needs mT, it is the main transforming protein of polyomavirus. T is comprised of 421 amino acid in the little hamster polyomavirus, can be divided at least three domains, and some of them and LT and sT share. The amino terminal domain comprises 79 amino acid in front, and described domain also is present in LT and sT. Between residue 80-192 contiguous it be the domain that also is present in polyoma sT, comprise two cysteine enrichment regions, Cys-X-Cys-X-X-Cys, this is also identified in the little T of SV40 to go out. The ability of mT transformant is eliminated in the sudden change of these cysteines. Remaining 229 amino acid are unique to mT, comprise the main Tyr phosphorylation site of mouse mT and the water repellent region (at about 20 amino acid of c-terminus) of the film location that participates in the activity of conversion indispensable protein.

The little T antigen of SV40 comprises 174 amino acid. Zone between residue 97-103 and PP2A (PP2A) interact. These interactions have reduced the ability of PP2A deactivation ERK1 and MEK1 protein kinase, cause stimulating the propagation of static MK cells. Little T antigen relies on test and also identifies the zone that other have the ability that strengthens cell transformation. These zones are positioned at the N end parts, and this is that the little and large T antigen of SV40 shares, and can play potentially Dna J domain. Little T antigen can also in conjunction with tubulin, show that this works in its biological function.

The applicant finds to have activation Ras cell active and little T antigen presentation and (therefore reduces little T antigen target protein activity, PP2A that for example reduces etc., perhaps strengthen ERK1 and MEK1) can by erastin and its analog by selective killing and wounding, may pass through the acellular mechanism of apoptosis. In preferred embodiments, basically Rb and/or the p53 (perhaps other E6/E7 albumen targets) of wild type level of cellular expression.

Therefore, in certain embodiments, the cancer cell of some specific gene type can killing and wounding by compound selective of the present invention. These can comprise the cancer with the PP2A of ERK1, MEK1 activity or the deactivation of the Ras sudden change that forms activation or the sudden change of Ras signal pathway and enhancing.

In certain other embodiments, the genotype of target cell can be by selective change (for example, express the little T antigen of SV40, express ERK1 or MEK1, perhaps suppress PP2A, etc.), like this before to erastin and the insensitive target cell of erastin analog now to this sensitivity of killing and wounding.

Particularly; the invention provides a kind of method of selective killing cancer cell; described cancer cell has active and little T antigen presentation (the little T antigen target protein activity that perhaps changes of Ras of rising; for example PP2A is active; the ERK1 that strengthens or MEK1 are active, and perhaps a kind of mechanism of imitating the sT effect includes but not limited to the sudden change in the PP2A regulator subunit); and protection does not have the relative normal cell of the Ras activity of rising, even these cells are also expressed little T antigen. This may be useful, because many cancers are with body cell Ras^V12Perhaps cause active other similar sudden changes that raise of Ras signal in the cancer cell, and the normal cell in same patient/individuality there is not identical Ras usually^V12Perhaps other Ras approach sudden change. Erastin and its analog can be used for optionally killing and wounding these cancer cells, if cancer cell is expressed little T antigen (the little T antigen target protein that perhaps has change is active) simultaneously. Even other normal cells among individuality/patient are also expressed little T antigen, the inventive method still can effectively be killed and wounded cancer cell, because normal cell may not have the Ras signal of rising active. Even individuality is not expressed little T antigen, little T antigen can flow to patient's (with form of albumen or vector encoded DNA) makes it have the sensitiveness of the erastin/erastin analog being killed and wounded cancer (rather than normal) cell.

Self be not enough to induce the side effect to the patient since be understood that little T antigen, the side effect for the treatment of (providing little T antigen to the patient) will be small or non-existent. In fact nearly 3,000 ten thousand Americans are considered to be exposed to SV40 by the polio vaccine inoculation between 1955 to 1963. SV40 enters vaccine by the RhMK cell that is used for the growth poliovirus. Those methods no longer are used, and the polio vaccine did not contain virus from 1963. The DNA research of the nineties is found SV40 in some human tumors. Yet the viral DNA in the tumor tissues causes the formation of tumour with the relevant virus that can not prove of somatoblast. In October, 2002, a science group of American Medical Association reaches a conclusion, and whether definite decades ago widely used polio vaccine that is polluted by simian virus SV40 of having no idea causes the increase of human cancer illness rate.

In some embodiments, the Ras of rising is active in

amino acid position

12,13 and/or 61 composition activation Ras (N-, H-, perhaps K-Ras) sudden change and show.

In some of the other embodiments, the rising of Ras activity shows by the activity that one or more downstream components of Ras pathway protein strengthen, includes but not limited to Raf, MEK, MAPK etc.

In other embodiments, little T antigen presentation can be attended by carrier to the infection of target cell, such as adenovirus or the retroviral vector (seeing below) of expressing the little T antigen of SV40.

Perhaps, little T antigen can directly offer target cell. For example, little T antigen can utilize the whole bag of tricks known in the art to import target cell (details vide infra). In one embodiment, little T antigen can offer target cell by the surface of liposome (for example lipofectins) that is captured in positive charge, the antibody labeling of optional available anti-target tissue cell surface antigen, for example, the antibody of anticancer cell surface antigen. In another embodiment, little T antigen can offer target cell by dysuria with lower abdominal colic (transcytosis) effect, utilize any " the internalization peptide " that can regulate this effect, (for example include but not limited to the N end structure territory of HIV albumen Tat, the residue 1-72 of Tat or its can promote the more small fragment of transcytosis), fruit bat antenopedia III albumen all or part of, enough parts of mastoparan (mastoparan) etc. (seeing below).

In other embodiments, the PP2A by carrying antibody, RNAi (siRNA, short hairpin RNA, etc.), antisense sequences or can obtaining to reduce to the special micromolecular inhibitor of this target protein.

The conveying of this target cell protein antagonist is well known in the art. Referring to, for example, WO04078940A2, EP1439227A1, WO04048545A2, US20040029275A1, WO03076592A2, WO04076674A1, WO9746671A1 all is incorporated herein by reference at this.

Another aspect of the present invention provides a kind of erastin/erastin of utilization analog and one or more by the reagent of Apoptosis mechanism killer cell or the conjoint therapy of therapy (for example radiotherapy). This reagent comprises Treated with Chemotherapeutic Drugs thing as described below.

It is believed that some albumen expression in the erastin sensitive cells raises. For example, a kind of like this albumen, VDAC3, when being exposed to erastin abundance rising 2-2.5 doubly, but the applicant do not wish to be bound by theory, it is requisite that the abundance of its existence and even increase is considered to killing and wounding of erastin mediation.

Therefore in another aspect of the present invention, the method of a kind of killer cell or reduction cell proliferation rate is provided, in the described cell VDAC for example the level of VDAC2 or VDAC3 raise, described method comprises target cell is contacted with erastin and/or the erastin analog of general formula I-IV.

In certain embodiments, target cell is strengthened the sensitiveness of killing and wounding or reduce the rate of increase by erastin and its functional analogue like this by the higher levels of VDAC of regulating and expressing for example VDAC2 or VDAC3.

For example, VDAC albumen can utilize the whole bag of tricks known in the art to import target cell (referring to following detailed description). In one embodiment, VDAC albumen can offer target cell by being captured in the liposome (for example lipofectins) with the positive charge surface, this liposome carries out mark with the antibody of the cell surface antigen of anti-target tissue alternatively, for example, the antibody of anticancer cell surface antigen. In another embodiment, VDAC albumen can offer target cell by transcytosis, utilize any " the internalization peptide " that can regulate this effect, (for example include but not limited to the N end structure territory of HIV albumen Tat, the residue 1-72 of Tat or its can promote the more small fragment of transcytosis), fruit bat antennapedia III albumen all or part of, enough parts of mastoparan etc. (seeing below).

In addition, the nucleic acid of encoding function VDAC can import this target cell, utilizes adenovirus or the retroviral vector of for example expressing VDAC.

In addition, (for example, VDAC3) activity can be stimulated by reagent endogenous VDAC, and described reagent stimulates VDAC to express, and perhaps suppresses the activity of VDAC inhibitor (transcribe or TI, perhaps promote the inhibitor that VDAC changes at transit cell).

In some aspects, method of the present invention also relates to the reagent of VDAC (for example, VDAC2, VDAC3) abundance in a kind of increase cell of administration. For increasing the reagent of VDAC abundance can, for example, comprise the polynucleotides of VDAC such as the VDAC3 that encodes; The VDAC albumen that cell is advanced in suitable transportation (for example, VDAC3), for example, merges with allos internalization domain or is prepared into Liposomal formulation.

In some aspects, method of the present invention also relates to the reagent of VDAC (for example, VDAC2, VDAC3) abundance in a kind of reduction cell of administration. Reagent for reducing the VDAC abundance can, for example, suppress (inhibit) endogenous VDAC (for example VDAC3) and express, (for example suppress (suppress) VDAC, VDAC3) express or strengthen VDAC (for example, the VDAC3) function of inhibitor.

Following chapters and sections are described some exemplary embodiment of the present invention, and their expections can be bonded to each other. In addition, described embodiment only is used for illustration purpose, is not that where face limits in office.

Through engineering approaches clone

On the one hand, the present invention relates to through engineering approaches tumorigenic cell system.

Report before shows, by introduce expressing hTERT and carcinogenic RAS albumen and other destroy the carrier of p53, RB and PP2A function, might be tumorigenic cell (people such as Hahn, 2002 with former generation people cell transformation, MoI Cell Biol 22,2111-23; The people such as Hahn, 1999, Nature 400,464-8; Hahn and Weinberg, 2002, Nat Rev Cancer 2,331-41; The people such as Lessnick, 2002, Cancer Cell 1,393-401). The applicant utilized a series of through engineering approaches people's tumorigenic cells and they precursor, these make up (Fig. 1) by introduce specific genetic elements in former generation HFF. The previous various features of having reported these through engineering approaches tumorigenic cells, the doubling time that comprises them, their anti-abilities that wears out and cultivate crisis that copies, their reaction to gamma-radiation, they are with the ability of non-anchorage dependence pattern growth, and they form ability (people such as Hahn, 1999, the preamble of tumour in immunodeficient mouse; The people such as Hahn, 2002, preamble; The people such as Lessnick, 2002, preamble).

In the through engineering approaches cell of a series, following genetic elements is sequentially introduced former generation BJ fibroblast: the catalytic subunit of people's telomerase (hTERT), the gene construct of coding simian virus 40 large (LT) and little T (sT) cancer protein, and the carcinogenic allele (RAS of HRAS^V12). The transformation cell lines that obtains is called after: BJ-TERT, BJ-TERT/LT/ST and BJ-TERT/LT/ST/RAS respectively^V12 In second series, complementary DNA (cDNA) construct of coding LT and ST is used for replacing the SV40 gene construct of this two-strain albumen of coding, thus the clone of constructing. In a rear series, ST in the end the stage be introduced into, make the applicant can ST exist or the situation of disappearance under detection compound. Rear a kind of through engineering approaches people's tumorigenic cell is called after BJ-TERT/LT/RAS^V12/ST。

In the 3rd series, use the clone of the people TIP5 human foreskin fibroblasts that derives from independent preparation, this is by introducing coding hTERT, LT, ST and RAS^V12The cDNA construct obtain (people such as Lessnick, 2002, Cancer Cell 1,393-401). These clones that obtain are called after: TIP5/TERT, TIP5/TERT/LT, TIP5/TERT/LT/ST and TIP5/TERT/LT/ST/RAS respectively^V12 In the 4th series, use to derive from the fibroblastic clone of TIP5, this is by introducing coding hTERT, E6, E7, ST and RAS^V12CDNA obtain. These clones are called after: TIP5/TERT/E6, TIP5/TERT/E6/E7, TIP5/TERT/E6/E7/ST and TIP5/TERT/E6/E7/ST/RAS respectively^V12 In these series, HPV E6 and E7, respectively deactivation p53 and RB, play with in the similar effect of LT in above-mentioned series. But by utilizing HPV E6 and E7, the applicant can observe respectively and the effect of deactivation p53 and RB independently. The result of large-scale screening compounds is described, these through engineering approaches tumorigenic cells systems of described compound exhibits selective killing in example subsequently.

The method of screening-gene type alternative cpd

In certain embodiments, the present invention relates to the Large-scale Screening compound, described compound exhibits selective killing or inhibition through engineering approaches tumorigenic cell system's growth (selectively toxicity). Term reagent used herein and medicine are used interchangeably. Term used herein " right ... toxic " refers to that reagent or compound kill and wound or suppress the ability of the growth of tumorigenic cell/propagation. Large-scale Screening comprises hundreds of compound is carried out high flux screening to the selection toxicity of through engineering approaches tumorigenic cell. In one embodiment of the invention, the vigor by cell to be measured after relatively contacting with candidate agent and control cells detects optionally toxicity, and described cell to be measured is the through engineering approaches tumorigenic cell. A kind of suitable contrast is the cell with cell same type to be measured, except control cells is not engineered to tumorigenesis. For example, control cells may be the parent's primary cell that derives cell to be measured. Control cells contacts candidate agent under the condition identical with control cells to be measured. Suitable contrast can be carried out simultaneously, perhaps can set up in advance (for example, in advance Criterion or reference). In certain embodiments, candidate agent is selected from library of compounds, such as combinatorial libraries. Cell viability can detect by the whole bag of tricks known in the art, comprises and utilizes dyestuff for example calcein acetyl methyl esters (calcein AM) and Alamar Blue. In certain embodiments of the invention, after processing with candidate agent, dyestuff is used to be measured and control cells such as calcein. In living cells, calcein AM is cut by born of the same parents' lactonase, forms anion fluorescent derivative calcein, and it can't diffuse out living cells. Therefore, when hatching with calcein AM, living cells shows green fluorescence, and dead cell does not show. The green fluorescence that living cells shows can be detected, thereby the method that detects cell viability is provided.

In certain embodiments of the invention, the further characteristic of indentifying substance in animal model, described reagent has been defined as the selective induction cell death in the through engineering approaches tumorigenic cell. Animal model comprises mouse, rat, and rabbit and monkey, they can be non-transgenic (for example wild type) or transgenic animals. The effect of the reagent of selective induction cell death can be evaluated any multi-effect in animal model in the through engineering approaches tumorigenic cell, for example the ability of its selective induction cell death in the animal tumorigenic cell and its overall toxicity to animal. For example, this method also is included in and evaluates reagent (for example medicine) in the suitable mouse model to the selection toxicity of tumorigenic cell.

The effect of the reagent of inducing cell death can be evaluated any multi-effect in animal model in the through engineering approaches tumorigenic cell, for example the ability of its inducing cell death in the tumorigenic cell of animal and its overall toxicity to animal. For example, this method also is included in and evaluates reagent (for example medicine) in the suitable mouse model to the toxicity of tumorigenic cell. In order to illustrate, further estimate reagent by using the tumor growth test, the ability of the implanted solid tumor growth of setting up in the described test evaluation test agent inhibition mouse. Described test is undertaken by the fat pad implantation tumour cell nude mice. Allow growth of tumour cell to arrive a certain size before the administration. Within several weeks, monitor gross tumor volume, for example, three weeks. In process of the test, also monitor the general health of tested animal.

In other embodiments of the present invention, further evaluate the mechanism of action of reagent in based on the test of cell, described reagent has been accredited as the reagent of a kind of selective killing or inhibition through engineering approaches tumorigenic cell growth/propagation. For example, reagent can detect its ability by front-apoptotic pathways inducing cell death in the apoptosis test. In other embodiments of the present invention, evaluation reagent is induced dead ability by the acellular apoptosis pathway in tumorigenic cell, and described reagent is induced death in tumour cell. For example, reagent can in apoptosis test, detect its by front-apoptotic pathways can not inducing cell death ability.

In other embodiments, the present invention relates to the method for a kind of indentifying substance (for example, medicine), described reagent is the selective cytotoxicity that suppresses in the through engineering approaches cell. In one embodiment, the present invention relates to the method that a kind of evaluation suppresses Cytotoxic reagent (medicine), comprise with candidate agent contact measured cell (for example, expressing the through engineering approaches cell of mutain); Detect the vigor of the cell to be measured that contacts with candidate agent; With the vigor of the vigor of cell to be measured and suitable contrast is made comparisons. If if the work of cell to be measured, then identifies the Cytotoxic reagent of selective inhibition (medicine) greater than the vigor of control cells. Suitable contrast is the cell identical with cell type to be measured, except control cells is not carried out through engineering approaches causes toxicity with expression albumen. For example, control cells may be the parent's primary cell that derives cell to be measured. Control cells contacts candidate agent under the condition identical with control cells to be measured. Suitable contrast can be carried out simultaneously, perhaps can set up in advance (for example, in advance Criterion or reference).

In certain embodiments, genotype of the present invention optionally compound (anti-tumor agent comprising salmosin) can be any chemicals (element, molecule, compound, medicine), though be synthesize, with recombinant technique preparation or separate from natural origin. For example, these compounds can be peptides, polypeptide, class peptide, sugar, hormone, perhaps nucleic acid molecules (such as antisense or RNAi nucleic acid molecules). In addition, these compounds can be little molecule or the more complicated molecule by combinatorial chemistry preparation, for example, and are combined into the library. These libraries can comprise, for example, and alcohol, alkyl halide, amine, acid amides, ester, aldehyde, the organic compound of ether and other types. These compounds can also be to separate the natural or genetic engineering product of--bacterium, animal or plant cell--pyrolysis product or growth medium from cell or can be product of cell lysis or growth medium self. The form of compound that can adopt the compound of separation or mixing with these compounds for detection of system, especially in initial screening step.

Genotype alternative cpd of the present invention

Result's proof of applicant may identify effectiveness and active compound with increase in the situation that specific genetic elements exists. Although previous report is pointed out may identify optionally compound (people such as Simons, 2001, Genome Res 11,266-73 of this genotype to interested a kind of genetic elements; The people such as Stockwell, 1999, Chem Biol 6,71-83; The people such as Torrance, 2001, Nat Biotechnol 19,940-5), work described herein provides a kind of utilization to surpass the system test of the synthetic lethal of 23,000 kinds of compounds and one or more cancer correlated inheritance elements.

9 kinds of alternative cpds identifying help clearly to introduce at normal cell the consequence of TERT and one or more LT, ST, E6, E7 and carcinogenic RAS. An effect of these hereditary changes is to improve cell proliferation rate, and responsive to suppressing the synthetic little molecule of DNA. Although determined that this reagent preferably is targeted to the tumour cell of quick copy, it is other conclusive evidence that this principle occurs without inclined to one side screening technique at this. In addition, the method can easily be distinguished those compounds that have the selective basis of clear and definite heredity and do not have.

The result shows that the expression of hTERT and E7 or LT makes cell responsive to the topoisomerase II toxin. Because the loss of RB or inactivation (Sellers and Kaelin, 1997, J Clin Oncol 15,3301-12; Sherr, 2001, Nat Rev MoI Cell Biol 2,731-7) and activation (Hahn and Weinberg, 2002, Nat Rev Cancer 2, the 331-41 of telomerase; Harley, 1994, Pathol Biol (Paris) 42 342-5) is found in most of human cancer, and the activity of these reagent in human tumor type on a large scale can be partly explained in these observations.

The applicant finds that camptothecine selectively causes death with the cell of ST and carcinogenic RAS, because the expression of these two kinds of gene pairs topoisomerase Is has the effect of combination. The tumour cell of division utilizes topoisomerase I to untie super coiled DNA so that continuous and fast cell division fast. When these two approach are changed simultaneously, topoisomerase I is raised, and may be indirectly, causes this tumour cell responsive to the topoisomerase I toxin.

These observations show ST and RAS^V12, LT and hTERT transformation of human cell ability an aspect can be ST and RAS^V12Effect to topoisomerase I. Sudden change among HRAS and the KRAS was described in many kinds of human cancers. In addition, be reported in recently the PP2A element arranged in colon and the lung neoplasm--the inactivation (people such as Wang of PPP2R1B, 1998, Science 282,284-7), and the sudden change in different PP2A subunits has description (people such as Calin, 2000 in melanoma, lung, mammary gland and colon cancer, Oncogene 19,1191-5; The people such as Kohno, 1999, Cancer Res 59,4170-4; The people such as Ruediger, 2001, Oncogene 20,1892-9; The people such as Ruediger, 2001, Oncogene 20,10-5). At present, change whether whether cancer occurred frequently or these two approach disorders shows that the susceptibility to these compounds increases in human tumor when not clear this two approach.

In addition, the applicant identifies a kind of novel compound, is called erastin (referring to Fig. 8), and it is to expressing ST and RAS^V12Cell cause death. Even when its working concentration ratio is being expressed RAS^V12When observing high 8 times of effect desired concn in the cell of ST, fail to kill and wound with this compound treatment cell and lack RAS^V12With the cell of ST, show certain specificity. In case obtain, the lethal effect of erastin is fast with irreversible.

Erastin can be used at any tumour cell inducing cell death, and wherein erastin contacts with tumour cell and causes cell death. Be not only the through engineering approaches tumorigenic cell by what comprise in the active tumorigenic cell that produces lethal of erastin, for example express ST and RAS^V12The through engineering approaches cell, and comprise in addition and do not rely on ST and RAS^V12The tumorigenic cell of the RAS approach of the activation of expressing.

The applicant has also detected the activity and selectivity of 135 erastin analogs in the relative normal cell of tumour cell. In these analogs 134 be do not have activated. One has activity and selectivity, but renders a service lower than erastin. This compound is named as erastin B (referring to Fig. 8). In certain embodiments of the invention, the present invention relates to compound, erastin. In other embodiments, the present invention relates to the analog of Verbindung rastin, wherein analog shows the selection toxicity to the through engineering approaches tumorigenic cell, such as through engineering approaches people tumorigenic cell. In one embodiment, showing the erastin analog of through engineering approaches people tumorigenic cell being selected toxicity, is erastin B. In certain embodiments, the present invention relates to the racemic mixture of the compounds of this invention, described mixture shows the selection toxicity to the through engineering approaches tumorigenic cell.

Concerning camptothecine (CPT) and erastin, the applicant has determined by RAS^V12And ST expresses the synergy between the approach that changes. RAS^V12Expression cause several activation of the clear signal pathway that characterizes, comprise the RAF-MEK-MAPK signal cascade, phosphatidylinositols 3-kinases (PI3K) signal pathway and Ral-guanine dissociation factor approach (Ral-GDS). Each of these approach is all relevant with human cancer, nearest studies show that these approach and cell transformation system echo mutually and work (people such as Hamad, 2002, Genes Dev 16,2045-57). In addition, the serine-threonine phosphatase PP2A of ST combination and a kind of wide expression of deactivation. Although when ST expresses the PP2A of multilated the certain enzyme target also do not know, in the approach that is changed by PP2A and RAS, exist in essence overlapping (people such as Millward, 1999, Trends Biochem Sci 24,186-91). Further understand by which kind of mechanism, erastin induces death in these two signal pathway cells that band changes, the overlapping essence of these two approach functions and the clue of degree can be provided.

Erastin analog of the present invention except erastin B and erastinA, represents with general formula I:

Wherein:

R ¹Be selected from H ,-Z-Q-Z ,-C_1-8Alkyl-N (R²)(R ⁴)、-C _1-8Alkyl-OR³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and C_1-4Aralkyl;

Each R that occurs²And R⁴All be independently to be selected from H, C_1-4Alkyl, C_1-4Aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are if work as R²And R⁴On identical nitrogen and R²Or R⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C_1-8Alkyl, aryl, C_1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C_1-4Alkyl, C_1-4Aralkyl, aryl and heteroaryl;

W is selected fromOr

Q is selected from O and NR² With

In some preferred embodiment, work as R²And R⁴In the time of on identical N atom, they can all be H, or different.

In certain embodiments, R¹H.

In certain embodiments, W is

。

In certain embodiments, R⁴Be selected from H or replacement or for get low alkyl group.

In certain embodiments, R¹Be H, W is，R ⁴Be selected from low alkyl group H or replacement or unsubstituted.

The example compound of general formula I comprises:

Other erastin analogs of the present invention, I represents with general formula I:

Wherein

Ar is the phenyl that replaces;

R ¹Be selected from H ,-C_1-8Alkyl ,-Z-Q-Z ,-C_1-8Alkyl-N (R²)(R ⁴)、-C _1-8Alkyl-OR³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and C_1-4Aralkyl;

Each R that occurs²And R⁴All be independently to be selected from H, C_1-4Alkyl, C_1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R²And R⁴On identical nitrogen and R²Or R⁴Be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C_1-8Alkyl, aryl, C_1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C_1-4Alkyl, C_1-4Aralkyl, aryl and heteroaryl;

R ⁵The 0-4 of representative on the ring of its a combination substituting group.

W is

Perhaps

Q is selected from O and NR² With

In some embodiments, R⁵Represent 1-4 substituting group such as halogen or nitro. In certain embodiments, R5 represents a substituting group, and such as halogen or nitro, especially chlorine is positioned in the carbonyl contraposition of quinazolone ring. In other embodiments, the upper unsubstituted (that is, all substituting groups are hydrogen atoms) of R5 representative ring.

In some embodiment, Ar is single replacement, and wherein substituting group is halogen, lower alkoxy, perhaps low alkyl group. In certain embodiments, Ar has a substituting group at the ortho position, and wherein substituting group is halogen, lower alkoxy, perhaps low alkyl group. In certain embodiments, Ar is that 2,6-two replaces, and like this a substituting group is halogen, lower alkoxy, and perhaps low alkyl group, second substituting group is halogen, lower alkoxy, perhaps low alkyl group.

In certain embodiments, the compound of general formula I I do not comprise those wherein the substituting group on the Ar be ethyoxyl, it is positioned at the compound of the position at nitrogen key ortho position on the quinazolone ring. In other embodiments, the compound of general formula I I do not comprise those wherein Ar be not positioned at lower alkoxy or the low-grade alkyl substituent at ortho position of the nitrogen key of quinazolone ring.

In some embodiment of the compound of general formula I I, Ar has a halogenic substituent at least. In certain embodiments, Ar has halogenic substituent at the ortho position. In preferred embodiments, the compound of general formula I I comprise those wherein Ar be the dibasic phenyl ring of 2,6-, wherein substituting group is halogen atom.

The exemplary compounds of general formula I I comprises:

Other erastin analogs of the present invention represent with general formula III:

Wherein

Ar replaces or unsubstituted phenyl;

R ³Be selected from H, C_1-4Alkyl, C_1-4Aralkyl, aryl and heteroaryl;

W is selected from

, perhaps

Q is selected from O and NR²。

In certain embodiments, R2 is with R4 or be hydrogen or different.

In some embodiment, R5 represents 1-4 substituting group on the ring of its combination, such as halogen or nitro. In certain embodiments, R5 represents a substituting group, and such as halogen or nitro, especially chlorine is positioned in the carbonyl contraposition of quinazolone ring. In other embodiments, the upper unsubstituted (that is, all substituting groups are hydrogen atoms) of R5 representative ring.

In certain embodiments, the compound of general formula III do not comprise those wherein the substituting group on the Ar be the compound that is positioned at the ethyoxyl of the position at nitrogen key ortho position on the quinazolone ring. In other embodiments, the compound of general formula III do not comprise those wherein Ar be not positioned at lower alkoxy or the former compound of low-grade alkyl substituent at ortho position of the nitrogen key of quinazolone ring.

In the preferred embodiment of the invention, Ar is the phenyl that replaces. In some embodiment of the compound of general formula III, Ar has a halogenic substituent at least. In certain embodiments, Ar has at the ortho position halogenic substituent. In preferred embodiments, the compound of general formula III comprise those wherein Ar be the dibasic phenyl ring of 2,6-, wherein substituting group is halogen atom.

The exemplary compounds of general formula III comprises:

Other erastin analogs of the present invention represent with general formula I V:

Wherein

Ar is that replace or unsubstituted phenyl;

R ¹Be-C_1-8Alkyl;

Each R that occurs²And R⁴All be independently to be selected from H and C_1-8Alkyl;

R ⁵The 0-4 substituting group of representative on the ring of its combination.

W is selected from

Perhaps

Q is selected from O and NR²。

In the preferred embodiment of the invention, Ar is substituted-phenyl. In certain embodiments, Ar is single replacement, and wherein substituting group is halogen, lower alkoxy, perhaps low alkyl group. In certain embodiments, Ar has substituting group at the ortho position, and wherein substituting group is halogen, lower alkoxy, perhaps low alkyl group. In certain embodiments, Ar is that 2,6-is dibasic, and like this a substituting group is halogen, lower alkoxy, and perhaps low alkyl group, second substituting group is halogen, lower alkoxy, perhaps low alkyl group.

In certain embodiments, the compound of general formula I V do not comprise those wherein the substituting group on the Ar be the compound that is positioned at the ethyoxyl of the position at nitrogen key ortho position on the quinazolone ring. In other embodiments, the compound of general formula I V do not comprise those wherein Ar be not positioned at the lower alkoxy at ortho position of nitrogen key of quinazolone ring or the compound of low-grade alkyl substituent.

In some embodiment of the compound of general formula I V, Ar has a halogenic substituent at least. In certain embodiments, Ar has halogenic substituent at the ortho position. In preferred embodiments, the compound of general formula I V comprise those wherein Ar be the dibasic phenyl ring of 2,6-, wherein substituting group is halogen atom.

The exemplary compounds of general formula I V comprises:

Other erastin analogs of the present invention represent with general formula V:

Wherein

R ¹Be selected from H and C_1-8Alkyl;

R ²Be selected from H and C_1-8Alkyl;

R ³Be selected from halogen, C_1-8Alkoxyl and C_1-8Alkyl;

R ⁴Be selected from H, halogen, C_1-8Alkoxyl and C_1-8Alkyl;

R ⁵Be selected from H, halogen and nitro; With

N is 1 or 2.

The exemplary compounds of general formula V comprises:

The compound of general formula I-V can be used for erastin and the erastin analog any means of being used for described herein.

Compound of the present invention comprises enantiomer and the diastereomer of compound disclosed herein. The present invention also comprises the salt of compound disclosed herein, especially the acceptable salt of pharmacy. In addition, the present invention includes the solvate of compound disclosed herein, hydrate and polymorph crystal form.

Suitable reagent can have aforesaid activity after existing form or completely or partially metabolism.

The present invention also provides the synthetic or preparation of the compounds of this invention.

The invention provides in certain embodiments compd A

Preparation, R wherein⁵And R¹Such as the description to structure I I-V. In certain embodiments, the synthesis step of compd A is

Compd B

With Compound C

Reaction.

In certain embodiments, the reaction of compd B and Compound C is to carry out in the aprotic solvent of polarity, for example acetonitrile, DMSO, diethyl ether, butanone, cyclohexanone, acetophenone, oxolane, acetone, carrene, sulfolane or dimethyl formamide. In preferred embodiments, solvent is carrene or dimethyl formamide. In certain embodiments, reaction is carried out in nitrogen. In certain embodiments, organic base is such as pyridine, diisopropylamine, 2,6-lutidines, trialkylamine are (for example, triethylamine), pyrrolidines, imidazoles or piperidines, be added in the solution of compd B, then Compound C is added in the solution that obtains. In preferred embodiments, organic base is amine alkali, such as trialkylamine triethylamine for example. In preferred embodiments, reaction is to carry out at 0-10 ℃.

The present invention also provides the compounds process for production thereof of structure D

R wherein⁵、R ¹With Ar such as the description to structure I I-V. In certain embodiments, the synthesis step of D is compd A and compd E, Ar-NH₂Reaction.

In certain embodiments, the reaction of compd A and compd E is to carry out in the aprotic solvent of polarity, for example acetonitrile, DMSO, diethyl ether, butanone, cyclohexanone, acetophenone, oxolane, acetone, carrene, sulfolane or dimethyl formamide. In preferred embodiments, solvent is acetonitrile. In certain embodiments, reaction is carried out in nitrogen. In certain embodiments, reaction is carried out under the condition that the trichlorine phosphine exists. In certain embodiments, reaction is kept a period of time for example 5-15 hour at 40-60 ℃. In other embodiment, the phosphoryl terchoride is added in the solution A and E of stirring, and the mixture that obtains added hot reflux a period of time, such as 1-5 hour.

The present invention also provides structure F's

Compounds process for production thereof

R wherein⁵、R ¹, Ar and W be such as the description to structure I I-V. In certain embodiments, the synthesis step of compound F 17-hydroxy-corticosterone is Compound D and HNR₂Reaction, HNR wherein₂Be equivalent to HW. In certain embodiments, reaction is to carry out under the condition that exists in potash and iodide source, such as cuprous iodide, and KI, cesium iodide, sodium iodide, perhaps tetrabutylammonium iodide is in polar proton inert solvent. In certain embodiments, Compound D and potash mix, and add HNR₂, then be the iodide sources. In preferred embodiments, solvent is acetonitrile. In certain embodiments, iodide reagent is tetrabutylammonium iodide; In certain embodiments, iodide reagent is sodium iodide. In some embodiments, mixture keeps a period of time such as 5-15 hour at 50-70 ℃.

HNR2 (HW) comprises second nitrogen-atoms in certain embodiments, and amine protecting group group is arranged on it. In some embodiments, blocking group can be t-butoxycarbonyl, benzyloxycarbonyl group, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, 2-(trimethyl silyl) carbethoxyl group, perhaps 2,2,2-trichlorine ethoxy carbonyl. In certain embodiments, reaction is carried out under the condition that alkali exists, such as potash, and sodium carbonate, pyridine, diisopropylamine, 2,6-lutidines, triethylamine, pyrrolidines, imidazoles, carry out in polar proton inert solvent in perhaps piperidines, and iodide source. In certain embodiments, alkali is potash or triethylamine. In certain embodiments, Compound D and alkali mix, and add HNR₂, then be the iodide sources. In preferred embodiments, solvent is acetonitrile or acetone. In certain embodiments, iodide reagent is tetrabutylammonium iodide; In certain embodiments, iodide reagent is sodium iodide. In some embodiments, mixture keeps a period of time such as 1-10 hour at 70-90 ℃. After addition reaction is finished, can remove blocking group from the product that obtains by suitable deprotection reaction. For example, when blocking group was t-butoxycarbonyl, blocking group can remove by add acid in compound solution (for example adding 4N HCl De dioxane solution in product De dioxane solution). In certain embodiments, before neutralise mixt, reactant liquor water and organic solvent diluting. In certain embodiments, mixture is adjusted to alkalescence by adding saturated aqueous sodium carbonate.

Expect whole embodiment of the present invention can with one or more other embodiment couplings, although they are described at different aspect of the present invention.

Term " acyl group " is well known in the art, refers to the group of general formula alkyl C (O)-representative, preferred alkyl C (O)-.

Term " acylamino-" is well known in the art, refers to that amine groups is replaced by acyl group group, can be for example with general formula alkyl C (O) NH-representative.

Term " acyloxy " is well known in the art, refers to general formula alkyl C (O) O-, the group of preferred alkyl C (O) O-representative.

Term " alkoxyl " refers to have the alkyl of oxygen and its combination, preferred low alkyl group. Typical alkoxyl comprises methoxyl group, ethyoxyl, propoxyl group, uncle-butoxy and analog thereof.

Term " alkoxyalkyl " refers to that alkyl replaces with alkoxyl, can use general formula alkyl-O-alkyl represent.

Term used herein " thiazolinyl ", refer to a kind of fat-based, comprise at least one two key, and comprise " unsubstituted thiazolinyl " and " thiazolinyl of replacement ", wherein the latter refers to that alkenyl part has substituting group, and described substituting group is replaced the hydrogen on the one or more carbon of alkenyl group. This replacement may occur on one or more carbon atoms, and described carbon atom comprises or is not included in one or more two strandss. In addition, this replacement comprises the replacement of the alkyl that all can expect, and is as described below, except instability is unallowed. For example, with one or more alkyl, carbocyclic ring, aryl, heterocycle or heteroaryl groups substituted alkenyl group are expected to obtain.

Term " alkyl " refers to the saturated fat group, comprises straight chained alkyl, branched alkyl, cycloalkyl (alicyclic ring) group, the group of naphthene base of alkyl-replacement and the alkyl of cycloalkyl-replacement. In preferred embodiments, straight chain or branched alkyl have 30 or still less carbon atom (for example, C1-C30 for straight chain, C3-C30 for side chain) at its skeleton, and more preferably 20 or still less. Similarly, preferred cycloalkyl has the carbon atom from 3 to 10 in their circulus, and more preferably has 5,6 or 7 carbon in circulus.

In addition, the term that uses in whole specification, embodiment and claim " alkyl " (perhaps " low alkyl group ") comprises " unsubstituted alkyl " and " alkyl of replacement ", wherein the latter refers to that moieties has substituting group, the hydrogen on one or more carbon of the skeleton of described substituting group replacement hydrocarbon. This substituting group can comprise, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxy carbonyl group; formyl, perhaps acyl group), thiocarbonyl (such as thioesters, thioacetate, perhaps dithioformate), alkoxyl; phosphoryl, phosphate, phosphonate, phosphinates, amino, acylamino-; amidine, imines, cyano group, nitro, azido; sulfydryl, alkylthio group, sulfate, sulfonate, sulfonamides; sulfonamido, sulphonyl, heterocycle, aralkyl, perhaps aromatic series or heteroaromatic part. They self also are can be substituted to those skilled in the art will know that on the hydrocarbon chain substituted part, if suitable. For example, the substituting group of substituted alkyl can comprise the amino that replaces and do not replace form, azido; imino group, acylamino-, phosphoryl (comprising phosphonate and phosphinates); sulphonyl (comprising sulfate, sulfoamino-group, sulfonamides and sulfonate); and silyl-group, and ether, alkylthio group; carbonyl (comprises ketone, aldehyde, carboxylate; and ester) ,-CF₃,-CN and analog. The alkyl of exemplary replacement is described below. Cycloalkyl can also be used alkyl, thiazolinyl, alkoxyl, alkylthio group, aminoalkyl, the alkyl of carbonyl substituted ,-CF₃,-CN and analog replace.

Term " C_x-y" when with chemical part acyl group for example, acyloxy, alkyl, thiazolinyl, alkynyl perhaps represents to comprise some groups during the alkoxyl coupling, described group comprises a carbon from X to Y at chain. For example, term " C_x-yAlkyl " refer to saturated hydrocarbons group that replace or unsubstituted, comprise straight chained alkyl and branched alkyl group, described group comprises from X to Y a carbon at chain, comprises alkylhalide group group for example trifluoromethyl and 2,2,2-trifluoroethyl etc. C₀Alkyl represents if being positioned at terminal position is hydrogen, if in inside, then is key. Term " C_2-yThiazolinyl " and " C_2-yAlkynyl " refer to unsaturated fat-based that replace or unsubstituted, length is similar and may replace abovementioned alkyl, but comprises respectively at least one two keys or triple bond.

Term used herein " alkylamino " refers to the amine groups with at least one alkyl replacement.

Term used herein " alkylthio group " refers to can be represented by general formula alkyl S-with the mercapto of alkyl replacement.

Term used herein " alkynyl ", refer to a kind of fat-based, comprise at least one triple bond, and comprise " unsubstituted alkynyl " and " alkynyl of replacement ", wherein the latter refers to that alkynyl partly has substituting group, and described substituting group is replaced the hydrogen on the one or more carbon of alkynyl group. This substituting group may appear on one or more carbon atoms, and described carbon atom comprises or is not included in one or more triple bonds. In addition, this replacement comprises the replacement to alkyl that all can expect, as mentioned above, and except instability is unallowed. For example, with one or more alkyl, carbocyclic ring, aryl, heterocycle or heteroaryl groups substituted alkynyl group are expected to obtain. Term used herein " acid amides " as using herein, refers to a kind of group

R wherein⁹And R¹⁰Represent independently of one another hydrogen or hydrocarbyl group, perhaps R⁹And R¹⁰Nitrogen-atoms with their combinations is formed on the heterocycle that has 4-8 atom on the ring structure.

Term " amine " and " amino " they are well known in the art, refer to amine and salt thereof unsubstituted and that replace, for example, and the part that can be represented by following structure

R wherein⁹、R ¹⁰And R¹⁰' represent independently of one another hydrogen or hydrocarbyl group, perhaps R⁹And R¹⁰Nitrogen-atoms with their combinations is formed on the heterocycle that has 4-8 atom on the ring structure.

Term used herein " aminoalkyl " refers to the groups with the amino group replacement.

Term used herein " aralkyl " refers to the groups with the aromatic yl group replacement.

Term used herein " aryl " comprises monocyclic aromatic base replacement or unsubstituted, and wherein each atom on the ring is carbon. Preferred ring is that 5-encircles to 7-unit, more preferably 6-unit ring. Term " aryl " also comprises the polycyclic system with two or more rings, and having two or more carbon is that two adjacent ring are public, and wherein at least one ring is aromatic series, for example, another ring can be cycloalkyl, cycloalkenyl group, cycloalkynyl radical, aryl, heteroaryl, and/or heterocycle. Aromatic yl group comprises benzene, naphthalene, phenanthrene, phenol, aniline and analog.

Term " carbaminate " is well known in the art, refers to group

R wherein⁹And R¹⁰Represent independently hydrogen or hydrocarbyl group.

Term used herein " carbocyclic ring ", " carbocylic radical " and " carbocyclic ring " refer to the saturated or unsaturated ring of non-aromatic, and wherein each atom on the ring is carbon.

Preferred carbocyclic ring comprises 3-10 atom, more preferably 5-7 atom.

Term used herein " carbocyclic ring alkyl " refers to the groups with the carbon ring group replacement.

Term " carbonic ester " is well known in the art, refers to group-OCO₂-R ⁹, R wherein⁹The representation hydrocarbyl group.

Term used herein " carboxyl " refers to by formula-CO₂The group of H representative.

Term used herein " ester " refers to group-C (O) OR⁹, R wherein⁹The representation hydrocarbyl group.

Term used herein " ether " refers to that hydrocarbyl group is connected to another hydrocarbyl group by oxygen. Therefore, the ether substituting group of hydrocarbyl group can be alkyl-O-. Ether can be symmetrical or asymmetric. The example of ether includes, but not limited to heterocycle-O-heterocycle and aryl-O-heterocycle. Ether comprises " alkoxyalkyl " group, can be by general formula alkyl-O-alkyl represent.

Term used herein " halogen " and " halogen " expression halogen comprise chlorine, fluorine, bromine and iodine.

Term used herein " assorted alkyl " and " heteroarylalkyl " refer to the groups with the heteroaryl groups replacement.

Term " heteroaryl " and " heteroaryl " comprise aromatic series single ring architecture replacement or unsubstituted, preferred 5-is to 7-unit ring, and preferred 5-is to 6-unit ring, and its circulus comprises at least one hetero atom, preferred one to four hetero atom, more preferably one to two hetero atom. Term " heteroaryl " and " heteroaryl " also comprise the polycyclic system with two or more rings, two or more carbon are that two adjacent ring are public, wherein at least one ring is heteroatomic, for example, another ring can be cycloalkyl, cycloalkenyl group, cycloalkynyl radical, aryl, heteroaryl, and/or heterocycle. Heteroaryl groups comprises, for example: pyrroles, furans, thiophene, imidazoles, oxazole, thiazole, pyrazoles, pyridine, pyrazine, pyridazine, and pyrimidine, and analog.

The atom of the arbitrary element beyond term used herein " hetero atom " expression de-carbon or the hydrogen. Preferred hetero atom is nitrogen, oxygen and sulphur.

Term " heterocyclic radical ", " heterocycle " and " heterocycle " refers to non-aromatic ring structure that replace or unsubstituted, preferred 3-is to 10-unit ring, preferred 3-is to 7-unit ring, its circulus comprises at least one hetero atom, preferred one to four hetero atom, more preferably one to two hetero atom. Term " heterocyclic radical " and " heterocycle " also comprise the polycyclic system with two or more rings, wherein two or more carbon are that two adjacent ring are public, wherein at least one ring is heterocycle, for example, another ring can be cycloalkyl, cycloalkenyl group, cycloalkynyl radical, aryl, heterocycle, and/or heterocyclic radical. Heterocyclic group comprises, for example, and piperidines, piperazine, pyrrolidines, morpholine, lactone, lactams, and analog.

Term used herein " Heterocyclylalkyl " refers to the groups with the heterocyclic group replacement.

Term used herein " alkyl " refers to the group by carbon atom Cheng Jian, described carbon atom do not have=O or=the S substituting group, usually have at least one hydrocarbon key and main carbon skeleton, but an optional hetero atom that comprises. Therefore; be considered to alkyl for the application's purpose as the group of methyl, ethoxyethyl group, 2-pyridine radicals and trifluoromethyl, but substituting group is connected with ethyoxyl by oxygen rather than connection carbon such as acetyl group (having=the O substituting group connecting carbon)) be not. Hydrocarbyl group includes, but are not limited to aryl, heteroaryl, carbocyclic ring, heterocycle, alkyl, thiazolinyl, alkynyl, and combination.

Term used herein " hydroxyalkyl " refers to the groups with the hydroxyl replacement.

Term " rudimentary " when with chemical part for example, acyl group, acyloxy, alkyl, thiazolinyl, alkynyl perhaps represents to comprise some groups during the alkoxyl coupling, described group has ten or non-hydrogen atom still less at substituting group, preferred six or still less. " low alkyl group " for example, refers to a kind of alkyl, and described alkyl comprises ten or carbon atom still less, preferred six or still less. In certain embodiments, the acyl group that herein defines, acyloxy; alkyl, thiazolinyl, alkynyl; perhaps alkoxy substituent is respectively lower acyl, low-grade acyloxy; low alkyl group, low-grade alkenyl, low-grade alkynyl; perhaps lower alkoxy is no matter they are jointly to exist separately or with other substituting groups, such as at hydroxyalkyl and aralkyl (in such cases; for example, when the carbon atom in the calculating alkyl substituent, do not calculate the atom in the aromatic yl group).

Term " many cyclic groups ", " many rings " and " many rings " refer to two or more rings (for example, cycloalkyl, cycloalkenyl group, cycloalkynyl radical, aryl, heteroaryl and/or heterocyclic radical), wherein two or more atoms are that two adjacent ring share, and for example, ring is " condensed ring ". Each ring in many rings can be replacement or unsubstituted. In certain embodiments, each ring of many rings comprises 3 to 10 atoms, preferred 5 to 7 in ring.

Term " replacement " refers to have the substituent part of replacing the hydrogen on one or more carbon on the skeleton. Very clear " replacement " or " using ... replace " comprises Implicit Conditions, be that this replacement is consistent with the chemical valence that atom and the substituting group of replacement allow, and replace and cause stable compound, for example, can spontaneously not transform for example by rearrangement, cyclisation, elimination etc. Term used herein " replacement " expection includes the replacement of all permissions of organic compounds. At relative broad range, the substituting group of permission includes the acyclic and ring-type of organic compounds, tributary and unbranched, carbocyclic ring with heterocycle, aromatic series and non-aromatic substituting group. To suitable organic compound, the replacement of permission can be one or more, can be identical or different. For purposes of the invention, hetero atom can have the organic compound of hydrogen substituting group and/or any permission described herein to replace such as nitrogen, and it satisfies heteroatomic chemical valence. Substituting group can comprise any substituting group described herein, for example: halogen, hydroxyl, carbonyl is (such as carboxyl; alkoxy carbonyl group, formyl, perhaps acyl group), thiocarbonyl is (such as thioesters; thioacetate, perhaps dithioformate), alkoxyl; phosphoryl, phosphate, phosphonate; phosphinates, amino, acylamino-; amidine, imines, cyano group; nitro, azido, sulfydryl; alkylthio group, sulfate, sulfonate; sulfonamides, sulphonyl ammonia, sulphonyl; heterocycle, aralkyl, perhaps aromatic series or heteroaromatic moiety. They self also are can be substituted to those skilled in the art will know that on the hydrocarbon chain substituted part, if suitable.

Unless be indicated as especially " unsubstituted ", the reference about chemical group herein is interpreted as the variant that comprises replacement. For example, about the reference of " aryl " group or part implicit comprise replacement and unsubstituted variant.

Term " sulfate " is well known in the art, refers to group-OSO₃H, the perhaps acceptable salt of pharmacy.

Term " sulfonamides " is well known in the art, refers to the group by following general formula representative

R wherein⁹And R¹⁰Represent independently hydrogen or alkyl.

Term " sulfoxide " is well known in the art, refers to group-S (O)-R⁹, R wherein⁹The representation hydrocarbyl group.

Term " sulfonate " is well known in the art, refers to group SO₃H, the perhaps acceptable salt of pharmacy.

Term " sulfone " is well known in the art, refers to group-S (O)₂-R ⁹, R wherein⁹The representation hydrocarbyl group.

Term used herein " alkylthio " refers to the groups with the mercapto replacement.

Term used herein " thioesters " refers to group-C (O) SR⁹Perhaps-SC (O) R⁹, R wherein⁹The representation hydrocarbyl group.

Term used herein " thioether " is equivalent to ether, and wherein oxygen is replaced with sulphur.

Term " urea " is well known in the art, can be by following general formula representative

R wherein ⁹And R ¹⁰Represent hydrogen or hydrocarbyl group independently.

Term " little organic molecule " is meant the non-polymer of molecular weight less than 2000amu.Usually, the molecular weight of this molecule is less than 1000amu, such as less than 500amu.

The method of identified gene type alternative cpd target

In certain embodiments, the present invention relates to utilize the genotype alternative cpd, (for example be also referred to as " part " herein, erastin), identify that the target that participates in change diseased cells phenotype (also is called as " cellular component " (for example, albumen herein, nucleic acid, perhaps lipid).

In one embodiment, the invention provides a kind of method that participates in tumorigenic cellular component of identifying, wherein tumorigenic cell such as through engineering approaches people tumorigenic cell, tissue, organ, organism or its split product or extract, contacts with described antineoplastic compound; After contact, differentiated with the cellular component of erastin effect (direct or indirect), participate in tumorigenic cellular component thereby can identify.In another embodiment, the invention provides a kind of method that participates in tumorigenic cellular component of identifying.In this method, (a) tumorigenic cell such as through engineering approaches people tumorigenic cell, tissue, organ, organism or its split product or extract, contacts with the erastin inhibitor and contacts with erastin; (b) differentiated that with the cellular component of erastin inhibitor effect (direct or indirect) these cellular components participate in tumours and take place.Cell can successively or contact erastin and erastin inhibitor simultaneously.Can utilize known method evaluation and erastin or the interactional cellular component of any reagent of the present invention.

As describe herein, the described compound of these methods (perhaps part) can prepare with any chemical process.In addition, described compound can be in the body or the naturally occurring biomolecules of external synthetic.Part is optional derived from another compound.A benefit of this change is, derivative compound can be used to promote part target mixture to collect or part is collected, for example, and behind isolating ligands and target.The limiting examples of deriveding group comprises vitamin H, fluorescein, digoxin, green fluorescent protein, isotropic substance, poly Histidine, magnetic bead, glutathione s-transferase, photoactivation linking agent or its combination arbitrarily.Deriveding group can also be used in conjunction with target (for example, erastin is conjugated protein) to help their detection.

According to the present invention, target (cellular component) can be in the body or the naturally occurring biomolecules of external synthetic.Target can comprise amino acid, nucleic acid, sugar, lipid, natural product or its arbitrary combination.A benefit of the present invention is the priori that does not need target character or function.

The interaction of part and target can be covalency or non-covalent.Optional, the part in part-target pairing can show avidity to other targets, perhaps also can not show avidity.Target in part-target pairing can show avidity to other parts, perhaps also can not show avidity.

For example, the combination between part and target can use external biochemical process to identify at protein level, comprises light-crosslinked, the part combination of labelled with radioisotope and affinity chromatography (people such as Jakoby WB, 1974, Methods in Enzymology 46:1).In addition, small molecules can be fixed on suitable solid support or affinity matrix for example on the agarose matrix, is used to screen the extract of different cell types and organism.Similarly, small molecules can exposing cell, tissue, and organ, organism or its split product or extract add solid support then to catch small molecules and bonded target protein.

Cloning by expression can be used for detecting the target (people such as King RW, 1997, Science 277:973) in the proteic little storehouse.Peptide (people such as Kieffer, 1992, PNAS 89:12048), nucleoside derivates (people such as Haushalter KA, 1999, Curr.Biol.9:174) and medicine-bovine serum albumin (binding substances of medicine-BSA) (people such as Tanaka, 1999, Mol.Pharmacol.55:356) be used in the cloning by expression.

Another is a phage display with the useful technology that the DNA of part and coding target is closely connected.Phage display is mainly used in the monoclonal antibody field, has created peptide or albumen library at virus surface, and is used for screening active (Smith GP, 1985, Science 228:1315).Wash in a pan the sieve phage with obtain to be connected to target on the solid phase (people such as Parmley SF, 1988, Gene73:305).An advantage of phage display is that cDNA is arranged in phage, does not therefore need the separating clone step.

A kind of limiting examples comprises the association reaction condition, and wherein part comprises marker biological example element, fluorescein, digoxin, green fluorescent protein, radio isotope, histidine mark thing, magnetic bead, enzyme or its combination.In one embodiment of the invention, target can screen in the test based on mechanism, such as the test that detects in conjunction with the part of target.This can comprise with the solid phase or the liquid phase of part or albumen or detected indicator and combines situation.Perhaps, the also uncertain proteic gene of encoding function can with reporting system (for example, beta-galactosidase enzymes, luciferase or green fluorescent protein) transfectional cell together, and screening library is preferably by high flux screening or with the separate member in library.Can use combination test, for example, detect biochemical test the enzymic activity effect based on other mechanism, test based on cell, target and reporting system (for example, luciferase or beta-galactosidase enzymes) transfered cell wherein, and detect free energy change in conjunction with test.Target is fixed on hole, pearl or the chip, perhaps catches by immobilized antibody, and perhaps capillary electrophoresis analysis carries out the combination test.The bonded part can use colorimetric or fluorescence or surface plasma resonance to detect usually.

In certain embodiments, the function by regulating the target (cellular component) that the present invention identifies (for example, active or express), the present invention has also expected the method for treatment or preventing disease (for example cancer).In order to illustrate, if target is identified the promotion tumor growth, therapeutical agent can be used for changing or reducing the function (active or expression) of target.Perhaps, if target is identified the inhibition tumor growth, therapeutical agent can be used for intensifier target target function (active or expression).Therapeutical agent includes, but not limited to antibody, nucleic acid (for example, antisense oligonucleotide or be used for the little inhibition of RNA interferential RNA), albumen, small molecules (for example, compound of the present invention) or simulating peptide.

The Erastin target

In certain embodiments, the invention provides the target of erastin and erastin analogue, be commonly referred to as the erastin target herein.The Erastin target can be directly or indirectly in conjunction with erastin or aforesaid erastin analogue.Alternatively, the erastin target can be regulated for example anti-tumor activity of erastin or erastin analogue of compound in cell.Exemplary erastin target includes, but are not limited to: VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a and Sec22b.

The PFP family that voltage dependence anion channel (VDACs) is encoded by different genes gives expression at least three kinds of protein products (VDAC1, VDAC2, and VDAC3) in mammalian tissues.The known major function of VDACs is the almost free permeation that allows outer plastosome film (ODF).Referring to, for example, people such as Shoshan-Barmatz, 2003, CellBiochem Biophys 39:279-92.In ODF and in plastosome, VDAC2 and VDAC3 may have selectable structure organization, different function (people such as Hinsch, 2004, JBiol Chem.279:15281-8).Different types of representative VDAC sequence has been stored in GenBank.For example, people VDAC1 amino acid and nucleotide sequence can find in GenBank accession number NP_003365 and NM_003374; People VDAC2 amino acid and nucleotide sequence can find in GenBank accession number NP_003366 and NM_003375; People VDAC3 amino acid and nucleotide sequence can find in GenBank accession number NP_005653 and NM_005662.

Prohibitin is the gene of the evolution conservative of omnipresence expression.It is believed that it is the negative conditioning agent of cell proliferation, can be used as tumor inhibitor (for example, people such as Fusaro, 2003, J.Biol.Chem.278:47853-47861; People such as Fusaro, 2002, Oncogene 21:4539-4548).Different types of representative prohibitin sequence has been stored in GenBank.For example, people prohibitin amino acid and nucleotide sequence can find in GenBank accession number NP_002625 and NM_002634.

Ribophorin (for example, I and II) seems to participate in rrna bonded albumen.They are glycoprotein a large amount of, high conservative, and single-minded target is (for example, people such as Fu, 2000, J.Biol.Chem.275:3984-3990 on the film of rough surfaced endoplasmic reticulum; People such as Crimaudo, 1987, EMBO is J.6:75-82).Different types of representative ribophorin sequence has been stored in GenBank.For example, human rebosomal can find in GenBank accession number NP_002941 and NM_002950 in conjunction with glycoprotein I amino acid and nucleotide sequence; Human rebosomal can find in GenBank accession number NP_002942 and NM_002951 in conjunction with glycoprotein I I amino acid and nucleotide sequence.

Sec61-alpha albumen it is believed that and insert in the endoplasmic reticulum at secretion peptide and membrane polypeptides and to work (referring to, for example, people such as Higy, 2004, Biochemistry 43:12716-22).The typical Sec61 alpha of different sorts sequence is stored among the GenBank.For example, people Sec61-alpha-I amino acid and nucleotide sequence can find in GenBank accession number NP_037468 and NM_013336; And the amino acid of people Sec61-alpha-II and nucleotide sequence can find in GenBank accession number NP_060614 and NM_018144.

Sec22-beta albumen shows transporting and form work in the mixture (for example, people such as Parlati, 2000, Nature407:194-198 with SNARE at endoplasmic reticulum-Golgi apparatus protein; People such as Mao, 1998, Proc.Natl.Acad.Sci.U.S.A.95:8175-8180).The typical Sec61-beta sequence of different sorts is stored among the GenBank.For example, the amino acid of people Sec61-beta and nucleotide sequence can find in GenBank accession number NP_004883 and NM_004892.

In certain embodiments, the present invention relates to by utilizing the erastin target to identify the method for candidate's anti-tumor therapeutic agent.In this method, combine with the erastin target or the test agent that increases or reduce erastin target function (for example, active or express or interact) can be defined as candidate's anti-tumor therapeutic agent.Candidate's anti-tumor therapeutic agent can also be in vivo or its anti-tumor activity of vitro detection.The method of evaluation candidate anti-tumor therapeutic agent can be finished by aforesaid sieve method similarly.

Carrying method

Use in certain embodiments of the present invention and carry the albumen (for example, little t antigen, VDAC, PP2A inhibitor or the like) or this proteic DNA that encodes to arrive the method for target cell, this can realize by arbitrary standards molecular biology and molecular medicine technology.Below illustrational embodiment just can be used for several in the technology of this purpose.

In one aspect of the invention, relevant albumen or produce the expression construct of antisense molecule, the effective carrier administration of available any biology, for example in vivo can be effectively with any preparation or the composition of recombination transfectional cell.Described method comprises inserts virus vector with related gene, and described virus vector comprises recombinant retrovirus, adenovirus, adeno-associated virus and hsv-1, perhaps recombinant bacteria or eucaryon plasmid.Virus vector can be used for the direct transfection cell; Plasmid DNA can be by means of cationic-liposome for example (for example, lipofectin) or deutero-(for example, antibodies), carrier is carried in poly-lysine binding substances, gramacidin S, artificial viral tunicle or other this classes born of the same parents, and gene construct directly injects or carries out CaPO in vivo ₄Precipitation.Should understand the transduction of suitable target cell and represent the first step crucial in the gene therapy, the selection of specific gene delivery system depends on some factors, and is such as pre-determined target target phenotype and route of administration, for example partial or whole body.

Introduce the preferable methods of encoding to cell in the body and be to use the virus vector that comprises nucleic acid about proteic nucleic acid, for example, the cDNA of encoding gene product.Advantage with the viral vector infection cell is that most of target cell can capture nucleic acid.In addition, the molecule of coding in the virus vector, for example, by the cDNA that comprises in the virus vector, can be in the cell of admitting virus vector nucleic acid effective expression.

Retroviral vector and gland relevant viral vector are considered to the selection of recombination delivery system usually, are used for foreign gene and are delivered in the body, especially in human body.These carriers can effectively be conveyed into cell with gene, are incorporated in host's the chromosomal DNA nucleic acid stability of transmission.Human Immunodeficiency Virus (HIV) and feline immunodeficiency virus (FIV) have been represented the retrovirus family subgroup that is called " slow virus ", and they are gained the name because be in latent period for a long time after integration.The carrier system that derives from these two kinds of viruses has been effective to preclinical models, has shown bright prospects in treatment is used.Different with most of retrovirus, HIV and FIV (with the carrier that the derives from them) Unseparated Cell of can transduceing (people such as Humeau, Mol Ther.2004,9 (6): 902-13; People such as Curran, Mol Ther.2000,1 (1): 31-8).According to this character of target cell type may be favourable.In addition, FIV can distinguish (people such as Curran, MoI Ther.2000,1 (1): 31-8) .2000,1 (1): 31-8) with other retrovirus by the transgenosis carrying capacity of its increase.The main prerequisite that retrovirus uses is to guarantee that it is safe in utilization, especially considers the possibility that wild-type virus is propagated in cell mass.The exploitation that only produces the retroviral specialization clone of replication defect type (being called " packing cell ") has promoted the application of retrovirus in gene therapy, the defective type retrovirus has been carried out further investigation (to be summarized referring to Miller with the transgenosis that is used for the gene therapy purpose, A.D.Blood 76:271,1990).Therefore, can make up recombinant retrovirus, wherein Partial Inverse is transcribed the encoding viral sequence (gag pol, env) the be encoded nucleic acid of relevant polypeptide is replaced, and causes replication defective retroviral.The packaged then virion that becomes of replication defect type retrovirus, described virion adopt standard technique by the helper virus target cell infection.

Produce in recombinant retrovirus and external or the body experimental procedure with this virus infected cell referring to Current Protocols in Molecular Biology, Ausubel, people such as F.M., (eds.), John Wiley﹠amp; Sons, Inc.Greene Publishing Associates, (2001), 9.9-9.14 chapter and other standard test handbooks.Suitable retroviral example comprises pLJ, pZIP pWE and pEM, and these all well known to a person skilled in the art.The example that is used to prepare the suitable package virus stock of having a liking for parent's property and amphotropic retrovirus system comprises ψ Crip, ψ Cre, ψ 2 and ψ Am.Retrovirus has been used in external and/or body range gene imported many different cell types, comprise that neurocyte, epithelial cell, endotheliocyte, lymphocyte, sarcoplast, liver cell, medullary cell are (referring to people such as for example Eglitis, Science230:1395-1398,1985; Danos and Mulligan, PNAS USA 85:6460-6464,1988; People such as Wilson, PNAS USA 85:3014-3018,1988; People such as Armentano, PNASUSA 87:6141-6145,1990; People such as Huber, PNAS USA 88:8039-8043,1991; People such as Ferry, PNAS USA 88:8377-8381,1991; People such as Chowdhury, Science254:1802-1805,1991; People such as van Beusechem, PNAS USA 89:7640-7644,1992; People such as Kay, Human Gene Therapy 3:641-647,1992; People such as Dai, PNASUSA 89:10892-10895,1992; People such as Hwu, J.Immunol.150:4104-4115,1993; U.S.Patent No.4,868,116; U.S. patent 4,980, and 286; PCT applies for WO89/07136; PCT applies for WO 89/02468; PCT applies for WO 89/05345; With PCT application WO 92/07573).

In addition, proved, can limit reversed record the infection scope of virus, thereby limited based on retroviral carrier (referring to the open text WO93/25234 of PCT, WO94/06920, and WO94/11524) by the viral packaging protein on the modification virus particle surface.For example, change the step that retroviral vector infects scope and comprise: the antibody coupling that the pair cell surface antigen is special is to viral env albumen (people such as Roux, PNAS USA 86:9079-9083,1989; People such as Julan, J.Gen Virol 73:3251-3255,1992; With people such as Goud, Virology 163:251-254,1983); Perhaps the cell surface ligand coupling is arrived viral env albumen (Neda et al, J.Biol.chem.266:14143-14146,1991).Coupling can be the form (for example, being coupled to lactose so that env albumen is converted into asialoglycoprotein) with albumen or other kind chemically crosslinkeds, and the coupling by producing fusion rotein (for example, single-chain antibody/env fusion rotein).These technology not only help restriction or in contrast, guidance can also be used for converting facultative carrier to having a liking for parent's property carrier to the infection of some types of organization.

To the useful another kind of virogene delivery system of the present invention is adenovirus deutero-carrier.The genome of adenovirus can be regulated and control, its interested gene product of encoding like this, but it does not have the ability of duplicating (referring to people such as for example Berkner, BioTechniques 6:616,1988 in normal virus cracking performance life cycle; People such as Rosenfeld, Science 252:431-434,1991; With people such as Rosenfeld, Cell 68:143-155,1992).Suitable adenovirus carrier derives from adenopathy strain Ad type 5 d1324 or well known to a person skilled in the art other adenopathy strains (for example Ad2, Ad3, Ad7 or the like).Recombinant adenovirus has superiority in some aspects, and they can not infect Unseparated Cell, can be used for infecting a large amount of cell types, comprise airway epithelia (people such as Rosenfeld, (1992) quote preamble), endotheliocyte (people such as Lemarchand, PNASUSA 89:6482-6486,1992), liver cell (Herz and Gerard, PNAS USA90:2812-2816,1993) and myocyte (people such as Quantin, PNAS USA 89:2581-2584,1992).In addition, virion is relatively stable, is easy to purifying and concentrated, and as mentioned above, can modify the scope that infects with influence.In addition, adenovirus DNA (with its foreign DNA that the comprises) unconformability that imports is to the genome of host cell, still remain episome, thereby potential problems have been avoided, promptly as being integrated into the result of the insertion mutagenesis that the position of host genome (for example, retrovirus DNA) takes place importing DNA.In addition, other gene delivery carriers relatively, the ability of adenoviral gene group delivery foreign DNA strong (to 8kb) (people such as Berkner, preamble; Haj-Ahmand and Graham J.Virol.57:267,1986).The great majority of current use are replication-defective adenoviral vectors, therefore the present invention also uses this carrier, cut all or part of viral E1 of described carrier and E3 gene, but keep similar 80% adenovirus genetic material (referring to people such as for example Jones, Cell 16:683,1979; People such as Berkner, preamble; With people such as Graham, at Methods in Molecular Biology, EJ.Murray, Ed. (Humana, Clifton, NJ, 1991) vol.7.pp.109-127).Insert Expression of Related Genes and for example can be subjected to, E1A promotor, major late promoter (MLP) and bonded leader sequence, viral E3 promotor or external source are added the control of promoter sequence.

The virus carrier system that another kind is used for the related gene conveying is adeno-associated virus (AAV).Adeno-associated virus is a kind of defective virus of natural generation, need another kind of virus, for example adenovirus or simplexvirus (are summarized referring to people such as Muzyczka so that effectively duplicate with the life cycle of fecund as helper virus, Curr.Topics in Micro.Immunol. (1992) 158:97-129,1992).It also is that a few can be incorporated into its DNA in the Unseparated Cell, and the virus that demonstrates high-frequency stable integration is (referring to people such as for example Flotte, Am.J.Respir.Cell.Mol.Biol.7:349-356,1992; People such as Samulski, J.Virol.63:3822-3828,1989; With people such as McLaughlin, J.Virol.62:1963-1973,1989).The carrier that only includes 300 base pairs of AAV can be packed and integrate.The space that is used for foreign DNA is limited to about 4.5kb.As people such as Tratschin, Mol.Cell.biol.5:3251-3260, the AAV carrier described in 1985 can be used for the transfered cell with DNA.Use the AAV carrier multiple nucleic acid to be imported different cell types (referring to people such as for example Hermonat, PNASUSA 81:6466-6470,1984; People such as Tratschin, Mol.Cell.biol.4:2072-2081,1985; People such as Wondisford, Mol.Endocrinol.2:32-39,1988; Tratschin et al, J.Virol.51:611-619,1984; With people such as Flotte, J.Biol.Chem.268:3781-3790,1993).

Other virus carrier systems that are used for gene therapy derive from simplexvirus, vaccinia virus and several RNA viruses.Especially, herpesvirus vector can provide a kind of unique strategies (people such as Pepose, Invest Ophthalmol Vis Sci 35:2662-2666,1994) that continues to keep relevant recombination in central nervous system is unified the cell of ocular tissue.

Except that such as top those illustrational viral delivering methods, non-viral method also can be used for causing the expression of relevant albumen in animal tissues.The most of non-viral method of transgenosis relies on the high molecular conventional mechanism of transportation in mammalian cell picked-up and the born of the same parents.In preferred embodiments, non-viral gene delivery system of the present invention relies on the endocytic pathway picked-up related gene of target cell.The exemplary gene delivery system of this type comprises liposome deutero-system, poly-lysine binding substances and artificial viral tunicle.

In a typical embodiment, the gene of relevant polypeptide of encoding can be caught by the liposome (for example lipofectins) that the surface has a positive charge, (choosing wantonly) described liposome is with the antibody labeling (people such as Mizuno of anti-target tissue cell-surface antigens, No Shinkei Geka20:547-551,1992; PCT announces WO91/06309; Japanese patent application 1047381; With European patent publication EP-A-43075).For example, can use the anti-glioma-liposome of antigen monoclonal antibody mark to carry out the liposome transfection (people such as Mizuno, Neurol.Med.Chir.32:873-876,1992) of neuroglial cytoma.

In another illustrative embodiment, gene delivery system comprises antibody or cell surface part, they and gene wedding agent for example poly-lysine crosslinked (referring to, for example, PCT announces WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180).For example, the solvable polynucleotide carrier that the related gene construct can be used for comprising antibody by use is the transfection specific cells in vivo, described antibodies polycation, for example poly-lysine (referring to United States Patent (USP) 5,166,320).The effective conveying that should understand by peptide-mediated endocytosis related nucleic acid construct can utilize reagent to improve, and described reagent enhancing gene is escaped from interior body structure.For example, the fusogenic peptide of whole adenovirus or influenza HA gene product can be as the part of delivery system with effective destruction of inducing the endosome that comprises DNA (people such as Mulligan, Science 260-926,1993; People such as Wagner, PNAS USA89:7934,1992; With people such as Christiano, PNAS USA 90:2122,1993).

Under clinical condition, gene delivery system can import the patient by one of arbitrarily big metering method well known in the art.For example, the pharmaceutical preparation of gene delivery system can general importing, for example, by intravenous injection, and the specialized transduction of construct mainly is derived from the specificity of transfection in the target cell, this is to be expressed by cell type or types of organization that the transcriptional regulatory sequences that gene delivery carrier, controlling gene are expressed causes, and perhaps its combination causes.In other embodiments, the initial delivery of recombination is very limited, and this is because limited to very much when importing animal.For example, gene delivery carrier can pass through conduit (referring to United States Patent (USP) 5,328,470) or Stereo target to injection (for example people such as Chen, PNAS USA 91:3054-3057,1994) importing.

In addition, about albumen can provide with the form of second kind of peptide with fusogenic peptide, described second kind of peptide promotes " transcytosis ", for example by target cell picked-up peptide.Illustrate, relevant albumen can be used as the part of fusion polypeptide, and described fusion polypeptide also comprises a whole or fragment in HIV albumen Tat N end structure territory, for example, and the residue 1-72 of Tat or its small segment more that can promote transcytosis.In other embodiments, about polypeptide can with the proteic all or part of formation fusion polypeptide of feeler foot III.Therefore synthetic peptide has been used for translocator, peptide and small molecules effectively and has passed the microbial film that comprises hemato encephalic barrier, can be used for the application (people such as Rothbard, Nat Med.2000,6 (11): 1253-7; People such as Rothbard, J MedChem.2002,45 (17): 3612-8).Although the synthetic proteins that provides transduction example series is characterised in that highdensity arginine residues, can also replace with other function classes molecule that seemingly structure is different or sequence.

For further illustrating, relevant polypeptide (perhaps simulating peptide) can the chimeric peptide form provide, comprising heterologous peptides sequence (" internalization peptide " or " internalization structural domain "), described peptide sequence drives sails the displacement that the outer form of relevant peptide sequence born of the same parents is passed cytolemma, to locate in the born of the same parents that promote relevant polypeptide.In this, the relevant polypeptide of therapeutic is activated in born of the same parents.Internalization peptide self can pass cytolemma with higher rate by for example transcytosis.Optional, but the internalization peptide is with cutting mode and relevant polypeptide fusion for example formation fusion rotein.With respect to independent activated polypeptides, the chimeric peptide that obtains is transported into cell with higher rate, promotes it to import the method for its institute's function cells thereby provide a kind of, for example, promotes the topical application of relevant polypeptide.Except that albumen and simulating peptide, a kind of pharmaceutical agent can be transported to a kind of compound coupling (for example, receptor-mediated compound is such as vitamin B12) of material with promotion.

In one embodiment, the internalization peptide originates from the fruit bat rqikiwfqnrrmkwkk, perhaps its analogue.Verified can the displacement of homeodomain of 60 amino acid longs of similar-albumen feeler foot passed microbial film, can promote the displacement with its link coupled heterologous polypeptide.Referring to for example, people such as Derossi (1994) J Biol Chem 269:10444-10450; With people (1992) J CellSci 102:717-722 such as Perez.Verified this albumen is little to be enough to drive internalization to 16 amino acid whose fragments.Referring to people such as Derossi (1996) J Biol Chem 271:18188-18193.

The present invention also provides polypeptide (little t antigen or VDAC) or simulating peptide sequence described herein, and at least a portion of rqikiwfqnrrmkwkk (perhaps its homologue), relevant relatively polypeptide or simulating peptide are enough to promote significantly that the film of striding of chimeric protein transports on statistical significance.This peptide species or its simulating peptide can be used to help effectively and the methods involving of specific killing cancer cells.

Another example of internalization peptide is HIV trans-activating factor (TAT) albumen.This albumen is divided into four structural domains (people (1989) Nucl.Acids Res.17:3551-3561 such as Kuppuswamy).The TAT albumen of purifying can be by the cellular uptake in tissue culture (Frankel and Pabo, (1989) Cell 55:1189-1193), for example corresponding to the segmental peptide of TAT residue 37-62 at external absorbed fast by cell (Green and Loewenstein, (1989) Cell 55:1179-1188).Highly the zone of alkalescence is regulated internalization and internalization and partly is targeted to nuclear people such as (, (1989) J.Virol.63:1-8) Ruben.

Another kind of exemplary transcellular polypeptide can be produced with enough parts of comprising mastoparan people such as (, (1990) J.Biol.Chem.265:14176) T.Higashijima strides the film transportation with what promote chimeric protein.

But without wishing to be bound by any particular theory, should be noted that by with the polypeptide coupling or be attached to transportable peptide, described transportable peptide can pass film by receptor-mediated transcytosis, the wetting ability polypeptide also can the transportation of physiology ground pass barrier film.Can use, for example, histone, Regular Insulin, Transferrins,iron complexes, alkaline albumin, prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) or other somatomedins all or part of produces such suitable internalization peptide.For example, have been found that a kind of insulin fragment, show the avidity to insulin receptor on the capillary cell, its lowering blood glucose efficiency ratio Regular Insulin is low, can transmembrane transport by receptor-mediated transcytosis, therefore can be used as about striding the internalization peptide of cell peptide and simulating peptide.Preferred somatomedin-deutero-internalization peptide comprises EGF (Urogastron)-derived peptide for example CMHIESLDSYTC and CMYIEALDKYAC; TGF-beta (transforming growth factor beta) derived peptide; Peptide derived from PDGF (Thr6 PDGF BB) or PDGF-2; Peptide derived from IGF-I (rhIGF-1) or IGF-II; And FGF (fibroblast growth factor)-derived peptide.

Another kind of displacement/internalization peptide shows the film combination that pH-relies on.For the internalization peptide that has helical conformation in the acid pH supposition, the internalization peptide obtains amphipathic character, and for example, it has hydrophobicity and wetting ability interface.More specifically, in the pH scope of about 5.0-5.5, the internalization peptide forms alpha-spiral, the amphipathic structure that promotes group to insert the target film.In the low pH environment in the intracellular for example, can find to induce the acid pH of alpha-spiral.By the picked-up of endocytosis mechanism, this internalization peptide can be used for promoting relevant polypeptide and simulating peptide from endosome compartment transporte to cells matter.

The film that preferred pH relies on comprises a high proportion of spiralization residue in conjunction with the internalization peptide, such as L-glutamic acid, and methionine(Met), L-Ala and leucine.In addition, preferred internalization peptide sequence comprises the ionization residue of pKa in the pH5-7 scope, like this has abundant uncharged film binding domains in the peptide when pH5 so that insert target cell membrane.

The film that particularly preferred in this respect pH relies on is aa1-aa2-aa3-EAALA (EALA) 4-EALEALAA-acid amides in conjunction with the internalization peptide, and this represents the modification (Biochemistry 26:2964,1987) of people's such as Subbarao peptide sequence.In these peptide sequences, the residue that first amino-acid residue (aa1) is preferably unique such as halfcystine or Methionin, promotes the Chemical bond of internalization peptide and target protein binding substances.Can select amino-acid residue 2-3 to regulate the avidity of internalization peptide to different films.For example, if

residue

2 and 3 all is Methionin or arginine, the internalization peptide will have in conjunction with having the film of negative surface charge or the ability of lipid.If residue 2-3 is a neutral amino acids, the internalization peptide will insert neutral film.

Other preferred internalization peptides comprise the peptide of apo-lipoprotein A-I and B; The peptide toxin, such as melittin, bombolittin, delta hemolysin and pardaxin (paradaxin); Antibiotic peptide is such as alamethicin; Peptide hormone, such as thyrocalcitonin, corticotropin releasing factor(CRF), beta endorphin, hyperglycemic-glycogenolytic factor, Rat parathyroid hormone 1-34, pancreatic polypeptide; With peptide corresponding to a lot of secretory protein signal sequences.In addition, exemplary internalization peptide can be modified by adding substituting group, and described substituting group strengthens the alpha-spinning behaviour of internalization peptide under acid pH.

Another kind ofly be suitable for internalization peptide of the present invention and be included in the hydrophobic structure territory that " hides " under the physiological pH, but under the low pH environment of target cell endosome, expose.Open and when exposing when pH induces the hydrophobic structure territory, this part is in conjunction with lipid bilayer and influence the covalently bound polypeptide into tenuigenin that is shifted.This internalization peptide is at for example Rhodopseudomonas exotoxin A, and clathrin perhaps can make up model after the order-checking evaluation in the diphtheria toxin.

Hole formation albumen or peptide also can be used as internalization peptide herein.The hole form albumen or peptide can available from or derived from, for example, C9 complement proteins, molten born of the same parents T cellular elements or NK cellular elements.These parts can form ring texture on film, enter cell interior thereby allow the bonded polypeptide to transport film.

Only the film of internalization peptide inserts just is enough to make relevant polypeptide or simulating peptide displacement to pass cytolemma.But displacement can be by improving (that is, " attached peptide ") on the substrate of internalization peptide attached to intracellular enzyme.Preferred attached peptide is attached to the part of internalization peptide, and described internalization peptide is projected into the kytoplasm surface from cytolemma.Attached peptide can advantageously be attached to the end of displacement/internalization part or anchor peptide.Accessory constituent of the present invention can comprise one or more amino-acid residues.In one embodiment, accessory constituent can provide the substrate (for example, attached peptide can comprise tyrosine residues) of cells phosphorylation effect.

Exemplary in this respect accessory constituent is the peptide substrates of N-mnyristoyl transferring enzyme, and for example (people such as Eubanks is at Peptides.Chemistry and Biology for GNAAAARR, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp.566-69).In these constructs, the internalization peptide will be incorporated into the C end of attached peptide, because N end glycine is to the unusual crux of the activity of accessory constituent.This hybridizes peptide, after C end is attached to E2 peptide or simulating peptide, by the N-myristylation, further anchor to target cell membrane, for example, it is used to increase the partial concn of peptide on cytolemma.

For further illustrating the use of attached peptide, but at first being covalently attached to the C of internalization peptide, holds the attached peptide of phosphorylation, be integrated into fusion rotein with relevant polypeptide or simulating peptide then.The peptide assembly of fusion rotein inserts the target cell plasma membrane, so the advance by leaps and bounds tenuigenin of target cell of film is passed in the displacement of attached peptide.At the kytoplasm face of plasma membrane, attached peptide is by the cell kinase phosphorylation in neutral pH.In case phosphorylation, attached peptide is irreversible to be anchored to fusion rotein on the film.Be targeted to cell surface membrane and can promote the polypeptide into tenuigenin that is shifted.

Suitable attached peptide comprises the peptide as kinase substrate, has the peptide of single positive charge and comprises the peptide of tunicle in conjunction with the glycosylated sequence of glycosyltransferase.Tunicle can comprise sequence x-NLT-x in conjunction with the glycosylated attached peptide of glycosyltransferase, and wherein " x " can be another peptide for example, a seed amino acid, coupler or hydrophobic molecule.When this hydrophobicity tripeptides and microsome vesicles were hatched, it passed the cyst film, at the chamber face by glycosylation, because its wetting ability is trapped in the vesicles (C.Hirschberg et ah, (1987) Ann.RRev.BBiochem.556:63-87).Therefore the attached peptide that comprises sequence x-NLT-x will promote that target cell keeps corresponding polypeptide.

In another embodiment of this respect of the present invention, attached peptide can be used for promoting the interaction of polypeptide or simulating peptide and target cell.Exemplary in this respect attached peptide comprises the peptide derived from the cell adhesion protein that comprises sequence " RGD ", perhaps derived from the peptide of the ln that comprises sequence C DPGYIGSRC.Extracellular matrix glycoprotein, such as fibronectin and ln, by receptor-mediated step in conjunction with cell surface.Tripeptide sequence, RGD, it is essential to have determined in conjunction with cell surface receptor.This sequence is present in fibronectin, vitronectin, complement C3bi, the von-Willebrand factor, EGF acceptor, transforming growth factor beta, collagen-type I, colibacillary lambda acceptor, Fibrinogen and Sindbis capsid protein (E.Ruoslahti, Ann.Rev.Biochem.557:375-413,1988).The cell surface receptor of identification RGD sequence has assembled the superfamily of the associated protein that is called " integrating plain "." RGD peptide " integrated the plain cell surface that will promote polypeptide that combines with cell surface and is detained, and finally is the displacement of polypeptide.

As mentioned above, by chemically crosslinked or fusion rotein form, internalization peptide and attached peptide can be independent separately is added to polypeptide or simulating peptide.In the example of fusion rotein, structureless peptide linker can be included between each peptide moiety.

Usually, the internalization peptide is enough to be used in the direct output of polypeptide.But,, may need to comprise secretory signal sequence so that directly export fusion rotein from its host cell when providing attached peptide for example during the RGD sequence.In preferred embodiments, secretory signal sequence is positioned at the N-terminal far-end, and (choosing wantonly) flank is the proteolyzing site of secretion signal and other parts of fusion rotein.

In an exemplary embodiment, polypeptide or simulating peptide be designed to comprise integrate plain in conjunction with RGD peptide/SV40 nuclear localization signal (referring to people such as for example Hart S L, 1994; J.Biol.Chem.269:12468-12474), nucleotide sequence coded by in the Ndel-EcoR1 fragment for example: catatggutgactgccgtggcgatatgttcggttgcggtgctcctccaaaaaagaa gagaaaggtagctggattc, its coding RGD/SV40 nucleotide sequence: MGGCRGDMFGCGAPPKKKRKVAGF.In another embodiment, albumen can with HIV-1 tat (1-72) polypeptide through engineering approaches, for example, provide as the Ndel-EcoR1 fragment: catatggagccagtagatcctagactagagccctggaagcatccaggaagtcagcc taaaactgcttgtaccaattgctattgtaaaaagtgttgctttcattgccaagtgt ttcataacaaaagcccttggcatctcctatggcaggaagaagcgagacagcgacga agacctcctcaaggcagtcagactcatcaagtttctctaagtaagcaaggattc, its coding HIV-1 tat (1-72) peptide sequence: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRR RPPQGSQTHQVSLS KQ.In another embodiment, fusion rotein comprises that (Elliott G.O ' Hare P (1997) Cell 88:223-233), is provided by the Ndel-EcoR1 fragment HSV-I VP22 polypeptide.In another embodiment, fusion rotein comprises the proteic C end structure of the VP22 territory from for example nucleotide sequence (Ndel-EcoR1 fragment).

In some example, need comprise the part of nuclear localization signal as relevant polypeptide.When generation comprises the fusion polypeptide of polypeptide, may need to comprise structureless joint so that guarantee the correct folding of different peptide domains.Multiple synthesizing with natural joint known in the art can be used for the present invention, comprises (Gly ₃Ser) ₄Joint.

Methods of treatment

In certain embodiments, the invention provides in individuality the method for treatment or preventing cancer.Term " cancer ", " tumour " and " tumorigenesis " can exchange use herein.The characteristics of the cancer of Shi Yonging (tumour or tumour form) are one or more following character herein: the cell growth is not regulated by normal biochemical and physiological effect in environment; Anaplasia (cytodifferentiation that for example, lacks normal regulating); With in some cases, shift.Cancerous disease comprises, for example, and anus cancer, bladder cancer, mammary cancer, cervical cancer, lymphocytic leukemia, chronic myelogenous leukemia, carcinoma of endometrium, hairy cell, head and neck cancer, lung (minicell) cancer, multiple myeloma, non-hodgkin ' s lymphoma, follicular lymphoma, ovarian cancer, cerebral tumor, colorectal carcinoma, hepatocellular carcinoma, Kaposi sarcoma, lung (non-small cell carcinoma), melanoma, carcinoma of the pancreas, prostate cancer, renal cell carcinoma, and soft tissue sarcoma.Other cancer illness can referring to, for example, people such as Isselbacher (1994) Harrison ' sPrinciples of Internal Medicine 1814-1877 is hereby incorporated by.

Usually, the cancer of aforesaid available method treatment described herein shows that uncontrolled VDAC expresses.In one embodiment, above-mentioned cancer comprises the sudden change of Ras signal pathway, causes the active rising of Ras signal.For example, sudden change can be the composition activation sudden change on the Ras gene, such as Ras V12.In other embodiments, cancer can comprise the sudden change that PP2A goes up loss of function, and/or the activation of MEK1 and/or ERK1 sudden change.In certain other embodiments, the characteristics of cancer are the little t cancer protein of cell expressing SV40, and are perhaps similar to the cell phenotype of expressing ST and/or carcinogenic HRAS.In certain preferred aspects, the cell expressing Rb of wild-type level (for example, about at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, perhaps 150% or the like) basically.

In one embodiment, the present invention relates to a kind of in individuality the treatment or the method for preventing cancer, the compound that comprises administration individual treatment significant quantity, described compound is to through engineering approaches people tumorigenic cell, and perhaps the cancer cells of specific gene type (the perhaps genotype of special change) shows selection toxicity.In certain embodiments, the characteristics of cancer are to comprise the cell of activatory RAS approach.In certain other embodiments, the characteristics of cancer are the little t cancer protein of cell expressing SV40, perhaps show the adjusting of the target of sT and/or carcinogenic RAS.

In related embodiment, the present invention expects method of the present invention and other antitumor therapies, and for example traditional embolic chemotherapy of setting up, combined utilization are shifted in solid tumor resisting and control.The administration of The compounds of this invention can be carried out after chemotherapeutic period or chemotherapy.This reagent usually is mixed with preparation with acceptable carrier, can intravenously, oral, cheek, parenteral, spray by sucking, by topical application or through percutaneous drug delivery.Reagent can also pass through the administration of topical mode.Preferably, one or more other reagent and anticancer disease chemotherapeutics (for example, compound of the present invention) Combined Preparation is to add up or the cooperative mode anticancer.

A large amount of conventional compounds has shown to have anti-tumor activity.These compounds have been used as pharmaceutical preparation and have dwindled solid tumor in chemotherapy, prevention is shifted and further growth, perhaps are reduced in the number of malignant cell in leukemia or the marrow malignant tumour.Although effectively, many antineoplastic compound are induced adverse side effect to chemotherapy in the various types of malignant tumours of treatment.In most cases, when two kinds or more of different treatment methods in conjunction with the time, treatment can act synergistically, and can reduce the dosage of every kind of therapy, thereby reduces the adverse side effect of every kind of compound when higher dosage.In other cases, treat unresponsive malignant tumour and may respond a kind of the therapies combination of two or more different treatments.

Therefore, compound of the present invention and pharmaceutical composition can with traditional antineoplastic compound Combined Preparation.Traditional antineoplastic compound comprises, only for illustrating: aminoglutethimide, amsacrine, Anastrozole, asparaginase, bacille Calmette-Guerin vaccine, bicalutamide, bleomycin, buserelin, busulfan, camptothecine, capecitabine, carboplatin, carmustine, Chlorambucil, cis-platinum, 2-chlorodeoxyadenosine, clodronate, colchicine, endoxan, cyproterone acetate, cytosine arabinoside, Dacarbazine, dactinomycin, daunorubicin, Retalon, stilboestrol, Japanese yew terpene, Zorubicin, epirubicin, estradiol, Emcyt, Zuyeyidal, Exemestane, filgrastim, fludarabine, fluohydrocortisone, Fluracil, fluorohydrocarbon methyltestosterone, Drogenil, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, Interferon, rabbit, Rinotecan, ironotecan, letrozole, formyl tetrahydrofolic acid, Leuprolide, L-tetramisole, lomustine, mustargen, medroxyprogesterone, megestrol, alkeran, purinethol, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, Nilutamide, R 17934, Sostatin, oxaliplatin, taxol, handkerchief is bent phosphonic acids disodium, pentostatin, Plicamycin, porfimer, procarbazine, Raltitrexed, riruximab, U-9889, Suramine, tamoxifen, Temozolomide, Vumon, testosterone, Tioguanine, plug is for group, titanocene dichloride, Hycamtin, trastuzumab, vitamin A acid, vincaleucoblastine, vincristine(VCR), vindesine, and vinorelbine.

In other embodiments, compound of the present invention and pharmaceutical composition can with traditional antineoplastic compound Combined Preparation, described traditional antineoplastic compound is selected from: epidermal growth factor receptor antagonists, arsenic sulfide, Zorubicin, cis-platinum, carboplatin, Cimitidine Type A/AB, carminomycin, the mustargen hydrochloride, pentamethylmelamine, plug is for group, Vumon, endoxan, Chlorambucil, de-methoxy Hypocrellin A A, left-handed L-Ala mustargen, ifosfamide, trofosfamide, Treosulfan, podophyllinic acid lactone or podophyllinic acid lactone derivative, Zuyeyidal phosphoric acid salt, Vumon, Zuyeyidal, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, methotrexate, ametycin, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, 1-methyl-4-phenylpyridinium ion, semustine, staurosporine, U-9889, phthalocyanine, Dacarbazine, aminopterin, methotrexate, Trimetrexate, Tioguanine, purinethol, fludarabine, pentastatin, carat bent guest, cytosine arabinoside (ara C), porfiromycin, 5 FU 5 fluorouracil, Ismipur, the Zorubicin hydrochloride, formyl tetrahydrofolic acid, mycophenolic acid, daunorubicin, desferrioxamine, floxuridine, doxifluridine, Raltitrexed, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, bleomycin sulfate, dactinomycin, Safranin, saframycins, quinocarcins, discodermolides, vincristine(VCR), vincaleucoblastine, preparing vinorelbine tartrate, vertoporfin, taxol, tamoxifen, raloxifene, tiazofurine, Tioguanine, virazole, EICAR, Emcyt, estramustine phosphate sodium, Drogenil, bicalutamide, buserelin, Leuprolide, pteridine, enediyne, L-tetramisole, aflacon, Interferon, rabbit, interleukin, rIL-2, filgrastim, sargramostim, Rituximab, bacille Calmette-Guerin vaccine, vitamin A acid, betamethosone, gemcitabine hydrochloride, verapamil, VP-16, hexamethyl melamine, thapsigargin (thapsigargin), oxaliplatin, iproplatin, four platinum, Lip river platinum, DCP, PLD-147, JM118, JM216, JM335, husky platinum, the Japanese yew terpene, deoxy taxol, TL-139,5 '-fall-F 81097 (below: 5 '-fall-vincaleucoblastine), camptothecine, Rinotecan (Camptosar, CPT-11), Hycamtin (Hycamptin), BAY 38-3441,9-nitrocamptothecin (Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C), gimatecan, homocamptothecins diflomotecan (BN-80915) and 9-aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), indole carbazole (indolocarbazole) is (for example, NB-506), protoberberine, intoplicine, idenoisoquinolones, benzene-azophenlyene or NB-506.

In another related embodiment, the present invention's expection is for example shone coupling with method of the invention process and other antitumor therapies.The term of Shi Yonging " irradiation " is to be used to comprise by the ionizing radiation treatment neoplastic cell of photon, neutron, electronics or other types or any therapy of relevant cell herein.This radiotherapy includes, but not limited to X ray, gamma irradiation, and perhaps heavy ion particle is such as alpha or beta particle.In addition, irradiation can be radioactive.The method of irradiation neoplastic cell is well known in the art in the patient, for example comprises external beam therapy and brachytherapy.

The method whether detection cancer (tumour or tumour form) has been cured is well known to a person skilled in the art, comprises, for example, the tumour cell number reduces (for example, cell proliferation decline or tumour size reduce).Be to be appreciated that treatment of the present invention can be durable and complete reaction, perhaps can comprise part or of short duration clinical response.Referring to example, people such as Isselbacher (1996) Harrison ' s Principles of Internal Medicine 13 ed.1814-1882 are hereby incorporated by.

The test of the sensitization of detection tumour cell or the death of increase is well known in the art, comprises, for example, the standard dose reaction test of evaluation cell viability; The flow cytometry of the dna break of the agarose gel electrophoresis of DNA extraction thing or detection expression necrocytosis feature; Detect the test of the polypeptide active of apoptosis involvement; With the test that detects the necrocytosis morphological specificity.Details elsewhere description herein about these tests.Other tests comprise that chromatin test (for example, counting the frequency of spissated nuclear chromatin) or the resistant test of for example describing are hereby incorporated by in people such as Lowe (1993) Cell 74:95 7-697.Also can also be hereby incorporated by referring to U.S. Patent number 5,821,072.

Pharmaceutical composition

The therapeutical agent of expection can be estimated so that detect them and be included in adaptability in the pharmaceutical composition.General detection method of this reagent is a therapeutic index, and this is the ratio of therapeutic dose to poisonous dosage.That the threshold value of therapeutic dose (effect) and poisonous dosage can be adjusted into is suitable (for example, therapeutic response or minimize the necessary amounts of toxic reaction).For example, therapeutic dose can be (with respect to one or more illnesss of treatment) of reagent treatment significant quantity, and poisonous dosage can be to cause that dead dosage (for example, LD50) or cause undesirable effect in the certain proportion curee.Preferably, the therapeutic index of reagent is 2 at least, and preferred is 5 at least, in addition preferred be 10 at least.Estimate the pharmacokinetics that therapeutical agent can also comprise detection reagent, when with different preparations and/or by the different approaches administration, detect its bioavailability and/or absorption.

Compound of the present invention, for example erastin or Antitubulin can be needed its individuality to use.In certain embodiments, individuality be Mammals such as the people, perhaps non-human mammal.When administration was individual, compound of the present invention can be used as the pharmaceutical composition administration, and described pharmaceutical composition comprises, for example, and compound of the present invention and pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier is well known in the art, comprises, for example, the aqueous solution is such as water or physiological buffer salt solution or other solvents, perhaps media ethylene glycol for example, and glycerine, finish is such as sweet oil or injectable organic ester.In preferred embodiments, when this pharmaceutical composition was used for the people and uses, the aqueous solution is pyrogen-free, and was perhaps pyrogen-free basically.Can select vehicle, for example, with release or one or more cells of selectivity target that delay reagent, tissue or organ.

Pharmaceutically acceptable carrier can comprise the physiology acceptable agents, and described reagent is used as, and is for example, stable or promote for example absorption of erastin or Antitubulin of compound.This physiology acceptable agents comprises, for example, carbohydrate, such as glucose, sucrose or dextran, antioxidant, such as xitix or gsh, sequestrant, low molecular weight protein (LMWP) or other stablizers or vehicle.The selection of pharmaceutically acceptable carrier comprises the physiology acceptable agents, depends on for example route of administration of composition.Pharmaceutical composition (preparation) can also be liposome or other polymeric matrixs, and this can be integrated into compound for example of the present invention.Liposome for example, is made up of phosphatide or other lipid, is nontoxic, the acceptable and metabolizable carrier of physiology, manufacturing that can be relatively easy and administration.

The pharmaceutical composition (preparation) that comprises The compounds of this invention can comprise by multiple route of administration administration, and is for example, oral; Intramuscular; Intravenously; Anus; Vagina; Parenteral; Nose; Intraperitoneal; Subcutaneous; And part.Composition can be by injection or by the incubation administration.

In certain embodiments, compound of the present invention (for example, erastin) can be individually dosed or with another kind of anti-tumor therapeutic agent Combined Preparation.The phrase of Shi Yonging " Combined Preparation " is meant any form of medication of the two or more different treatment compounds of coupling herein, second kind of compound of administration (for example when like this treatment compound of administration formerly was still effective in vivo, two kinds of compounds work in patient's body simultaneously, and this can comprise the synergistic effect of two kinds of compounds).For example, different treatment compounds can administration in same preparation or the preparation that is separating, perhaps simultaneously or successively.Therefore, the individuality of accepting this treatment can be benefited from different synergistic effects for the treatment of compounds.

Expect that compound of the present invention (for example erastin) is with treatment significant quantity (dosage) administration patient (for example Mammals, preferably people)." treatment significant quantity " is meant that compound concentrations is enough to obtain required result of treatment (for example, the healing of illness, the death of neoplastic cell).The significant quantity that should be appreciated that compound can be according to patient's weight, sex, and the age changes with the treatment history.Other factors that influence significant quantity may include, but not limited to the severity of patient's illness, the disease of receiving treatment, the stability of compound and, if desired, with the another kind of therapeutical agent of compound administration of the present invention.Usually, to the scope of human patients significant quantity from about 0.001mg/kg body weight to about 50mg/kg body weight.Can carry more total dose by reagent administration repeatedly.The method that detects effect and dosage is well known by persons skilled in the art.Referring to for example, people such as Isselbacher (1996) Harrison ' s Principles of InternalMedicine 13 ed.1814-1882 are hereby incorporated by.

Embodiment

General introduction the present invention now is more readily understood the present invention with reference to the following example, comprises that the following example just to illustrating some aspect of the present invention and embodiment, is not to be used to limit the present invention.

Embodiment 1 differentiates to have to strengthen under the relevant allelotrope existence condition of specific cancer and renders a service or active compound

Work described herein is to be used for identifying at hTERT LT, ST, E6, E7 or RAS ^V12Having enhanced under the existence condition renders a service or active compound.Although work described herein utilizes hTERT, LT, ST, E6, E7 and RAS ^V12As transforming gene, research in the future can utilize multiple cancer in conjunction with allelotrope, utilizes this method so that determine to participate in the signal network of many oncogenes and tumor inhibitor.Through engineering approaches clone with these genetic elements is used to screen 23,550 kinds of compounds, comprise from 20 of combinatorial library, 000 kind of compound, 1,990 kind of compound and 1 from the cancer association diversity gleanings of country, 540 kinds of known compounds that biologic activity is arranged, described known compound are that the applicant selects and buy and make the set that can supply screening.Primary dcreening operation detects (in quadruplicate) and measures with every kind of compounds for treating tumorigenesis BJ-TERT/LT/ST/RAS ^V12The effect of through engineering approaches tumorigenic cell, described compound was 4 μ g/mL mass actions 48 hours, and corresponding to the compound 10 μ M of molecular weight 400, this approximately is the intermediate value molecular weight in library.Use dyestuff fluorexon acetyl methyl esters (fluorexon AM) detect cell viability (people such as Wang, 1993, Hum.Immunol.337,264-270), this is a kind of non-fluorescent chemicals that can freely diffuse into cell.In viable cell, fluorexon AM is cut by born of the same parents' lactonase, forms anion fluorescent derivative fluorexon, and it can not diffuse out viable cell.Therefore, when hatching with fluorexon AM, viable cell shows green fluorescence, and dead cell does not show.For at BJ-TERT/LT/ST/RAS ^V12The compound of demonstration 50% or stronger inhibition dyestuff fluorexon AM dyeing vigor in the cell is then at BJ and BJ-TERT/LT/ST/RAS ^V12Compound described in the cell carries out the twice serial dilution to identify the compound that shows synthetic lethal, and this compound is lethality in tumorigenic cell, but in the homology primary cell is not.Calculate the IC of each compound in each clone ₅₀Value (suppressing 50% fluorexon AM signal desired concn) (table 1).The result identifies the kind compound coldest days of the year end (Fig. 2), and these compounds are at BJ-TERT/LT/ST/RAS ^V12In the tumorigenic cell than BJ primary cell activity high at least 4 times (in the BJ primary cell, need 4 times of high concentration at least and suppress) so that obtain 50% of identical fluorexon AM signal.It below is the more detailed analysis of these nine kinds of compounds.

Three kinds of (Zorubicins in these compounds, daunorubicin and mitoxantrone) now clinical in cancer therapy drug, a kind of (camptothecine) is the natural product analogue of clinical use anticarcinogen (Hycamtin and Rinotecan), and a kind of (Quinomycin A) carrying out the II clinical trial phase recently.All these nine kinds of compounds carry out the multiple hole of multiple doses then and detect in each group through engineering approaches cell, also be present in the multiple independent deutero-clone (Fig. 1 and table 1) to confirm observed selectivity.

The applicant has developed a kind of detection compound IC in two kinds of different clones ₅₀The selectivity measurement Law that (suppressing the required concentration of 50% vigor signal) changes.For calculating two selective values between clone, the IC of compound in a kind of clone ₅₀Divided by the IC of same compound in second kind of clone ₅₀Therefore, it is high 4 times to have second kind of clone of concentration ratio that the compound of selective value 4 uses in a kind of clone.Calculate " the tumor-selective value " of each compound, by using the IC of compound in the former generation BJ of parent cell ₅₀Value divided by compound at through engineering approaches BJ-TERT/LT/ST/RAS ^V12IC in the cell ₅₀Value, through engineering approaches BJ-TERT/LT/ST/RAS ^V12Cell comprises the required whole four kinds of genetic elements (table 1) of generation tumorigenic cell.

The viral cancer protein that these through engineering approaches tumorigenic cell utilizations play a major role is such as LT, ST, E6 and E7.These viral proteins may participate in cell transformation in the cancer of particular form, the cancer of described particular form is simian virus 40 inductive malignant mesothe (Testa and Giordano, 2001, Semin Cancer Biol 77,31-8) and human papillomavirus inductive cervical cancer (people such as Bosch, 2002, J Clin Pathol 55,244-65), and the function that these viral proteins have been used to destroy p53 and pRB is with transformant (people such as Elenbaas in vitro and in vivo, 2001, Genes Dev 15,50-65; People such as Jorcyk, 1998, Prostate 34,10-22; People such as Perez-Stable, 1997, Cancer Res 57,900-6; People such as Rich, 2001, CancerRes 61,3556-60; People such as Sandmoller, 1995, Cell Growth Differ 6,97-103).The applicant utilizes two kinds of diverse ways as killed cells albumen, (they detect LT and based on the effect of E6/E7 deactivation pRB and p53) in case control to the observed atopy effect of specific virus albumen.The selectivity of these compounds also mainly confirms that described p53 and pRB are not derived from viral element in the clone of negative inhibitor expressing p53 and pRB.This expression of cell lines (i) destroys the p53 clipped form (p53DD) of endogenous p53 tetramerization, (ii) anti-p16 ^INK4AAnd p15 ^INK4BThe CDK4 that (the main down regulator of CDK4) suppresses ^R24CMutant and (iii) cyclin D1.Detect the effect of nine kinds of genotype alternative cpds in these cells in multiple concentration range, described cell is meant BJ-TERT/p53DD/CDK4 ^R24C/ D1/ST/RAS ^V12Cell (table 1).The result is presented at when detecting in these cells, and all there is active overall moderate reduction in all compounds.But the total result of utilizing non-virus protein to analyze in these clones does not change (table 1).

Embodiment 2 measures the hereditary basis of compound selective

The applicant has managed to detect the hereditary basis of every kind of compound selective.In brief, to every kind of compound, attempt determining to cause the combination (table 1) of cell to the gene or the gene of compound sensitivity.The result shows that these nine kinds of compounds can be divided into 3 groups, promptly (i) shows optionally compound of no simple heredity, (ii) the cell that has TERT and nonactive RB is shown compound optionally and (iii) is the compound of the existence that shows carcinogenic RAS of lethality needs and ST.

Compound in the group (i), sangivamycin, bouvardin, NSC146109 and Quinomycin A, the tumorigenic cell selectivity does not have clear and definite hereditary basis.For example, when each genetic elements imports active some rising of Quinomycin A (Fig. 3 a).The applicant has been noted that when these genetic elements all import cell proliferation rate increases.Therefore, the compound of organizing probably in (i) is only selective to quick splitted cell.Support the fact of this explanation to be, all these compounds it is reported and work by suppressing DNA or protein synthesis that its demand in quick splitted cell is bigger.For example, it is reported the effect of Quinomycin A be as the dna double intercalating agent (Van Dyke and Dervan, 1984, Science 225,1122-7; Waring and Wakelin, 1974, Nature252,653-7), bouvardin is as protein synthesis inhibitor (people such as Zalacain, 1982, FEBS Lett 148,95-7), sangivamycin is nucleotide analog (Rao, 1968, J MedChem 11,939-41), NSC146109 structurally is similar to DNA intercalating agent (Fig. 2).It should be noted that and report that sangivamycin is as pkc inhibitor (Loomis and Bell, 1988, J Biol Chem 263,1682-92), although this activity as if greatly can not with this spot correlation because other pkc inhibitors show non-selectivity in this system.The applicant can identify just active higher compound in quick splitted cell, for example organizes the compound in (i), because they do not show clear and definite selectivity hereditary basis.These compounds are not further worked.Therefore, they can concentrate on the mechanism research that shows optionally group compound (ii) and (iii).

The compound of group in (ii), mitoxantrone, Zorubicin and daunorubicin are the topoisomerase II toxin, they are in conjunction with topoisomerase II and DNA and prevent reconnecting of double-stranded DNA fracture that topoisomerase II causes.These compounds and anthracycline antibiotics, had the formation (ROS) that is reported in induced activity oxygen in some cell types (Laurent and Jaffrezou, 2001, Blood 98,913-24; People such as Muller, 1998, Int J MoI Med 1 491 4; People such as Richard, 2002, Leuk Res 26,927-31), although the applicant does not observe the formation of ROS in these through engineering approaches cells under the condition that these three kinds of compounds exist.They find to be imported into and when RB is inactivated by importing LT or HPV E7 these compound vigor higher (activity is also arranged) as hTERT under lower concentration.In cell, E7 imports after E6, so the effectiveness that these compounds increase in having the cell of E7 may also depend on the existence of E6, even the increase that E6 self does not cause these compounds to be renderd a service.The importing of hTERT and the inactivation of RB cause increase (Fig. 5 a), the just very limited increase of topoisomerase II alpha expression of topoisomerase II alpha expression.The importing of carcinogenic RAS causes the further increase of topoisomerase II alpha expression, although the applicant does not observe under the situation that carcinogenic RAS exists for the further sensitizationization of topoisomerase II alpha toxin.

(iii) compound of group is a camptothecine (CPT) and from the new compound of combinatorial library, and the applicant is with its called after erastin because it be the remover of expressing the cell of RAS and ST ( eRadicator of RAS and ST-expressing cells) (Fig. 2).Effectively CPT induces that necrocytosis needs ST and RAS with the erastin inductive ^V12Have (Fig. 3,4 and table 1).Although CPT has identical selectivity hereditary basis with erastin, their mechanism of action difference.CPT is the part activatory in the cell that lacks RB function (by the expression of E7), and erastin is not, and CPT need cause BJ-TERT/LT/ST/RAS in two days ^V12Middle dead, and erastin was 100% effective (Fig. 3 and 4) in 18 hours.It is to have the BJ primary cell of resistance to CPT sensitization (Fig. 5 E) in other cases that the inhibitors of phosphatases okadaic acid can make, may be because okadaic acid raises TOP1 (Fig. 5 F).Okadaic acid can not cause BJ or BJ-TERT cell to the erastin sensitivity, with CPT and erastin by different mechanism action model unanimities.In addition, the applicant finds lethal compound podophyllinic acid lactone, a kind of Antitubulin, do not make BJ or BJ-TERT cell to the CPT sensitivity, confirm that BJ cell that okadaic acid causes is special to the sensitization of CPT, be not two kinds and have and add up but the faint necrocytosis of incoherent effect stimulates on the function result.

Express RAS for understanding ^V12The molecular basis that CPT susceptibility is increased with the cell of ST, the applicant has detected the expression level of topoisomerase I in the through engineering approaches cell, and topoisomerase I is CPT target (people such as Andoh, 1987 of generally acknowledging, Proc Natl Acad Sci U S A 84,5565-9; People such as Bjornsti, 1989, Cancer Res 49,6318-23; Champoux, 2000, Ann N YAcad Sci 922,56-64; People such as D ' Arpa, 1990, Cancer Res 50,6919-24; People such as Eng, 1988, MoI Pharmacol 34,755-60; People such as Hsiang, 1989, Cancer Res 49,5077-82; Hsiang and Liu, 1988, Cancer Res 48,1722-6; People such as Liu, 2000, Ann N Y Acad Sci 922,1-10; Madden and Champoux, 1992, Cancer Res 52,525-32; People such as Tsao, 1993, Cancer Res 53,5908-14).They find that the cell of expressing RASV12 and ST raises TOP1 (Fig. 5 B).Because the generally acknowledged mechanism of action that CPT acts in other cell types relates to the acquisition of function, promptly the mode that relies on TOP1 import the double-stranded DNA fracture (people such as Liu, 2000, Ann N Y Acad Sci 922,1-10), the rise of TOP1 can be explained and express RAS ^V12With of the increase of ST cell to CPT susceptibility.For supporting these explanations, they find at BJ-TERT/LT/ST/RAS ^V12The TOP1 genetic inactivation that causes with siRNA (siRNA) in the cell cause partial resistance to CPT (Fig. 5 C, D).

The applicant has detected activity and the selectivity of other erastin analogues in the relative normal cell of tumour cell in addition.Another similar compound has been confirmed as activity and selectivity, but specific activity erastin is low.This compound is named as erastin B (referring to Fig. 8).The BJELR cell is BJ-TERT/LT/ST/RAS ^V12Cell, BJEH cell are the BJ-TERT cells.

Further compound test is as follows in BJELR and BJEH cell:

The sign of embodiment 3 necrocytosiss is identified

The applicant manages to have identified at tumorigenesis BJ-TERT/LT/ST/RAS ^V12CPT and erastin inductive necrocytosis type in the cell.In other documents, have been found that the apoptosis-induced cell death of CPT (people such as Traganos, 1996, Ann N Y Acad Sci 803,101-10), its characteristics are that the change of karyomorphology comprises pyknosis, nucleorhexis and/or chromatinic limit (Majno and Joris, 1995, Am J Pathol 146,3-15).Whether apoptosis-induced in its system for detecting erastin or CPT, the applicant uses the fluorescent microscopy monitoring to accept the karyomorphology of the tumorigenic cell of CPT and erastin processing.Although can obvious nucleorhexis in the cell that CPT handles and chromatinic limit, in the cell that erastin handles, do not see this morphological change (Fig. 7 A).Because nuclear morphology variation is that apoptotic cell is required, applicant's erastin inductive necrocytosis right and wrong of reaching a conclusion are apoptotic.What further support this conclusion is to observe CPT, rather than erastin, inducing DNA fracture (forming the DNA band), general caspase inhibitor (50M Boc-Asp (Ome)-methyl fluoride ketone, Sigma#B2682 (people such as Chan, 2001, Neuroreport 12,541-545)), part is blocked CPT rather than the necrocytosis of erastin inductive, and CPT rather than erastin cause the appearance (Fig. 7 C) of the painted increase of Annexin V (Fig. 7 B) and cutting, activatory caspase3.In addition, the tumour cell center of handling at erastin be kept perfectly (Fig. 9).

Erastin is expressing ST and RAS ^V12The ability of inducing non-apoptotic cell death in the cell is optionally.The processing of Erastin longer time and higher concentration are to lacking RAS ^V12Perhaps the cytoactive of ST influence is very little, this confirm erastin optionally qualitative property (Fig. 6 A, C).Because the cell that erastin handles does not experience apoptosis, the applicant strives to corroborate the certain inducing cell death of erastin, rather than cell detachment.They use detect reduction potential in the born of the same parents dyestuff AlamarBlue under the condition that erastin exists the quantitative assay cell viability (people such as Ahmed, 1994, J.Immunol.Methods 170,211-224).In homogeneity Alamar Blue vitality test, with respect to BJ-TERT cell Erastin at tumorigenesis BJ-TERT/LT/ST/RAS ^V12Show selectivity lethality (Fig. 6 B) in the cell.With 18 hours BJ-TERT/LT/ST/RAS of erastin processing ^V12Cell rounding and disengaging (Fig. 6 C) can not be got rid of the vital dye trypan blue, and the loss of display line plastochondria membrane potential in potential determination dyestuff JC-1 detects has the minicell size of dead cell feature.The applicant detects, and erastin inductive vigor loss is in case to finish be exactly irreversible, because handle 24 hours BJ-TERT/LT/ST/RAS with erastin ^V12Cell rounding, disengaging can not recover when being re-applied on the medium that does not contain erastin.Therefore, erastin is with ST and RAS ^V12The dependence mode is induced fast (12-24 hour), irreversible, non-apoptotic cell death.

Study formation (referring to Figure 10) in addition with proof erastin induced activity oxygen.

Screen and seek the active containment agent of erastin (inhibitor).Identify four kinds and suppress the active antioxidant of erastin, one of them is an antioxidant, alpha-tocopherol.

Following method and material are used for embodiment described herein.

Construct and retrovirus

HTERT, LT, ST, SV40 early region and HRAS ^V12Expression construct by previous described use (people such as Harm, 1999, preamble; People such as Hahn, 2002, preamble).HTERT-pWZL-Blast ε, E6-pWZL-zeo ε and E6E7-pWZL-Zeo ε were before existing to be described (people such as Lessnick, 2002, preamble).E6 and LT cDNAs are cloned into pWZL-Hygro ε retroviral vector (J.Morgenstern, Millenium Pharmaceuticals is so kind as to give).Preparation blister stomatitis virus-G glycoprotein pseudotype retrovirus infects by preceding method and carries out (people such as Lessnick, 2002, preamble).

Clone

TIP5 inoblast of former generation (people such as Lessnick, 2002, preamble) the newborn foreskin preparation from abandoning makes cell immortalityization with hTERT-pWZL-blast ε or hTERT-pBabe-hygro retroviral infection, respectively with blasticidin or hygromycin selection.The BJ cell is so kind as to give by Jim Smith.HTERT-immortalization inoblast is selected the appropriate flags thing with specifying retroviral infection.All BJ derivatives are cultivated in 1: 1 mixture of DMEM that adds 15% inactivated fetal bovine serum, penicillin and Streptomycin sulphate (pen/strep) and M199.The TIP5 cell is grown in the DMEM that comprises 10%FBS and pen/strep.The all cells culture comprises 5%CO at 37 ° of C ₂Moistening incubator in hatch.

Library of compounds

Comprise 1, the mark library of compounds (ACL) of 540 kinds of compounds, the NCI diversity group of 1, the 990 kind of compound that obtains from national cancer association and comprise 20, (Comgenex International Inc.) is used for the synthetic lethal screening of tumor-selective for 000 kind of combination of compounds library.All library of compounds are prepared as the 4mg/ml solution (row 3-22) that is dissolved among the DMSO ,-20 ℃ of preservations in 384 hole polypropylene boards.Camptothecine (cat#_C9911, MW 348.4), Zorubicin (cat#_D1515 MW 580.0), daunorubicin (cat#_D8809, MW 564.0), mitoxantrone (cat#_M6545, MW 517.4), okadaic acid (cat#_O4511, MW 805.0), Quinomycin A (cat#_E4392, MW 1101), sangivamycin (cat#_S5895, MW 309.3) is available from Sigma-Aldrich Co.Bouvardin (MW 772.84) and NSC146109 (MW280.39) are available from national cancer association development treatment project.Erastin (MW 545.07) is available from Comgenex International, Inc.

Fluorexon AM vitality test

Fluorexon acetyl methyl esters (AM) is that cytolemma is permeable, the compound of non-fluorescence, and it is formed negatively charged ion, impermeable, the fluorescent chemicals fluorexon of cell by the cutting of born of the same parents' lactonase.Viable cell is dyeed by fluorexon, because the existence of born of the same parents' lactonase and because complete plasma membrane stops fluorescence fluorexon emigrated cell (people such as Wang, 1993, preamble).Cell uses ZymarkSciclone ALH to be seeded in 384 orifice plates, handled two days with all cpds 4 μ g/mL are triplicate during primary dcreening operation, on the 384 hole cleaning machines of Packard Minitrak,, hatched four hours with 0.7 μ g/mL fluorexon (Molecular Probes) with the phosphate buffered saline buffer washing.Total fluorescence intensity in each hole of PackardFusion plate counter records by deduction instrument background and the average signal that obtains need not any compound treatment the time divided by cell, changes it inhibiting rate of signal into.

Alamar Blue vitality test

Alamar Blue is by the activity of cyclophorase in viable cell reduction, cause colorimetric and change in fluorescence (people such as Nociari, 1998, J.IImmunol.MMethods, 13,157-167).Use a large amount of dispensers of injecting type (Zymark) that cell is inoculated in 384 hole black, dianegative with the density (50 μ l) of 6000 cells in every hole.Use 384 permanent sleeve tube heads to take out 10 μ l, make the ultimate density in the maximum concentration hole reach 20 μ g/ml from the erastin plate (6 * ultimate density) of twice serial dilution.Plate was hatched 24 hours.1: 10 dilution back of Alamar Blue (BiosourceInternational) adds each hole, hatches 16 hours at 37 ℃.Use excites filter disc to concentrate on the Packard Fusion plate counter that 535nm and emission filter disc concentrate on 590nm to come fluorescence intensity.Calculate the average inhibiting rate of each concentration.Error bars shows a standard deviation.Alamar Blue test does not relate to washed cell.

Screening

Xerox daughter board with Zymark Sciclone ALH preparation, by integrating Twister II for 50 times, to obtain to have in the daughter board compound concentration of the 80 μ g/ml of 2%DMSO with the substratum dilution motherboard that does not contain serum and pen/strep.Use a large amount of dispensers of injecting type with 1-23 row (every hole 6000 cells, 57 μ ls) the preparation check-out console of cell inoculation in black, clear bottom 384 orifice plates.The compound treatment of 3-22 row from the library daughter board shifts 3 μ l by the Guan Zhen that uses 384 stationkeeping from the library daughter board.Therefore final compound concentration is 4 μ g/ml in the test board.Test board comprises 5%CO at 37 ℃ ₂Moistening incubator in hatched 48 hours.Use is carried out the plate treatment step from the integration Minitrak/Sidetrak robot system of Packard Bioscience (Perkin Elmer) and is used to analyze fluorexon AM vigor.Test board washs with phosphate buffered saline buffer, and 20 μ l fluorexon AM (0.7 μ g/ml) are added in every hole.Plate was incubated at room 4 hours.Excite filter disc and the Packard Fusion plate counter that concentrates on the emission filter disc of 590nm that use concentrates on 535nm come fluorescence intensity.

The revision test of compound in serial dilution

The compound of revision test is bought from manufacturer.Reserve is dissolved among the DMSO with 1mg/ml concentration in 384 hole polypropylene boards and prepares, and each compound has 16 points, twice thinner discharge curve in every row, and is duplicate.Row 1-2 and 23-24 stay into blank in contrast.By in 384 hole deep-deep orifice plates, diluting 66.6 times (4.5 μ l transfer among the 300 μ l), prepare the revision test daughter board with the revision test plate that stores with DMEM.Cell is with the density plantation of 6000 cells of every hole 40 μ l, and 20 μ l add from the revision test daughter board.Plate is at 37 ℃ of 5%CO ₂Under hatched two days.

Data analysis

The average RFU (relative fluorescence unit) of untreated cell calculates by the value of

average row

1,2 and 23 (cell is arranged but do not have the hole of compound).The fluorexon background is calculated by the value of average row 24 (fluorexon being arranged but acellular hole).The inhibiting rate in every hole calculates by [1-(contrast of RFU-fluorexon)/(untreated cell-fluorexon contrast) * 100].By at former generation of BJ and BJ-TERT/LT/ST/RAS ^V12Detection in the cell in the finite concentration scope determines to cause in primary dcreening operation the compound of fluorexon dyeing at least 50% inhibition to BJ-TERT/LT/ST/RAS ^V12The selectivity of through engineering approaches tumour cell.Alternative cpd is revision test in all through engineering approaches clones.

The karyomorphology test

200,000 tumorigenesis BJ-TERT/LT/ST/RAS of inoculation 2mL on the glass cover slide in every hole of six orifice plates ^V12Cell is used in growth medium respectively blank (NT), and 9 μ M erastin or 1.1 μ M camptothecine (CPT) were handled 18 hours, at 37 ℃ of 5%CO ₂Under hatch.Nuclear is observed with oily mirror 100 * object lens on fluorescent microscope with 25 μ g/mL Hoechst 33342 (Molecular Probes) dyeing.

The cell size is measured

200,000 BJ-TERT/LT/ST/RAS ^V12The cell kind has only 2mL growth medium (non-processor) in six orifice plates, has 9 μ M erastin or has 1.1 μ M camptothecine (CPT).After 24 hours, cell digests release with trypsinase/EDTA, is diluted to 10mL in growth medium, detects the cell size distribution of each sample on Coulter-counter.

The active cell counting test of camptothecine

BJ-TERT/LT/ST/RASV12 cell kind is in 6 orifice plates (200 000 cells/well; Every hole 2ml), utilize Oligofectamine (Life Technologies) transfection in the substratum of serum-free and antibiotic-free, every hole 100nM siRNA, cumulative volume 1ml.500 μ l comprise substratum interpolation in 4 hours after transfection of 30%FBS.Cell was handled 30 hours with the camptothecine of prescribed concentration after the transfection.5 * solution of the required camptothecine concentration of 500 μ l is added to each hole.With trypsinase-EDTA peptic cell, Hematocyte Counter is counted after 75 hours in transfection.Control experiment shows that transfection efficiency approximately is 10%.

Western Blot analyzes

caspase-3

In the 60mm ware, plant 5 * 10 before the experiment ⁵BJ-TERT/LT/ST/RAS ^V12Cell.Cell was handled 2,4,6,8 or 10 hours with 5 μ g/ml erastin (9 μ M).A ware is handled (0.4 μ g/ml 24 hours) as positive control with camptothecine.Cell is at lysis buffer (50mM HEPES KOH pH 7.4,40nM NaCl, 2mM EDTA, 0.5%Triton X-100,1.5mM Na behind each time point ₃VO ₄, 50mM NaF, the 10mM trisodium phosphate, 10mM beta-Sodium Glycerophosphate and proteolytic enzyme suppress sheet (Roche)) middle cracking.With Biorad protein assay reagent quantitative protein content.Equal protein is separated on the 16%SDS-polyacrylamide gel.The albumen of electrophoretic migration is transferred on the pvdf membrane, with 5% milk sealing, with the polyclonal antibody of the anti-activation caspase-3 of dilution in 1: 1500 4 ℃ of overnight incubation.The anti-rabbit HRP (Santa Cruz Biotechnology) of film and dilution in 1: 3000 was hatched 1 hour then, used enhanced chemiluminescence mixture (NEN life science, Renaissance) colour developing again.For detecting sample on every swimming lane equivalent, spot is cut into bar, and sealing is with anti-eIF-4E antibody (the BD Transduction laboratories) detection of dilution in 1: 1000.

Topoisomerase-II α

BJ, BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RAS ^VI2, BJ-TERT/LT/RAS ^V12And BJ-TERT/LT/RAS ^V12/ ST cell kind is gone into the 60mm ware, every ware 1 * 10 ⁶Individual cell.At 37 ℃ of 5%CO ₂After the overnight incubation, cell is cracking as stated above, and albumen separates on 10% polyacrylamide gel.The anti-human topoisomerase II α p170 antibody (TopoGEN) of film and dilution in 1: 1000 is hatched with anti-mouse HRP (Santa Cruz Biotechnology) then 4 ℃ of overnight incubation.

Topoisomerase 1 (TOP1)

21 nucleotide double siRNA (Nucleotide 2233-2255 at TOP1; count from initiator codon; Genbank accession number J03250) is synthesized (Dharmacon; purifying and desalination/deprotection), in six orifice plates, use oligofectamine (Life Technologies) transfection (100nM) BJ-TERT/LT/ST/RAS ^V12Cell.After 75 hours, cell is cleaved, detects the expression level (Topogen, Cat#_2012-2, dilution in 1: 1000) of TOP1 by Western blot.Redeterminate same blob by slitting with the antibody of anti-eIF (BD Biosciences, Cat#_610270, dilution in 1: 500), sample level on the albumen is detected.In addition, 1 * 10 ⁶The cell kind is in the 60mm ware, at 37 ℃ of 5%CO ₂Grow overnight under the condition, cracking in 150 μ l lysis buffers then.Cell scrapes with scraper, transfers to Eppendorf tube, hatches on ice 30 minutes.With the protein content in the Biorad protein assay reagent quantitative cleavage thing.Equal protein is splined on 10% gradient SDS-polyacrylamide gel.Electrophoretic migration albumen is transferred on the pvdf membrane.After the sealing of 5% milk powder, film and mouse anti human topoisomerase I antibody (Pharmingen) are hatched with anti-mouse peroxidase coupling antibody (Santa Cruz Biotechnology) then 4 ℃ of overnight incubation.

The test of Annexin V-FITC apoptosis

Plant 1 * 10 in each 100mm ware ⁶Individual BJ-TERT/LT/ST/RAS ^V12Cell, grow overnight.Cell was handled 6,8 or 11 hours with erastin (5 or 10 μ g/ml).Keep camptothecine to handle (0.4 μ g/ml), when inoculation, handled 20 hours.After the processing, cell is gathered in the crops with trypsinase/EDTA, and with comprising the washing of fresh serum substratum once, uses the phosphate buffered saline buffer washed twice then.Cell suspends again with 1 * binding buffer liquid (BD Pharmingen), and concentration is 1 * 10 ⁶Cell/ml.100 μ l (1 * 10 under room temperature lucifuge condition ⁵Individual cell) hatched 15 minutes with 5 μ l Annexin V-FITC (BD Pharmingen) and propidium iodide.Add 400 μ l, 1 * binding buffer liquid then, by flow cytometer (BectonDickinson) analysis of cells.Use Cellquest software to obtain and analytical data.Use the FL1 passage only to analyze the Annexin V-FITC dyeing of viable cell, described viable cell is not painted by propidium iodide.

ROS analyzes: the flow cytometry that utilizes H2DCF-DA

2 ', 7 '-the two hydrogen fluorescein diacetates (H2DCF-DA) of two chlorine are the permeable compounds of non-fluorocyte.Intracellular endogenous esterase cutting diacetate part, it is emigrated cell no longer.Therefore it accumulates in cell.H2DCF and ROS reaction forms fluorescence dichlorofluorescein (DCF) then, and this can detect at the FL1 passage with flow cytometer.

1. plant 3 * 10 in each 60mm ware ⁵Individual cell, grow overnight.

2. handle different time (1-10 hour) with testing compound.

3. each time point is kept a untreated cell respectively, the ware of compound treatment cell and positive control (hydrogen peroxide treatment).

4. hatched 10 minutes at 37 ℃ of cells and 10 μ M H2DCF-DA.

5. to positive control cell,, add 500 μ M hydrogen peroxide, hatched again 5 minutes adding H2DCF-DA after 5 minutes.

6. trysinization harvested cell.

7. with cold PBS washed twice.

8. resuspended precipitation in 100 μ l PBS is transferred to 5ml FACS pipe.

9. add 5 μ l iodate, third ingot (50 μ g/ml), ice bath is 10 minutes under the lucifuge condition.

10. add 400 μ l PBS, analyze by flow cytometer (Becton-Dickinson).

11. use the Cellquest software program to obtain and analytical data.

Carry out the DCF staining analysis 12. only choose propidium iodide negative cells (viable cell), use the FL1 passage, PI makes quadrantal diagram at the FL3 passage.

Screening ACL can suppress the active compound of erastin in the BJELR cell in the library

Method:

The ACL library and all compounds that comprise 1,540 kind of compound are prepared as the 4 μ g/ml solution that are dissolved among the DMSO ,-20 ℃ of preservations in 384 hole polypropylene boards.Use ZymarkScilone ALH to prepare the photomechanical printing daughter board in each library.Daughter board dilutes 50 times with DMEM, and the compound concentration in the daughter board is 80 μ g/ml, has among the 2%DMSO.Compound from daughter board in test board dilutes 20 times with cell suspension, so the ultimate density of each compound is 4 μ g/ml.

The BJELR cell with 6000 cells/well (57 μ l) (be used for co-processing screening) and 5000 cells/well (57 μ l) (being used for pre-treatment screens) with a large amount of dispenser kinds of injecting type in 384 hole black, dianegative.To the screening of co-processing inhibition, cell is handled with 5 μ g/mlerastin simultaneously with the compound treatment (ultimate density is at 4 μ g/ml in the test board) of 3 μ l from the daughter board in ACL library.The compound transfer is carried out in use 384 fixedly tube head.Plate is at 37 ℃ of 5%CO ₂Hatched in the incubator 48 hours.To the pre-treatment screening, cell uses the compound preincubate from ACL library daughter board to spend the night, and handles 48 hours with 5 μ g/ml erastin then again.Use the MiniTrak/SideTrak robot system of PackardBioScience that plate is carried out the fluorexon test.Test board washs with PBS, at room temperature hatches 4 hours with fluorexon AM (0.7 μ g/ml).Excite filter disc and the emission filter disc that concentrates on 535nm that use concentrates on 485nm come fluorescence intensity with Fusion plate counter.The BJELR cell is BJ-TERT/LT/ST/RAS ^V12Cell.

Table 1 shows the effectiveness of the compound of tumor-selective in the through engineering approaches clone.Nine kinds of tumor-selective compounds carry out the revision test of 16 points, twice thinner discharge curve in all-work clone kind.Table is listed in each clone each compound and reaches fluorexon AM dyeing 50% and suppress (IC ₅₀) concentration (μ g/mL).IC in former generation BJ cell ₅₀Divided by BJ-TERT/LT/ST/RAS ^V12IC in the tumorigenic cell ₅₀The tumour that obtains each compound is selected ratio.Recently examine and determine the selectivity of compound by calculating in the series each right selection of clone subsequently to each genetic elements.It is selective to PP2A (target of little T cancer protein) that little T cancer protein-alternative cpd is considered to, and the E6-alternative cpd is considered to selective to the p53 disappearance, and it is selective to the disappearance of RB that the E7-alternative cpd is considered to.

Quinomycin A	BJ	BJ-TERT	BJ-TERT/	BJ-TERT/	BJ-TERT/	BJ-TERT/	BJ-TERT/	Tumor-selective	The selectivity hereditary basis
	BJ	BJ-TERT	BJ-TERT/	BJ-TERT/	BJ-TERT/	BJ-TERT/	BJ-TERT/	Tumor-selective	The selectivity hereditary basis			LT/	LT/	LT/	LT/	p53DD/
			ST	ST/	Ras ^V12	Ras ^V12/	CDK4 ^R2C/					LT/	LT/	LT/	LT/	p53DD/
			ST	ST/	Ras ^V12	Ras ^V12/	CDK4 ^R2C/						Ras ^V12		ST	cyclinD 1/
							ST/Ras ^V12						Ras ^V12		ST	cyclinD 1/
							ST/Ras ^V12			＞5	0.312	0.0048	0.0012	0.0048	0.0012	0.078	＞8333	Non-special
	Sangivamycin	0.312	0.039	0.195	0.078	0.078	0.078	0.078	4	＞5	0.312	0.0048	0.0012	0.0048	0.0012	0.078	＞8333	Non-special	Non-special
NSC146109	Sangivamycin	0.312	0.039	0.195	0.078	0.078	0.078	0.078	4	＞5	5	2.5	2.5	5	2.5	5	＞4	Non-special	Non-special
NSC146109	Bouvardin	0.312	0.078	0.0195	0.078	0.078	0.0195	0.156	4	＞5	5	2.5	2.5	5	2.5	5	＞4	Non-special	Non-special
Mitoxantrone	Bouvardin	0.312	0.078	0.0195	0.078	0.078	0.0195	0.156	4	5	1.25	0.312	0.312	1.25	0.312	1.25	16	TERT/RB	Non-special
Mitoxantrone	Zorubicin	＞5	1.25	0.312	1.25	1.25	1.25	1.25	＞8	5	1.25	0.312	0.312	1.25	0.312	1.25	16	TERT/RB	TERT/RB
Daunorubicin	Zorubicin	＞5	1.25	0.312	1.25	1.25	1.25	1.25	＞8	5	1.25	0.312	0.312	1.25	0.625	0.625	16	TERT/RB	TERT/RB
Daunorubicin	Camptothecine	＞5	＞5	1.25	0.0195	1.25	0.0195	1.25	＞512	5	1.25	0.312	0.312	1.25	0.625	0.625	16	TERT/RB	RAS ^V12/PP2A/RB
Erastin	Camptothecine	＞5	＞5	1.25	0.0195	1.25	0.0195	1.25	＞512	＞5	＞5	＞5	1.25	＞5	1.25	2.5	＞8	RAS ^V12/PP2A	RAS ^V12/PP2A/RB

	TIP5-	TIP5-	TIP5-	TIP5-	TIP-	TIP5-	TIP5-	TIP5-
	TIP5-	TIP5-	TIP5-	TIP5-	TIP-	TIP5-	TIP5-	TIP5-		TERT	TERT/	TERT/	TERT/	TERT/	TERT/	TERT/	TERT/
		LT	LT/	LT/	E6	E6E7	E6E7/	E6E7/		TERT	TERT/	TERT/	TERT/	TERT/	TERT/	TERT/	TERT/
		LT	LT/	LT/	E6	E6E7	E6E7/	E6E7/				ST	ST/			ST	ST/
				Ras ^V12				Ras ^V12				ST	ST/			ST	ST/
				Ras ^V12				Ras ^V12	Quinomycin A	＞5	5	0.0048	0.0024	＞5	0.048	0.048	0.0048
Sangivamycin	1.25	0.312	0.039	0.078	0.156	0.078	0.078	0.078	Quinomycin A	＞5	5	0.0048	0.0024	＞5	0.048	0.048	0.0048
Sangivamycin	1.25	0.312	0.039	0.078	0.156	0.078	0.078	0.078	NSC146109	＞5	＞5	5	2.5	5	2.5	2.5	2.5
Bouvardin	＞5	0.312	0.039	0.039	0.078	0.039	0.039	0.078	NSC146109	＞5	＞5	5	2.5	5	2.5	2.5	2.5
Bouvardin	＞5	0.312	0.039	0.039	0.078	0.039	0.039	0.078	Mitoxantrone	＞5	1.25	0.625	1.25	1.25	0.625	0.625	1.25
Zorubicin	＞5	1.25	0.625	0.625	5	1.25	1.25	1.25	Mitoxantrone	＞5	1.25	0.625	1.25	1.25	0.625	0.625	1.25
Zorubicin	＞5	1.25	0.625	0.625	5	1.25	1.25	1.25	Daunorubicin	＞5	1.25	0.625	0.625	5	0.625	0.625	0.625
Camptothecine	＞5	＞5	0.156	0.156	＞5	0.625	0.156	0.156	Daunorubicin	＞5	1.25	0.625	0.625	5	0.625	0.625	0.625
Camptothecine	＞5	＞5	0.156	0.156	＞5	0.625	0.156	0.156	Erastin	＞5	＞5	＞5	＞5	＞5	＞5	＞5	5

Table 2 shows the effectiveness of tumor-selective compound in through engineering approaches clone.Tabular goes out in each clone each compound to the painted inhibition of fluorexon (negative % value) or strengthen (positive % value) (IC ₅₀).

Table 2

In the RJELR cell in the BJEH cell

Average inhibition/enhancing % on average suppresses/strengthens

％

Molecule

The evaluation and the sign of embodiment 4 Erastin binding partners

With the erastin binding partners in the Pull-down test identification of cell, fixed erastin and product of cell lysis are used in described Pull-down test at first.Initial pull-down experiment is carried out with HEK293, BJEH and the full product of cell lysis of BJELR.In these experiments, the methylamino-derivative (ERA-A6) of erastin is fixed to Affigel 10, hatches with split product under standard pull-down condition.The washing pearl is used 100 μ M erastin or 0.8% dodecanoyl sarkosine (sarkosyl) wash-out then.Elutriant is used for mass spectroscopy.

The analysis of Erastin pull-down test-results finds to identify plastosome in HEK293 or BJEH and BJELR split product first round pull-down test or the proteic frequency of occurrences of endoplasmic reticulum is higher.This shows that fenestra may be the target of erastin or its analogue.But these results do not get rid of following possibility, and promptly erastin may be in addition takes turns not certified other and/or different targets in the pull-down experiment at this.

In the pull-down test, observe several albumen repeatedly with erastin or erastin analogue.These albumen comprise VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a and Sec22b, suppose that they are targets of erastin.

Because it is quite complicated that full product of cell lysis mixture remains, those albumen only detect in the sarkosyl elutriant of pull-down experiment.This has increased the complicacy of mass spectroscopy potentially.Therefore, for simplifying mixture, some albumen that use different separation method separation or pre-concentration in full product of cell lysis pull-down experiment, to identify.

Because Prohibitin and VDAC isoform all are mitochondrial proteins, the applicant is by coming enrichment potential erastin target from the product of cell lysis separate mitochondria.Isolating plastosome extract is used for erastin pull-down experiment then.Utilize in the erastin pull-down experiment of plastosome extract at those, Prohibitin, VDAC and ribophorin are also identified by Western blot.Figure 14 shows the Western blot of pull-down, and wherein activity (A6) and inactive (B1) erastin derivative on the pearl is fixed in the contact of plastosome extract.The Pull-down experiment is carried out for the 0.25mg total protein with the plastosome extract.Pearl was hatched 1.5 hours with extract at 4 ℃, washed then several times.With fixed erastin derivative bonded albumen with 50 μ L, 0.8% dodecanoyl sarkosine eluant solution.Western blot identifies albumen, described western blot use anti--ribophorin ,-Sec6 ,-mixing of Prohibitin and anti--VDAC antibody.Albumen is also identified by mass spectroscopy.

Ribophorin and Prohibitin are acidic proteins, and the PI value of calculating is respectively 5.57 (Prohibitin) and 5.96 (ribophorin I).The applicant utilizes the MonoQ post by ion exchange chromatography the stronger albumen (VDAC isoform, Sec22 and Sec61a) of these two albumen and alkalescence to be separated under the pH6.8 condition.Use Prohibitin or ribophorin content in these fractions of antibody test then.Also detect these fractions they with comprise the BIACORE of fixed ERA-A6 and ERA-B1 compound ^TMThe combination on surface.At BIACORE ^TMShow in the experiment in bonded level part and found Prohibitin and ribophorin.Be enjoyably, the 45kDa albumen of observing a kind of the unknown dyes in the SDS-PAGE gel at silver and combines with ERA-A6 or ERA-B1 pearl, described agnoprotein not with the antibody response of any use.

For confirming these discoveries, ERA-A6 and ERA-B1 are carried out the pull-down experiment from MonoQ elutriant level part.Prohibitin and ribophorin are confirmed as once more in conjunction with the Erastin pearl from several grades of parts.Following viewpoint is clearly supported in these experiments, and promptly VDAC isoform and Prohibitin and ribophorin are in conjunction with erastin and erastin analogue.Therefore these albumen all are potential target/binding partners of erastin in vivo.

The expression level of embodiment 5 different VDAC isoforms

When utilizing the BJELR split product to carry out pull-down, with the split product comparison from the BJEH cell, the pull-down of the success of the ERA-A6 analogue of erastin produces higher VDAC3 all the time based on mass spectral evaluation value.When considering comparable total protein concentration, the value of VDAC isoform is high more, and consistent is that these isoforms are higher with respect to the abundance in the BJEH split product at the BJELR split product with it.The different level of target proteins may influence the selectivity of Erastin.

For this point is described, the applicant uses quantitative PCR (Q-PCR) to detect relative expression's level of different VDAC isoforms.Other available methods comprise Western blot and mass spectrometry.

Carry out the relative quantity (as the substitute marker of genetic expression) of different genes mRNA in quantitative PCR (Q-PCR) experiment detection " normally " BJEH clone and the tumorigenesis BJELR system.To each VDAC isoform (VDAC1,2 and 3), two zones of mRNA are used to amplification.These zones are called 1 and 1-2,2-1 and 2-2, and 3-1 and 3-2.The Q-PCR signal of the mRNA fragment amplification of each gene of interest and a series of confidential reference items relatively make a drawing to scale with respect to GAPDH mRNA deutero-signal in the target cell.The result shows shown in Figure 11, and the expression of VDAC3 is with respect to raising significantly in the BJEH cell in the BJELR cell.This finds that the result who observes with other several genes is opposite, is suppressed to described gene with respect to the BJEH cell observation in the BJELR cell.

Identical Q-PCR data are made Figure 12 among use Figure 11, but Figure 12 focuses on relative expression's level of VDAC isoform in the target cell.The segmental Q-PCR signal of each mRNA that increases and a series of confidential reference items relatively are expressed as the signal with respect to VDAC1 mRNA in the target cell, and this is defined as 100%.As shown in figure 11, two amplification region of each VDAC isoform (VDAC1,2 and 3) mRNA are called 1,1-2,2-1,2-2,3-1 and 3-2.The result shows the high 2-2.5 of expression ratio in the BJEH cell times of VDAC3 in the BJELR cell.

To sum up, these find to point out explanation BJELR cell erastin to be handled a kind of possibility mechanism of different susceptibility.

Embodiment 6 Erastin are to the functional evaluation of different VDAC isoforms

Function test helps to confirm that the albumen of identifying is the function target of erastin.In certain embodiments, isolating plastosome can be used to judge whether erastin has effect or phenotype effect to mitochondrial function.For example, the phenotype effect can be by microscopic examination, when the detection line mitochondrial membrane potential changes, perhaps by using some dyestuff to observe the release that erastin handles the back active oxygen, this known in the art be to be used for detection of active oxygen (ROS).

In some other embodiment, confirm that experiment can comprise with azido--erastin derivative, perhaps erastin analogue or be coupled to bidentate chelate affinity labelling linking agent (for example SBED) or the derivative of the linking agent that can cut comes the photoaffinity labeling target proteins.

Still in other embodiments, recombinating and crossing expressed proteins to be used for some in vitro tests, is used to evaluate any possible effect that erastin has its function.This in vitro tests can include, but are not limited to: directly in conjunction with (external or BIACORE ^TM), or can detect the test that effluxes of VDAC isoform channel properties.

Still in other embodiments, can use the knockout mutant strain (cell or organism) of those target proteinses.With wild-type relatively, these mutant strains can become erastin is had resistance or super quick.Those knock out clone also can be used for high flux screening (HTS) to detect and/or to estimate the specificity of erastin or its analogue.

Still in other embodiments, can also test any phenotype (for example, erastin resistance or hypersensitivity) of evaluating after erastin handles with the RNAi of VDACs, Prohibitin and ribophorin.According to this embodiment of the present invention, respectively with VDAC1, VDAC2 and VDAC3 SMARTPOOL as target ^SiRNAs available from Dharmacon (Lafayette, CO).Optimize transfection conditions then, for example, use FUGENE TM and oligofectamine and fluorescently-labeled siRNA duplex at 384 orifice plates.This program causes～90% transfection efficiency.The ELR tumour cell can detect the dose-response to erastin with the siRNAs transfection of anti-VDAC1, VDAC2 or VDAC3 then.

The inhibition of embodiment 7 cells growth

Detection compound suppresses the ability of BJELR and the growth of BJEH cell.Compound is analyzed by the Sytox primary dcreening operation, and this is the phenotypic assay of a kind of monitoring as compound treatment result's cell survival-propagation change.It is designed to identify the high throughput method of the compound of special change cell growth potential, and described cell has the pathogenic sudden change of finding in the cancer patients, and this sudden change does not influence Normocellular growth.This test depends on the reading of the impermeable fluorescence dye of a kind of cheap, simple and reliable film (Sytox is from Molecular Probes), described fluorescence dye bind nucleic acid.In healthy cell, do not detect signal, because cytolemma is complete, dyestuff can not enter.But,, will detect and be subjected to the similar proportional fluorescent signal of number that influences cell if cytolemma is as apoptosis or downright bad result and impaired.Utilize two step readings (final reading under the stain remover existence condition is with the mark all cells), this test can be identified and produce cytostasis, cytotoxicity and/or mitogenetic compound.For the first time reading or " dead cell " reading, number dead or dying cell is assessed the toxicity of given compound in the culture when showing test.For the second time reading or " total cell " reading obtain cytotoxicity in the storage effect that reduces aspect the cell mass size, and during nontoxicity testing compound to the inhibition cell or the anti-proliferative effect of the arbitrary cell of cell mass to be measured.

Be the screening purpose, previously described BJ-TERT is called " normally " with reference to clone, BJ-TERT/LT/ST/RAS ^V12Cell is a tumorigenic cell system.The cell kind is spent the night in 96 orifice plates, and planting density allows in 72 hours metapores 95% to merge under the situation of being untreated.Several days subsequently, cell was accepted the testing compound of serial dilution and was handled 48 hours.After this section incubation period, add Sytox reagent to culture by manufacturer's recommended density, read dead cell fluorescence reading.After finishing these measurements, add the stain remover Saponin/TSM and film is changed processing thoroughly to each hole of culture, to allow Sytox reagent, stay total cellular score in the culture thereby be convenient to measure to entering each cell.

Synthesizing of embodiment 8 3-(2-phenelyl)-2-(piperazine-1-ylmethyl) quinazolines-4 (3H)-ketone (compound 5)

Step 1:2-(chloromethyl)-4H-benzo [the d] [preparation of 1,3] oxazine-4-ketone (compound 2)

Method 1:

In nitrogen, (compound 1 15.3g) is dissolved in 300mL methylene dichloride (CH to anthranilic acid ₂Cl ₂).Add triethylamine (TEA, 1.1 equivalents) then, cooling mixture in ice bath.Drip the dichloromethane solution (150mL) of chloroacetyl chloride (1.1 equivalent), be warmed to the stirring at room mixture two hours.(after interpolation, can remove ice bath or mixture and can be warmed to room temperature above two hours.) by the filtering separation solid, usefulness cold water washing (2 *) is the hexane solution washing of 5% diethyl ether (Et2O) then, and is air-dry, obtains the white powder solid (22.5g, quantitative yield) of compound 2.The feature of the finished product is LC/MS m/z MH+196.13; Purity＞95%; 1HNMR.

Method 2 dimethyl formamides (DMF) are used as reaction solvent:

10 gram anthranilic acids are dissolved in 300mL DMF.Add TEA (1.5 equivalent) then, cooling mixture in ice/water-bath.The DMF solution (100mL) of chloroacetyl chloride (1.3 equivalent) is added dropwise in the refrigerative reaction mixture.Remove ice/water-bath, reaction mixture stirred 2 hours.Reaction mixture injects frozen water (200-300mL), extracts with ethyl acetate (EtOAc, 3 * extraction).Merge organic layer, water and salt water washing are with sodium sulfate (Na ₂SO ₄) drying.Concentrate and produce solid, this solid and 100mL 10%Et ₂The O/ hexane grinds, and obtains the white powder (12g of compound 2; 85% yield).The finished product characterize with LC/MS.

Step 2:2-(chloromethyl)-3-(2-phenelyl) quinazolinone-4 (3H)-ketone (compound 3) Preparation

Method 1 is used trichlorine phosphine (PCl ₃):

In nitrogen, compound 2 (8.8g) is dissolved in 440mL acetonitrile (CH ₃CN), add 2-phenetidine (1.5 equivalent), stir.In these well-beaten reaction mixtures, drip PCl ₃(2 equivalent).The slurry that obtains be heated to 50 ℃ 6-12 hour.Reaction mixture injects saturated Na ₂CO ₃In/ice the mixture, stirred 30 minutes, (3 * 300mL) extract with EtOAc.The organic layer that merges is with (minimum) water and salt water washing, with sodium sulfate (Na ₂SO ₄) drying.Concentrated solution, thick solid/oil mixt and 3%Et ₂(2 * 100mL) grind the O/ hexane, to remove the overwhelming majority in the unreacted amino phenyl ethyl ether.The solid filtering that obtains separates, and crosses silicagel column (20%EtOAc/ hexane) purifying then.Isolated compound 3 be white powder (11g, 75-80%).The feature of the finished product is LC/MS m/z MH+315; Purity＞98%.

Method 2 is used phosphinylidyne trichlorine (POCl ₃):

In nitrogen, compound 2 (1.2g; 6.0mmol; 1.0 equivalent) and 2-phenetidine (1.2mL; 9.0mmol, 1.5 equivalents) and be dissolved in 30mL CH ₃CN.Drip POCl to this solution ₃(1.1mL, 12mmol, 2.0 equivalents), mixture reflux 3 hours.Reactant is cooled to room temperature, injects ice/saturated NaHCO ₃In the slurry, (3 * 200mL) extract with EtOAc.Na is used in the organic layer water and the salt water washing that merge ₂SO ₄Dry.With LC/MS crude product mixture is analyzed, confirmed the existence of required product compound 3 (97%) and minor by-products compound 3c (2-3%).MH+m/z among the LC/MS (333/334/335) is consistent with the diamide structure of compound 3c.Usually the required product of chromatogram chromatographic separation (described in the method 1 of preceding step 2) on silica gel, the compound 3 that obtains is white powder (1.3g, 80-85% yield).

Step 3:3-(2-phenelyl)-2-(piperazine-1-ylmethyl) quinoline azoles-4 (3H)-ketone compound 5) preparation

Method 1:

In nitrogen, compound 3 (5g) is dissolved in CH ₃CN (0.08-0.2M compound concentration) is to wherein adding salt of wormwood (K successively ₂CO ₃, 1.2 equivalents, commercialization powder), piperazine (2 equivalent) and tetrabutylammonium iodide (0.2 equivalent).Mixture was 60 ℃ (bath temperature) heating 8-10 hour.Reaction can be undertaken by any in the following two lines: (a) evaporate 80% solvent, add water (20mL), (4 * 60mL) extract mixture with EtOAc; Perhaps (b) reaction mixture is with 400mL EtOAc dilution, water (3 * 20mL) washings.The organic layer salt water washing that merges is condensed into yellowish oil.In upward using, silica (10-25% ethanol/methylene) press the compound 5 that obtains behind chromatography (CombiFlash ) purifying to be white powder (80-85% yield).The feature of product is 1HNMR and LC/MS MH+365.

Step 4: tertiary butyl 4-(3-2-phenelyl)-4-oxo-3,4-dihydroquinazoline-2-yl) Methyl) preparation of piperazine-1-carboxylicesters (compound 4)

Method 1:

In nitrogen, compound 3 (4.0g, 12.7mmol, 1.0 equivalents) is dissolved in 60mLCH ₃CN adds K successively in this solid sample solution ₂CO ₃(2.1g, 15mmol, 1.2 equivalents), the solid sample of tertbutyloxycarbonyl-piperazine (4.73g, 25mmol, 2.0 equivalents) and sodium iodide (NaI, 570mg, 3mmol, 0.3 equivalent).Mixture (suspension) was 80 ℃ of heating 3-6 hour.Obtain white suspension.Utilize TLC and LC/MS detection reaction, show that compound 3 is converted into compound 4 fully.Remove about 30mL CH by distillation under the reduced pressure ₃CN adds 60mL water in the slurry that obtains, (4 * 60mL) extract mixture with EtOAc.The organic layer water that merges, ammonium chloride saturated solution (removing unreacted tertbutyloxycarbonyl-piperazine), NaHCO ₃Na is used in saturated solution and salt water washing ₂The SO4 drying.Solid and hexane grind, and concentrating after filtration becomes white solid (5.6g, volume production).This material need not to be further purified and just can be used for follow-up deprotection steps.The feature of final compound is by 1HNMR, and LCMS describes.

Step 5:3-(2-phenelyl)-2-(piperazine-ylmethyl) quinazoline-4 (3H)-ketone (compound 5) preparation

Method 1:

Compound 4 under the room temperature (2.7g, 5.8mmol, 1.0 equivalents) is suspended in the no Shui diox of 15mL.Drip 4N HCl/ dioxane solution (17mL, 12 equivalents) under the room temperature; React and add 17mL 4N HCl/ diox after 30 minutes again, with LC/MS monitoring reaction progress.(this is a kind of thermopositive reaction, observes gas evolution.Reaction system must be open, with relief pressure, keeps anhydrous condition simultaneously.If) the unreacted compound 4 of remaining any amount, can add 8-10mL 4N HCl/ diox again and finish to drive reaction.40mL water and CH are added in reaction at last respectively ₂Cl ₂In reactant, by adding capacity saturated aqueous Na ₂CO ₃It is alkalescence that solution is regulated mixture, reaches pH8-9.Layer is separated, water layer CH ₂Cl ₂(3 * 60mL) extract.Merge organic layer, water (4 * 10mL is approximately neutrality up to the pH of water extract) and salt water washing are at Na ₂SO ₄Middle dry.Concentrate to produce yellowish oil, this oil silica (10-25% ethanol/methylene) go up with in press chromatography (CombiFlash ) purifying, grinding the compound 5 that the back produces with the hexane that comprises the 5-10% diethyl ether is white powder (85-95% yield).The feature of compound 5 is described by 1H NMR and LC/MS.

Conventional: analytical procedure

Following LC condition is used for analysis of compounds 5:

Post:Be used for mass spectral XTerra ; C18,3.5 μ m

Size:2.1 * 150mm

Gradient:75%CH ₃CN (comprising 0.08% formic acid)/25% water (comprising 0.1% formic acid)-90%CH ₃CN (comprising 0.08% formic acid)/10% water (comprising 0.1% formic acid)

All compounds carry out LC/MS with following colonnade and mobile phase condition and analyze:

Post:Be used for mass spectral XTerra ; C18,3.5 μ m

Size:2.1 * 150mm

Gradient	Time	Water/0.1%FA	CH3CN/0.08％FA
Gradient	Time	Water/0.1%FA	CH3CN/0.08％FA	0:00	95	5
7:00	5	95		0:00	95	5
7:00	5	95	8:00	5	95
9:00	95	5	8:00	5	95
9:00	95	5	13:00	95	5

Embodiment 9 Sytox primary dcreening operations

The Sytox primary dcreening operation is a kind of phenotypic assay, and monitoring is as the change of compound treatment result's cell survival-propagation.It is designed to identify the high throughput method of the compound of special change cell growth potential, and described cell has the disease cause mutation of finding in the cancer patients, and this sudden change does not influence Normocellular growth.

This test depends on cheap, the simple and reliable reading of the impermeable fluorescence dye of a kind of film (Sytox is from MolecularProbes), described fluorescence dye bind nucleic acid.In healthy cell, do not detect signal, because cytolemma is complete, dyestuff can not enter.But,, will detect and be subjected to the similar proportional fluorescent signal of number that influences cell if cytolemma is as apoptosis or downright bad result and impaired.Utilize two step readings (final reading under the stain remover existence condition is with the mark all cells), this test can be identified and produce cytostasis, cytotoxicity and/or mitogenetic compound.For the first time reading or " dead cell " reading, number dead or dying cell is assessed the toxicity of given compound in the culture when showing test.For the second time reading or " total cell " reading obtain the storage effect that cytotoxicity reduces the cell mass size, and testing compound suppresses or anti-proliferative effect the arbitrary cell of cell mass to be measured during nontoxicity.

Be the screening purpose, previously described BJ-TERT is called " normally " with reference to clone, and the BJ-TERT/LT/ST/RASV12 cell is a tumorigenic cell system.The cell kind is spent the night in 96 orifice plates, and planting density allows in 72 hours metapores 95% to merge under the situation of being untreated.Several days subsequently, cell was accepted the testing compound of serial dilution and was handled 48 hours.After this section incubation period, add Sytox reagent to culture by manufacturer's recommended density, read dead cell fluorescence reading.After finishing these measurements, add the stain remover Saponin/TSM and film is changed processing thoroughly to each hole of culture, to allow Sytox reagent, stay the cells in culture sum thereby be convenient to measure to entering each cell.

Be data evaluation, we do not distinguish the showed cell poison or suppress the compound of cytological effect.Detect for being used for other, compound must satisfy the standard of two strictnesses:

I. dead cell or total cell reading (perhaps both) change the signal that tumor cell line produces 2 standard deviation sizes at least

Ii. the signal that " normally " control cells is produced less than 1 standard deviation size changes.

Referring to table 3 and 4 and Figure 15-17 corresponding to the vitro data of The compounds of this invention.

Compound number	IC ₅₀ in BJELR(□M)
Compound number	IC ₅₀ in BJELR(□M)	5	＜0.100
6	≥1.000	5	＜0.100
6	≥1.000	7	≥0.100
8	≥1.000	7	≥0.100
8	≥1.000	9	≥1.000
14	≥0.100	9	≥1.000
14	≥0.100	15	≥1.000
10	≥1.000	15	≥1.000
10	≥1.000	11	≥0.100
16	≥0.100	11	≥0.100
16	≥0.100	17	≥0.100
12	＜0.100	17	≥0.100
12	＜0.100	13	＜0.100

The clone title	Cell type	VDAC2 &3	The Ras state	Compound 5:IC50 (nM)
The clone title	Cell type	VDAC2 &3	The Ras state	Compound 5:IC50 (nM)	MCF7	Mammary gland	+	The activatory path	≥1000
BT-549	Mammary gland	+	The activatory path	≥100	MCF7	Mammary gland	+	The activatory path	≥1000
BT-549	Mammary gland	+	The activatory path	≥100	COLO205	Colon	+	B-raf V600E mut，ras wt
SW-620	Colon	+	K-ras G12V sudden change, wt B-raf		COLO205	Colon	+	B-raf V600E mut，ras wt
SW-620	Colon	+	K-ras G12V sudden change, wt B-raf		DLD-1	Colon		K-ras G13D sudden change, wtB-raf, (the p53 antigen of generation has c-＞T sudden change to the p53 antigen presentation positive, causes 241 Ser-＞Phe)	≥100
LS174T	Colon		K-ras2 G12D sudden change		DLD-1	Colon			≥100
LS174T	Colon		K-ras2 G12D sudden change		HCT116	Colon		K-ras G12D	≥100
HT1080	fibrosarcoma		The n-ras sudden change	＜100	HCT116	Colon		K-ras G12D	≥100
HT1080	fibrosarcoma		The n-ras sudden change	＜100	MALME-3M	Melanoma	+	B-rafV600E mut，ras wt	≥100
SK-MEL-28	Melanoma	+	B-rafV600E mut，ras wt	≥100	MALME-3M	Melanoma	+	B-rafV600E mut，ras wt	≥100
SK-MEL-28	Melanoma	+	B-rafV600E mut，ras wt	≥100	SK-MEL-5	Melanoma	+	The B-rafV600E heterozygous mutation	≥100
A375	Melanoma		Wtras, composition activatory MEK1		SK-MEL-5	Melanoma	+	The B-rafV600E heterozygous mutation	≥100
A375	Melanoma		Wtras, composition activatory MEK1		HOP-62	Non-small cell lung	+	K-ras G12V	≥100
HOP-92	Non-small cell lung	+	Wtras	≥1000	HOP-62	Non-small cell lung	+	K-ras G12V	≥100
HOP-92	Non-small cell lung	+	Wtras	≥1000	NCI-H322M	Non-small cell lung	+
OVCAR-3	Ovary	+	The K-ras amplification	≥100	NCI-H322M	Non-small cell lung	+
OVCAR-3	Ovary	+	The K-ras amplification	≥100	SK-OV-3	Ovary	+		≥100
OVCAR-5	Ovary	+	K-ras G12V	＜100	SK-OV-3	Ovary	+		≥100
OVCAR-5	Ovary	+	K-ras G12V	＜100	DU-145	Prostate gland	+	Wtras	≥100
PC-3	Prostate gland	+		≥1000	DU-145	Prostate gland	+	Wtras	≥100
PC-3	Prostate gland	+		≥1000	BJEH	Inoblast, former generation	+	normal
BJELR	Inoblast, former generation	+	Ras V12 sudden change and normal	＜100	BJEH	Inoblast, former generation	+	normal
BJELR	Inoblast, former generation	+	Ras V12 sudden change and normal	＜100	PANC-1	Pancreas		K-ras V12 sudden change	＜100
MIA-PaCa-2	Pancreas		K-ras suddenly change (G12C)	≥1000	PANC-1	Pancreas		K-ras V12 sudden change	＜100

B:PRLX compound 6@50mg/Kg, QD * 5 day, IP, n=8

C:PRLX compound 5@100mg/Kg, QD * 5 day, IP, n=8

D:PRLX compound 5@50mg/Kg, QD * 5 day, IP, n=8

E: media contrast, QD * 5 day, IP, n=8

F: not treatment contrast, n=8

Treatment plan:

When mean tumour volume reaches～200mm ³The time, continuing to the 5th day, every animal single IP of administration every day is injected a kind of in the above-mentioned treatment, 5 dosage altogether.

Tumour transplatation and by stages:

70 every of mouse all transplant 1 * 10 ⁷Individual HT-1080 cell is by injecting the 0.1cc inoculum at right hind side SC.Use 25G * 5/8 pin number.Use HT-1080 cell (ATCC separates, the 6th the frozen thing that goes down to posterity) preparation tumor cell inoculation thing, described HT-1080 cell is at DMEM[Gibco, No.10569-010]+10%FCS[Gibco, No.F-2442] in cultivate.When cell harvesting, cell grows into the degrees of fusion of 95-100%.The HT-1080 inoculum prepares in aseptic DMEM substratum+10%FCS, density 1.0 * 10 ⁸Cell/ml.After tumour transplatation+9 days, animal grouping coupling is treatment and control group, is made up of 8 mouse for every group.22 outliers are rejected from experiment altogether, because tumour is too little or too big.This is considered to test the 1st day, and treatment is from this day.

The preparation of injection liquid:

Following injection liquid during 5 days of compound administration every day prepared fresh:

100mg/kg dosage (group A and C)

At first, the liquid storage for preparing each compound by dissolving 35mg PRLX compound 6 or PRLX compound 5 in 0.35ml solvent (0.25%Tween-80,0.1% phenylcarbinol and 350mM acetate).Then by (100mM potassium phosphate buffer and 32mM sucrose pH6.8) mix to come and dilute the liquid storage that obtains at 1: 10 and prepare final injection liquid with each liquid storage and 3.15ml thinner.Solution is filter sterilization (0.45 μ m film) then.The solution that obtains comprises PRLX compound 6 or PRLX compound 5, final concentration 10.0mg/ml, pH=6.5.

50mg/kg dosage (group B and D)

To two kinds of compounds, by adding 1.0ml thinner (100mM potassium phosphate buffer and 32mM sucrose, pH 6.8), the 10mg/ml injection liquid (as mentioned above) of 1: 2 dilution 1.0ml.The final concentration that the solution that obtains comprises PRLX compound 6 or PRLX compound 5 is 5.0mg/ml pH=6.5.

Media contrast (group E)

Use then thinner (100mM potassiumphosphate buffer carrier contrast and 32mM sucrose, pH6.8) 1: 10 diluting solvent (0.25%Tween-80,0.1% phenylcarbinol and 350mM acetate) prepare media and contrast.The ultimate constituent of media contrast is included in the 0.025%Tween-80 in 100mM potassiumphosphate and the 32mM sucrose, 0.01% phenylcarbinol and 35mM HOAc, final pH=6.8.

The dosage general introduction:

Experimental group	Treatment	The concentration of injection solution	The quantity of given compound (mg)	The volume of given injection solution
Experimental group	Treatment	The concentration of injection solution	The quantity of given compound (mg)	The volume of given injection solution	A	PRLX compound 6@100	10.0mg/ml	2.0	0.2ml
B	PRLX compound 6@50	5.0mg/ml	1.0	0.2ml	A	PRLX compound 6@100	10.0mg/ml	2.0	0.2ml
B	PRLX compound 6@50	5.0mg/ml	1.0	0.2ml	C	PRLX compound 5@100	10.0mg/ml	2.0	0.2ml
D	PRLX compound 5@50	5.0mg/ml	1.0	0.2ml	C	PRLX compound 5@100	10.0mg/ml	2.0	0.2ml
D	PRLX compound 5@50	5.0mg/ml	1.0	0.2ml	E	The media contrast	0mg/ml	Has only media	0.2ml
F	Not treatment contrast	0	0	0	E	The media contrast	0mg/ml	Has only media	0.2ml

Measurement of tumor:

Since the 1st day, all animals of every other day weighing was measured tumor size (length (L) and width (W)).Use following formula that the measurement of tumor result is converted to gross tumor volume (mm ³):

Gross tumor volume=L * W * W/2.

Calculate the mean value of the gross tumor volume that each experimental group of each time point obtains, the time is mapped.Difference table be shown the mean value standard deviation (± SEM).

Figure 18-20 shows these result of experiment.

Embodiment 11 PANC-1 oncotherapies research: the anti-tumor activity evaluation of PRLX compound 6 and PRLX compound 5

Preparation of PANC-1 heterograft and transplanting

The experimental plan of PANC-1 research is basic identical with listed HT-1080 research in the foregoing description 10, except following difference: the go down to posterity right side of the subcutaneous implantation immune deficiency of tumor tissues fragment nude mice of about 30-40mg PANC-1.Monitor tumor growth every day, when tumour reaches about 100mm ³The time, the animal that has similar big or small tumour is grouped coupling, the administration of beginning compound dosage.Give drug compound 5 once continuous 5 day every day by following listed metering.In the PANC-1 heterograft, gemcitabine is with the administration of model maximum tolerated dose, with comparing.The gemcitabine therapy is to carry out administration 180mg/kg every day 3 times in per 3 days in 9 days.

Figure 21-22 shows these result of experiment.

Incorporated by reference

The whole publications herein mentioned and patent be this complete being incorporated herein by reference, just as each independent publication or patent are special and are introduced separately into as a reference.Under conflict situations, will comprise any definition herein with the application, be as the criterion.

Equivalent

When particular of the present invention was discussed, above-mentioned specification sheets was an illustrative and unrestriced.Many variations of the present invention are apparent to those skilled in the art when knowing this specification sheets and following claim.Four corner of the present invention should decide with reference to the four corner of claim and equivalent and specification sheets and this variation.

Claims

1. a sanatory method in Mammals comprises the compound with formula I structure or its pharmacologically acceptable salts to Mammals drug treatment significant quantity,

Wherein:

R ¹Be selected from H, Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl and C _1-4Aralkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴On identical nitrogen-atoms and when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

With

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl,

Wherein the characteristics of illness are that cell has the activity that enhanced Ras signal is active and the antigenic cell target albumen of the little t of SV40 changes.

2. method according to claim 1, wherein the characteristics of illness also are the Rb activity of wild-type level basically.

3. method according to claim 1, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

4. method according to claim 1, wherein aryl is optional with being selected from C _1-6Alkyl, CF ₃, hydroxyl, C _1-4Alkoxyl group, aryl, aryloxy, halogen, NR ²R ⁴, nitro, carboxylic acid, the group of carboxylicesters and alkylsulfonyl replaces.

5. a sanatory method in Mammals comprises the compound with formula I structure or its pharmacologically acceptable salts to Mammals drug treatment significant quantity,

Wherein:

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴On identical nitrogen-atoms and when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R ²And R ⁴One of be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

Q is selected from O and NR ²With

6. method according to claim 5, wherein the characteristics of illness also are the Rb activity of wild-type level basically.

7. method according to claim 5, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

8. method according to claim 7, wherein R ⁴Be selected from C _1-4Aralkyl and acyl group.

9. method according to claim 8, wherein R ⁴It is acyl group.

10. method according to claim 9, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from H, alkyl, alkoxyl group, aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

11. method according to claim 9, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

12. method according to claim 9, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy and heteroaryloxy.

13. according to arbitrary described method among the claim 1-12, wherein said compound passes through acellular apoptosis mechanism killer cell in Mammals.

14. method according to claim 13, wherein said cell have enhanced Ras signal activity.

15. method according to claim 13, wherein said cell are crossed the little t antigen of expression SV40.

16. method according to claim 13, wherein said cell have the Phosphoric acid esterase PP2A activity that essence reduces.

17. method according to claim 13, wherein said cell is crossed expression VDAC.

18. according to arbitrary described method among the claim 1-12, wherein said illness is a cancer.

19. method according to claim 13, wherein said cell is by the little t antigen of abduction delivering SV40.

20. method according to claim 19, wherein by using the described cell of the antigenic viral vector infection of the little t of expression SV40, described cell is by the little t antigen of abduction delivering SV40.

21. method according to claim 20, wherein said virus vector are retroviral vector or adenovirus carrier.

22., also comprise to a kind of reagent of described Mammals Combined Preparation by apoptosis mechanism killer cell according to arbitrary described method among the claim 1-12.

23. method according to claim 22, wherein said reagent is chemotherapeutics.

24. method according to claim 23, wherein said chemotherapeutics is selected from: epidermal growth factor receptor antagonists, arsenic sulfide, Zorubicin, cis-platinum, carboplatin, Cimitidine Type A/AB, carminomycin, mustargen hydrochloric acid, pentamethylmelamine, plug is for group, Vumon, endoxan, Chlorambucil, de-methoxy Hypocrellin A A, alkeran, ifosfamide, trofosfamide, Treosulfan, podophyllinic acid lactone or podophyllinic acid lactone derivative, Zuyeyidal phosphoric acid salt, Vumon, Zuyeyidal, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, megestrol, methotrexate, ametycin, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, the 1-methyl-4-phenylpyridinium ion, semustine, staurosporine, U-9889, phthalocyanine, Dacarbazine, aminopterin, methotrexate, Trimetrexate, Tioguanine, purinethol, fludarabine, pentastatin, 2-chlorodeoxyadenosine, cytosine arabinoside (ara C), methylmitomycin, 5 FU 5 fluorouracil, Ismipur, the Zorubicin hydrochloride, formyl tetrahydrofolic acid, mycophenolic acid, daunorubicin, desferrioxamine, floxuridine, doxifluridine, Raltitrexed, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, bleomycin vitriol, dactinomycin, Safranin, saframycins, quinocarcins, discodermolides, vincristine(VCR), vincaleucoblastine, preparing vinorelbine tartrate, vertoporfin, taxol, tamoxifen, raloxifene, tiazofurine, Tioguanine, virazole, EICAR, Emcyt, estramustine phosphate sodium, Drogenil, bicalutamide, buserelin, Leuprolide, pteridine, enediyne, L-tetramisole, aflacon, Interferon, rabbit, interleukin, rIL-2, filgrastim, sargramostim, Rituximab, bacille Calmette-Guerin vaccine, vitamin A acid, Betamethasone Valerate, the gemcitabine hydrochloride, verapamil, VP-16, hexamethyl melamine, the t thapsigargin, oxaliplatin, iproplatin, four platinum, Lip river platinum, DCP, PLD-147, JM118, JM216, JM335, husky platinum, the Japanese yew terpene, deoxy taxol, TL-139,5 '-fall-F 81097 (hereinafter: 5 ' vincaleucoblastine falls), camptothecine, Rinotecan (Camptosar, CPT-11), Hycamtin (Hycamptin), BAY 38-3441,9-nitrocamptothecin (Orethecin, rubitecan), exatecan (DX-8951), lurtotecan (GI-147211C), gimatecan, homocamptothecins diflomotecan (BN-80915) and 9-aminocamptothecin (IDEC-13 '), SN-38, ST1481, karanitecin (BNP1350), benzazole and carbazole (for example NB-506), protoberberine, intoplicine, idenoisoquinolones, phenonaphthazine and NB-506.

25. a killer cell, the method for promotion necrocytosis or inhibition cell proliferation comprises the pair cell administration:

(1) compound with formula I structure or its pharmacologically acceptable salts of significant quantity,

Wherein:

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from With

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl and

(2) a kind of reagent that increases VDAC abundance in the cell.

26. method according to claim 25, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

27. method according to claim 25, wherein aryl is optional with being selected from C _1-6Alkyl, CF ₃, hydroxyl, C _1-4Alkoxyl group, aryl, aryloxy, halogen, NR ²R ⁴, nitro, carboxylic acid, the group of carboxylicesters and alkylsulfonyl replaces.

28. method according to claim 25, wherein VDAC is VDAC1, VDAC2 or VDAC3.

29. a killer cell, the method for promotion necrocytosis or inhibition cell proliferation comprises the pair cell administration:

Wherein:

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen-atoms with when being not hydrogen simultaneously, they are different, and work as R ²And R ⁴On identical nitrogen and R ²And R ⁴One of be acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

Q is selected from O and NR ²With

(2) a kind of reagent that increases VDAC abundance in the cell.

30. method according to claim 29, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

31. method according to claim 30, wherein R ⁴Be selected from C _1-4Aralkyl and acyl group.

32. method according to claim 31, wherein R ⁴It is acyl group.

33. method according to claim 32, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from H, alkyl, alkoxyl group, aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

34. method according to claim 32, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

35. method according to claim 32, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy and heteroaryloxy.

36. method according to claim 29, wherein VDAC is VDAC1, VDAC2 or VDAC3.

37. according to arbitrary described method among the claim 25-35, wherein said cell is a cancer cells.

38. according to arbitrary described method among the claim 25-35, wherein said reagent comprises the polynucleotide of the VDAC that encodes.

39. according to arbitrary described method among the claim 25-35, wherein said reagent is to be fit to the VDAC albumen that cell is advanced in transhipment.

40. according to the described method of claim 39, wherein said reagent is the VDAC albumen that merges with allos internalization structural domain.

41. according to the described method of claim 39, wherein said reagent is to comprise the proteic Liposomal formulation of VDAC.

42. according to arbitrary described method among the claim 25-35, wherein said reagent strengthens or suppresses the expression of endogenous VDAC.

43. according to the described method of claim 42, wherein said reagent stimulates VDAC to express.

44. according to the described method of claim 42, wherein said reagent suppresses the function of VDAC inhibitor.

45. a killer cell, the method for promotion necrocytosis or inhibition cell proliferation comprises the pair cell administration:

Wherein:

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from With

Q is selected from O and NR ²With

(2) a kind of reagent that reduces VDAC abundance in the cell.

46. have compound or its pharmacologically acceptable salts of formula I structure,

Wherein:

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

With

Q is selected from O and NR ²With

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl.

47. according to the described compound of claim 46, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

48. according to the described compound of claim 46, wherein aryl is optional with being selected from C _1-6Alkyl, CF ₃, hydroxyl, C _1-4Alkoxyl group, aryl, aryloxy, halogen, NR ²R ⁴, nitro, carboxylic acid, the group of carboxylicesters and alkylsulfonyl replaces.

49. have compound or its pharmacologically acceptable salts of formula I structure,

Wherein:

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

Q is selected from O and NR ²With

50. according to the described compound of claim 49, wherein R ¹Be selected from Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, aryl, heteroaryl and C _1-4Aralkyl.

51. according to the described compound of claim 50, wherein R ⁴Be selected from C _1-4Aralkyl and acyl group.

52. according to the described compound of claim 51, wherein R ⁴It is acyl group.

53. according to the described compound of claim 52, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from H, alkyl, alkoxyl group, aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

54. according to the described compound of claim 52, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy, aryl, heteroaryl, heteroaryloxy and cycloalkyl.

55. according to the described compound of claim 52, wherein R ⁴Be-C (O)-C _1-3Alkyl-Y, Y are selected from aryloxy and heteroaryloxy.

56. a method of identifying candidate's anti-tumor agent comprising salmosin comprises:

A) under conditions suitable, cell is contacted with the capacity test agent; With

B) whether the detection test agent strengthens or suppresses the level of erastin protein binding mixture component or the level of the nucleic acid of the erastin protein binding mixture component of encoding.

57. according to the described method of claim 56, wherein erastin is conjugated protein is VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a or Sec22b.

58., also comprise according to the described method of claim 56:

A) test agent is contacted with tumour cell; With

B) detect the growth whether test agent suppresses tumour cell.

59. a method of identifying candidate's anti-tumor agent comprising salmosin comprises:

A) with test agent and erastin is conjugated protein or express the protein-bonded cells contacting of erastin; With

60. according to the described method of claim 59, wherein erastin is conjugated protein is VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a or Sec22b.

61., also comprise according to the described method of claim 59:

A) test agent is contacted with tumour cell; With

B) detect the growth whether test agent suppresses tumour cell.

62. according to claim 56 or 59 described methods, wherein test agent is little organic molecule.

63. according to claim 56 or 59 described methods, wherein test agent is peptide, albumen, carbohydrate or nucleic acid.

64., wherein described method is repeated in the library of different test agent according to claim 56 or 59 described methods.

65. according to the described method of claim 59, the detectable marker mark of the conjugated protein usefulness of test agent or erastin wherein.

66. according to the described method of claim 65, wherein detectable marker is a vitamin H, fluorescein, digoxin, green fluorescent protein (GFP), isotropic substance, poly Histidine, magnetic bead, perhaps glutathione s-transferase (GST).

67. a method that increases tumour cell to chemotherapeutics susceptibility comprises the compound of tumour cell with minimizing or the conjugated protein abundance of increase erastin contacted.

68. according to the described method of claim 67, wherein erastin is conjugated protein is VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a or Sec22b.

69. according to the described method of claim 67, wherein chemotherapeutics is the described compound of claim 46.

70. a method that reduces normal cell to chemotherapeutics susceptibility comprises the compound of normal cell with minimizing or the conjugated protein abundance of increase erastin contacted.

71. according to the described method of claim 70, wherein erastin is conjugated protein is VDAC1, VDAC2, VDAC3, Prohibitin, ribophorin, Sec61a or Sec22b.

72. according to the described method of claim 70, wherein chemotherapeutics is the described compound of claim 46.

73. a method of identifying the candidate therapeutic agent that suppresses unwanted cells propagation comprises:

(a) test agent is mixed with VDAC albumen or albumen composition, described albumen composition comprises at least a VDAC albumen and chooses any one kind of them or multiple other albumen;

(b) whether detect test agent in conjunction with VDAC albumen; With

(c) if test agent in conjunction with VDAC albumen, then with test agent and cells contacting, and detects the propagation whether test agent changes cell.

74. according to the described method of claim 73, wherein test agent is little organic molecule, peptide, albumen, simulating peptide, nucleic acid or antibody.

75., wherein detect combining of VDAC albumen and test agent by physical bond test according to claim 73 or 74 described methods.

76. a method that reduces the tumor growth rate comprises that administration is enough to reduce a certain amount of therapeutical agent of tumor growth rate, wherein therapeutical agent is:

(a) reagent of enhancing or inhibition VDAC protein level;

(b) reagent of enhancing or inhibition VDAC protein-active;

(c) with the protein bound reagent of VDAC;

(d) in conjunction with, regulate or in conjunction with and regulate and comprise at least a VDAC and choose any one kind of them or the reagent of multiple other proteic protein complexes;

(e) comprise the reagent of VDAC polypeptide or its functional variant; Perhaps

(f) comprise the reagent of the nucleic acid of coding VDAC polypeptide or its functional variant.

77. a method for the treatment of the cancer patients comprises a kind of therapeutical agent of administration patient, described therapeutical agent is selected from:

(a) reagent of enhancing or inhibition VDAC protein level;

(b) reagent of enhancing or inhibition VDAC protein-active;

(c) in conjunction with the proteic reagent of VDAC;

(e) comprise the reagent of VDAC polypeptide and its functional variant; With

78. according to claim 76 or 77 described methods, wherein VDAC albumen is VDAC1, VDAC2 or VDAC3.

79. according to claim 76 or 77 described methods, wherein test agent is little organic molecule, peptide, albumen, simulating peptide, nucleic acid or antibody.

80. according to claim 76 or 77 described methods, wherein reagent is mixed with preparation with a kind of pharmaceutically acceptable carrier.

81. according to claim 76 or 77 described methods, wherein said reagent by intravenously, oral, cheek, parenteral, by suck spraying, by topical application or through percutaneous drug delivery.

82. according to claim 76 or 77 described methods, wherein reagent is by the administration of topical mode.

83. according to claim 76 or 77 described methods, also comprise at least a other anti-cancer chemotherapeutic agents of administration, described chemotherapeutics and described therapeutical agent are with accumulation or synergistic mode anticancer.

84. have compound or its pharmacologically acceptable salts of formula II structure,

Wherein

Ar is the phenyl that replaces;

R ¹Be selected from H, C _1-8Alkyl ,-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl, and C _1-4Aralkyl;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴On identical nitrogen and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, aryl and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

R ⁵0-4 the substituting group of representative on its ring that links to each other.

W is

Perhaps

Q is selected from O and NR ²With

85. have compound or its pharmacologically acceptable salts of formula III structure,

Wherein

Ar is the phenyl that replaces;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴At identical nitrogen and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C ^1-8Alkyl, aryl, C ^1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

R ⁵0-4 the substituting group of representative on its adjacent ring.

W is selected from Perhaps

Q is selected from O and NR ²With

86. have compound or its pharmacologically acceptable salts of formula IV structure,

Wherein

Ar is the phenyl that replaces;

R ¹Be-C _1-8Alkyl;

W is selected from

, perhaps

Q is selected from O and NR ²

87. have compound or its pharmacologically acceptable salts of formula V structure,

Wherein

R ¹Be selected from H and-C _1-8Alkyl;

R ²Be selected from H and-C _1-8Alkyl;

R ³Be selected from halogen, alkoxyl group and-C _1-8Alkyl;

R ⁴Be selected from H, halogen, alkoxyl group and-C _1-8Alkyl;

R ⁵Be selected from H, halogen and nitro; With

N is 1 or 2.

88. a killer cell promotes necrocytosis or suppresses the method for cell proliferation, comprises the compound with formula I structure or its pharmacologically acceptable salts of pair cell effective dosage,

Wherein:

R ¹Be selected from H ,-Z-Q-Z ,-C _1-8Alkyl-N (R ²) (R ⁴) ,-C _1-8Alkyl-OR ³, 3-is to 8-unit carbocyclic ring or heterocycle, aryl, heteroaryl and C _1-4Aralkyl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

With

Q is selected from O and NR ²With

89. a killer cell promotes necrocytosis or suppresses the method for cell proliferation, comprises the compound with formula II structure or its pharmacologically acceptable salts of pair cell effective dosage,

Wherein

Ar is the phenyl that replaces;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is Perhaps

Q is selected from O and NR ²With

90. a killer cell promotes necrocytosis or suppresses the method for cell proliferation, comprises the compound with formula III structure or its pharmacologically acceptable salts of pair cell effective dosage,

Wherein

Ar is the phenyl that replaces;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴All be that hydrogen or they are different in identical nitrogen-atoms and they, and work as R ²And R ⁴At identical nitrogen and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

, perhaps

Q is selected from O and NR ²

Each Z that occurs is independently selected from C _1-6Alkyl, C _2-6Thiazolinyl and C _2-6Alkynyl; With

Wherein Ar nitrogen key adjacent position on the quinazolinone ring does not have the oxyethyl group substituting group.

91. a killer cell promotes necrocytosis or suppresses the method for cell proliferation, comprises the compound with formula IV structure or its pharmacologically acceptable salts of pair cell effective dosage,

Wherein

Ar is the phenyl that replaces;

R ¹Be-C _1-8Alkyl;

W is selected from

Perhaps

Q is selected from O and NR ²

92. a killer cell promotes necrocytosis or suppresses the method for cell proliferation, comprises the compound with formula V structure or its pharmacologically acceptable salts of pair cell effective dosage,

Wherein

R ¹Be selected from H and-C _1-8Alkyl;

R ²Be selected from H and-C _1-8Alkyl;

R ³Be selected from halogen, alkoxyl group and-C _1-8Alkyl;

R ⁴Be selected from H, halogen, alkoxyl group and-C _1-8Alkyl;

R ⁵Be selected from H, halogen and nitro; With

N is 1 or 2.

93. one kind increases tumour cell or normal cell to the method for chemotherapeutics susceptibility, comprises with described cell and claim 46 84,85,86 or 87 described compounds contacts.

94. in Mammals, treat method for cancer for one kind, comprise claim 46,84,85,86 or 87 described compounds or its pharmacologically acceptable salts to Mammals drug treatment significant quantity.

95. a killer cell, the method for promotion necrocytosis or inhibition cell proliferation comprises the pair cell administration:

(1) claim 85 of significant quantity, 85,86 or 87 described compounds or its pharmacologically acceptable salts and

(2) reagent of VDAC abundance in the increase cell.

96. a killer cell, the method for promotion necrocytosis or inhibition cell proliferation comprises the pair cell administration:

(2) reagent of VDAC abundance in the reduction cell.

97. the method for the compound of a preparation formula F representative

Comprise compound

With amine HNR ₂Reaction

Wherein,

HNR ₂Be equivalent to HW.

Ar is the phenyl that replaces;

Each R that occurs ²And R ⁴All be independently to be selected from H, C _1-4Alkyl, C _1-4If aralkyl, aryl, heteroaryl, acyl group, alkyl sulfonyl and arylsulfonyl are R ²And R ⁴In the time of on identical nitrogen, they or all be hydrogen or different, and work as R ²Or R ⁴In the time of on identical nitrogen, and R ²And R ⁴One of when being acyl group, alkyl sulfonyl or arylsulfonyl, another is selected from H, C _1-8Alkyl, aryl, C _1-4Aralkyl, and heteroaryl;

R ³Be selected from H, C _1-4Alkyl, C _1-4Aralkyl, aryl and heteroaryl;

W is selected from

Perhaps

Q is selected from O and NR ²