CN102323247A - Method for screening drug for treating or preventing cancer - Google Patents

Abstract

The present invention relates to a method for screening a drug for treating or preventing cancer. The drug comprises an effective dose of a PC4 antagonist. The PC4 antagonist is modified or nonfunctional PC4 polypeptide, or modified or nonfunctional PC4 protein. The method comprises the following two steps, wherein the two steps are sequentially performed, (1) providing a test for detecting the transcriptional level of the PC4 protein; (2) adding a candidate drug to the test, wherein the candidate drug for reducing the transcriptional level of the PC4 protein is the drug for treating or preventing the cancer. With the screened drug provided by the present invention, the purpose of treatment or prevention of the cancer through inhibiting the expression level or the function of the PC4 can be achieved.

Description

A kind of method of screening the medicine of treatment or prophylaxis of cancer

The present invention be that September 7, application number in 2008 are 2008101608911 the applying date, name is called the dividing an application of Chinese invention patent application of " method and the medicine of treatment or prophylaxis of cancer ".

Technical field

The present invention relates to treat or method and the medicine and the method for screening anticancer medicine of prophylaxis of cancer.

Background technology

It is a kind of single-stranded DNA binding protein that the people transcribes positive co-factor 4 (PC4 also claims the homology analog of p14, p15, Sub1 etc.), and this albumen is made up of 127 amino acid residues and near the N-end several serine enrichment regions is arranged.PC4 by the clone and be accredited as a kind of can through with many sequence-specifics and cell-specific regulate the direct general positive co-factor that is used for mediating many gene transcription activation of son (referring to document Ge etc., Cell (1994) 78:513-523; Kretzschmar, M. etc., Cell (1994) 78:525-534).Direct acting these of PC4 are regulated necessary important factor in the pathological process of generating process and other human diseasess that son comprises many nuclear hormone receptors, tumor suppression, cancer protein and tumour.

Tumor suppressor protein P53 directly and PC4 albumen interact at transcriptional level, thereby controlled the expression of PC4.In addition, PC4 as P53 unique activate that son can be regulated many participations cell cycle, apoptosis, DNA repairs and the gene transcription of other cell responses (referring to document Kishore A.H. etc., Biochem.J. (2007) BJ20070390; Banerjee, S. etc., Mol.Cell Biol. (2004) 24:2052-2062).The activity of PC4 receives the further adjusting of posttranslational modification, and the type of this posttranslational modification comprises phosphorylation modification and acetylation modification at least.PC4 is by after the phosphorylation modification, the activity inhibited of itself and the effect of target activity factor and can oppositely mediate the function of its co-activator.Mass spectrometry results shows: the ultra phosphorylation modification of PC4 in the body is mainly mediated and is occurred over just N-terminal filament propylhomoserin enrichment region (referring to document Ge etc., Proc.Natl.Acad.Sci.USA (1994) 91:12691-12695) by casein kinase i I.The acetylation modification of PC4 is mediated and is received the inhibition (Kumor, P.B.R. etc., J.Biol.Chem. (2001) 276:16804-16809) of phosphorylation modification by P300.

Up to the present, whether everybody not clear pc4 role in regulatory gene is relevant with the generation of cancer or tumour.

Summary of the invention

The inventor has adopted the different model organism system that comprises xenopus leavis oocytes system, tissue, other mammal cell lines; Be surprised to find PC4 have cancer protein function and cell differentiation, tumour grow and the process of pathology in play an important role; See the multiple function of the PC4 shown in Figure 10; All these functions, especially Figure 10 acceptance of the bid pour down the function of line, the basis of all relevant with pathogenesis of cancer mechanism or pathogenesis of cancer.Particularly; The inventor discovers in a lot of malignant tumor tissues; As in cancerous lung tissue, Bladder Cancer, colon cancer tissue, breast cancer tissue, endometrium cancerous tissue, thyroid cancer tissue, carcinoma of small intestine tissue, find that all PC4 is activated at protein level.On the contrary, the protein level of the PC4 in the normal tissues that does not live through accordingly quick growth does not raise.In cancerous cell line and other transformation cell lines, also found the rising of PC4RNA level, but in primary cell line, do not found.

For example, the inventor has measured the expression of the PC4 in the development by metamorphosis process of Africa xenopus.Assay method is following: the whole lysate of collecting the Africa xenopus of different developmental phases; Monitor the protein content of each lysate; Choose the total protein in each stage lysate of 50ug, last SDS-polyacrylamide gel electrophoresis is analyzed, and then the albumen on the gel is forwarded on the nitrocellulose membrane; Carry out western blot analysis at last, promptly adopt the polyclonal antibody of anti-PC4 to detect the albumen that is transferred on the nitrocellulose membrane.Measure the result shown in Fig. 5 A, Fig. 5 B and Fig. 5 C, the expression of PC4 is relevant with Africa xenopus bufonid toad development by metamorphosis process, and when current development by metamorphosis began, PC4 was in 56～58 these significantly activation of stages quilt.

The inventor is relevant with cell death through transfection test experience proof PC4.After the code area fusion with people PC4 code area (people PC4 code area wild type or serine enrichment region saltant) and green fluorescent protein (GFP); Subclone is to including in the CMV promoter mammalian expression vector (pcDNA3.1) again; To obtain the synthetic vectors transient transfection again to HeLa cell (referring to Ge etc., Proc.Natl.Acad.Sci.USA (1994) 91:12691-12695).After the transfection 24 hours, observation sample under laser confocal microscope can be observed PC4 role in the HeLa of transfection cell.The result is shown in Fig. 6 A, Fig. 6 B, Fig. 6 C; Fig. 6 A is the fluoroscopic examination result of control group (the HeLa cell of untransfected); The fluoroscopic examination result of the HeLa cell of Fig. 6 B is transfection wild type PC4; The fluoroscopic examination result of the HeLa cell of Fig. 6 C is transfection N-terminal filament propylhomoserin region mutation type PC4 (PC4-Δ S, its can not by phosphorylation, so should be complete activation).Three width of cloth figure contrast can find out that PC4 is arranged in nucleus and can causes chromosome condensation and Apoptosis, especially can judge that from Fig. 6 C the overexpression of PC4 has caused apoptosis, and this point is consistent with the effect of cancer protein.

In addition, the inventor also adopts the western blot analysis method to measure the PC4 level in many tissues.Collect the different tissue (being designated as of malignant tumor tissue " T ", normal tissues is designated as " N ", is provided by NIH) of operation back excision, and fast refrigeration in-80 ℃ of environment until use.Organize the preparation process of lysate following: after in organizing solution, adding lysis buffer (lysis buffer volume be cumulative volume 1/10), it to be uniformly dispersed with the miniature high-speed stirrer.The solution part is collected in centrifugal back.Detect the protein concentration of collected solution part lysate, can adopt Bradfort method of protein detection (BioRad Protein Detection appearance).For measuring the expression of PC4; The lysate of organizing of getting 50ug normal tissues or malignant tissue carries out the analysis of SDS-polyacrylamide gel electrophoresis; Albumen on the gel is forwarded on the nitrocellulose filter; Carry out western blot analysis at last, promptly adopt the polyclonal antibody of anti-people PC4 to detect the albumen that is transferred on the nitrocellulose membrane.The result is shown in Fig. 8 B; This result is illustrated in a lot of body tumor tissues; Like cancerous lung tissue, Bladder Cancer, colon cancer tissue, breast cancer tissue, endometrium cancerous tissue, thyroid cancer tissue and carcinoma of small intestine tissue; All find PC4, but in corresponding normal human tissue, all do not detected PC4.

The inventor also adopts immunohistochemical analysis to detect the expression of PC4 albumen in normal lung tissue and the cancerous lung tissue.The slide specimen of normal lung tissue that provides with NIH or cancerous lung tissue section is done immunohistochemical analysis.The immunohistochemical analysis process is following: handle the microslide of histotomy earlier with xylene, and 2 times, each 15 minutes; Then it is inserted the ethanol of (from 100%～70%) that concentration successively decreases successively, hatched 5 minutes at every turn; Oven dry is after 5 minutes down at 95 ℃, and water and PBS rinsing successively carried out mounting with 3% milk confining liquid then; Add one anti-(i.e. the antibody of anti-PC4), incubated at room 1 hour; Flush away unnecessary one anti-after, tangential section the two anti-of fluorescence of labelling, incubated at room 30 minutes; At last, the rinsing microslide is placed under the fluorescent microscope and can detects PC4 albumen.Shown in Fig. 9 A and Fig. 9 B, do not detect PC4 albumen in the normal lung tissue, and in all cancer cells of cancerous lung tissue, all find the aggegation of PC4 albumen.

The inventor (has detected the mRNA of PC4 at all through transformation cell lines (cos, HeLa, 293) and three breast cancer cell lines that detect in (MCF7, MD231, MDA468); But do not detect the mRNA of PC4 in former generation NIH3T3 clone and nonmalignant rat tissue (brain tissue m-Brain, testis tissue m-Testis).Detection method (Northern engram analysis) is as follows: from above-mentioned these different clones or tissue, extract total RNA and separate through formaldehyde-Ago-Gel of 1%, after the RNA on the gel forwards on the nitrocellulose membrane, will be marked with again ³²The people PC4 DNA (h-PC4probe) of P and people p52 DNA (h-p52 probe) are hybridized (placing the probe solution environment to hatch a period of time film) with the total RNA that is extracted respectively as probe; Detect the expression of PC4 mRNA and p52 mRNA among the total RNA of each sample respectively with ethidium bromide (EtBr) decoration method; I.e. 28S after the observation dyeing and 18S rRNA (referring to document Ge etc., EMBO J. (1998) 17:6723-6729).Testing result is shown in Fig. 7 A, Fig. 7 B, Fig. 7 C.Described p52 is the another kind of transcriptional coactivator that in above-mentioned various clones and tissue, exists.

The inventor also further connects activation and the activation of DNA topoisomerase I of PC4 in tumor tissues, shown in Fig. 8 A, Fig. 8 B.Described DNA topoisomerase I is a kind of enzyme that DNA is untwisted and is some cancer therapy drugs such as NB-506, J-I09, and J-382, the target spot of DB-67 (referring to document Pilch, B. etc., Cancer Res. (2001) 61:6876-6884; Chen, A.Y. etc., Mol.Cancer Thera. (2005) 4:317-324).

Many oncogenes and tumor suppressor gene encoded protein, for example C-myc, P53, DNA topoisomerase I, also be known can be as the albumen of transcription regulaton factor, the inventor finds that after deliberation PC4 can improve the activity of these albumen.

The above fact can draw a conclusion: except playing the effect of transcriptional coactivator, the people transcribes positive co-factor 4 (PC4) can also come active cell growth, differentiation, vicious transformation and tumor invasion growth as a kind of cancer protein.In fact, some adopt in other researchs of DNA array approach, have found that also PC4 is at activation (Das, C. etc., Mol.Cell.Biol. (2006) 26:8303-8315 of rna level in the cancerous lesion process; KIeivi, K. etc., Mol.Cancer (2007) 6:1-16).

Based on found that PC4 can play an important role as a kind of cancer protein in the forming process of dissimilar people's tumours; The inventor is the method that the research target spot has been invented a series of cancer diagnosis with PC4; Prevention, treatment method for cancer and medicine, and the method for screening cancer therapy drug.

The invention provides the method for diagnosis experimenter's cancer or other malignant tumours; This method comprises following content: collect suitable tissue appearance or liquid-like on one's body from the experimenter; Detect in the collected sample expression as the PC4 of biomarker; If the expression of PC4 wherein explains that than the height of control samples sample is cancerous tissue or malignant tissue.For further making a definite diagnosis, this method can also include the step of the level that detects other relevant biomarkers, for example detects the expression of DNA topoisomerase I.

The present invention further provides a kind of experimenter of detection whether to suffer from the method that whether has malignant cell in cancer or the body; This method comprises following content: collect suitable sample on one's body from the experimenter; Detect in the sample expression as the DNA topoisomerase I of biomarker; If the level of DNA topoisomerase I wherein explains that than the height of control samples sample is cancerous tissue or malignant tissue.

Above-mentioned " experimenter " comprises the people, other may cancered animal.

The invention provides a kind of function and prevent or treat the medicine of cancer, comprised the suitable PC4 antagonist of effective dose in this medicine through PC4 level in the reduction cell or inhibition PC4.

Accordingly, according to inventive concept of the present invention, the present invention also provides a kind of treatment method for cancer, uses the above-mentioned medicine that has comprised the suitable PC4 antagonist of effective dose promptly for the experimenter who suffers from cancer.Show that through inventor's research this type medicine corresponding methods of treatment in other words can be used to treat lung cancer, carcinoma of urinary bladder, colon cancer, breast cancer, carcinoma of endometrium, thyroid cancer, carcinoma of small intestine and other malignant tumours (referring to Fig. 8 B).

The above-mentioned medicine that includes suitable PC4 antagonist not only can also can be used to prevent purpose owing to treatment, prevents the generation of people's tumour.

Above-mentioned treatment method for cancer comprises chemotherapy, radiotherapy, immunotherapy and complex treatment.

The medicine of above-mentioned prevention or treatment cancer can use, also can unite other one or more cancer therapy drugs separately and use, and reaches the effective dose of prevention or treatment cancer jointly.

Can add the carrier of pharmaceutically accepting in the medicine of above-mentioned prevention or treatment cancer.

Based on the prior biological theory and technology, the present invention further provides some suitable PC4 antagonists: the antibody of PC4 polypeptide that modified or non-functional or albumen, anti-PC4, suitable antisense nucleoside acid molecule, suitable small molecules interference RNA molecule (siRNA).

PC4 polypeptide that modified or non-functional or albumen

That above-mentioned PC4 antagonist can be selected to have modified or do not have function PC4 polypeptide or albumen.PC4 is a kind of multifunctional protein of called optical imaging, and verified its bound DNA and when binding DNA, with other albumen interactional ability taken place.Everybody has recognized that a kind of PC4 mutant that modified or non-functional or part fragment wherein possibly suppressed by PC4 institute wild type or unmodified dominance, perhaps possibly be present in the cell with recessive state at present.Perhaps, mutant that modified or non-functional, analog or part fragment wherein maybe with the protein competition of wild type or unmodified, thereby weaken the function of the albumen of wild type or unmodified effectively.For example, because the PC4 of phosphorylation is inactivation (the especially PC4 inactivation of N-terminal filament propylhomoserin enrichment region after by phosphorylation modification), it also can be used to unphosphorylated PC4 (activity form) thereby competition reaches therapeutic purposes; In addition; Research at present shows; Wherein include the phenylalanine single amino acids sudden change (F77P) in the 77th site or lost the activity with the DNA binding with the PC4 albumen of the sudden change of this sudden change equivalence or polypeptide; Therefore according to inventive concept of the present invention, can infer that this no function PC4 polypeptide or albumen have the pharmaceutical use of anticancer aspect.They have the simulating peptide or the simulating peptide composition of this similar functions may become the treatment of cancer medicament that a kind of urgent need remains to be researched and developed, owing to can prevent that immune system of animal or protein metabolism mechanism from being destroyed.

The present invention also further provides some saltant PC4 albumen as PC4 antagonist (treatment depends on the medicine of the tumour of PC4).

Reported N-terminal filament propylhomoserin enrichment region is essential for the activity of PC4 albumen before, and receives the adjusting of the phosphorylation of EGFR-TK II, and deletion N-petiolarea causes the auxilliary activation of PC4 albumen to completely lose (Ge etc., 1994a; 1994b; Kretzschmar etc., 1994).The inventor has located some important amino acids that concerning the activity of PC4 albumen, are absolutely necessary through making a series of single amino acids sudden change.Thus, the inventor filters out 7 kinds of saltant PC4 albumen, and is as shown in Figure 2, mt-1 (K23I/K29A), mt-2 (K35I/K41A), mt-3 (R27A/K28I/K29A), mt-4 (R47N/K35I/K41A), mt-5 (F77P), mt-6 (K29A), mt-7 (K41A).Prove through experimental study; The PC4 that single amino acids sudden change (F77P) taken place except the 77th known site has lost ability and the transcriptional activity that combines DNA; The activity that the PC4 albumen of dual sudden change (K23I/K29A and K35I/K41A) and triple mutant (R27A/K28I/K29A) takes place at N-end group local area also greatly reduces; Therefore PC4 mutant: F77P, K23I/K29A, K35I/K41A, R27A/K28I/K29A can be used as cancer therapy drug, reach the effect that treatment depends on the malignant tumour of PC4 through competing with endogenous wild type PC4.

The present invention also provides short polypeptide with the amino acid sequence homologous in the activation structure territory of PC4 as the PC4 antagonist.

PC4, as a general transcriptional coactivator, its activity shows as and other general transcription factors, activating transcription factor, dna profiling between interaction, so the activation structure territory realizes that for PC4 its function is necessary.Thus, the inventor designs and has synthesized two polypeptide, and their sequence is consistent with two PC4 activation structure domain amino acid sequences respectively, and these two polypeptide can combine other to activate son with wild type PC4 competition, thereby reaches the active effect of PC4 that suppresses.These two peptide sequences are: 16-30 amino acid (DSDSEVDKKLKRKKQ sees SEQ ID NO.3) and 26-40 amino acid (KRKKQVAPEKPVKKQ sees SEQ ID NO.1) sequence in the amino acid sequence of PC4.

The present invention also provides the simulation activation structure domain polypeptide that can combine with PC4 as the PC4 antagonist.

Because the carcinogenic activity of PC4 is (specificity of PC4 and said oncogene, growth factor or other carcinogens activates the son interaction and activates their expression) of realizing through its expression as the whole or most oncogene of transcriptional coactivator deactivation, growth factor or other carcinogens; Therefore, suppressing PC4 albumen goes to combine endogenous activation can make PC4 albumen lose its auxilliary son activity that activates.The inventor has found that after deliberation has 15 amino acid whose amphipathic helix polypeptide (amphipathic helix peptide; AH peptide) can simulate a kind of activation structure territory and PC4 strong interaction takes place; Thereby sealed the activation minor structure territory of PC4; Suppress the effect of PC4 and endogenous activation, suppressed the function of PC4, reach therapeutic purposes.The sequence of this polypeptide is: ELQELQELQALLQQQ, see SEQ IDNO.5.

As further improvement of the present invention, the PC4 antagonist also comprises polypeptide or the protide PC4 antagonist that is connected with the migration domain.

In order to promote polypeptide or protide PC4 antagonist to get into tumour cell; Make it to examine interior cancer protein PC4 by target; Can (translocation domain TLD) can suppress fusions such as active albumen of PC4 or polypeptide with recombinant monoclonal antibodies, saltant PC4 albumen, the polypeptide with the amino acid sequence homologous in the activation structure territory of PC4, the simulation activation structure territory that can combine with PC4 or other and realize through moving domain.

The present invention adopted a kind of include 12 amino acid whose short polypeptide as the migration domain, its sequence is: PLSSIFSRIGDP, see SEQ ID NO.7, molecular weight is 1288.47, isoelectric point pI=6.27.It is reported this peptide species (Oess and Hildt 2000 that for the cell permeability of the infection of hepatitis type B virus and some other albumen, is absolutely necessary; Stoeckl et al., 2006).

Antibody

The invention provides the neutralizing antibody that some can suppress PC4 albumen or suppress the BA of PC4 albumen and DNA topoisomerase I (also claiming target protein below the albumen for these two).The anti-PC4 antibody of antibody of the present invention for accessing based on existing biology techniques, preferred monoclonal anti PC4 antibody also can select to include the polyclonal antibody of said monoclonal anti PC4 antibody.Described monoclonal anti PC4 antibody has comprised the subtype specificity antibody of PC4.

According to inventive concept of the present invention; Above-mentioned monoclonal anti PC4 antibody is the antibody that can specificity combines target protein; Fab in the antibody is also claimed the antigenicity fragment, and the antigenicity fragment can be Fab fragment, F (ab) 2 fragments, Fab ' fragment, F (ab ') 2 fragments, Fd fragment, Fd ' fragment or Fv fragment.Comprised the antigenicity fragment in the general monoclonal anti PC4 antibody, monoclonal anti PC4 antibody also can be selected these antigenicity fragments (being univalent antibody) certainly.Preferably select epitope, promptly after antibody gets in the body by the site of antigen recognizing.The first-selected mouse monoclonal antibody of monoclonal anti PC4 antibody or wherein antigenicity fragment, epitope.Except univalent antibody, domain antibodies (dAbs) (list of references Holt etc., Trends in Biotechnology (2003) 21:484-490) also is applicable to anti-PC4 antibody of the present invention.

If the treatment people should adopt humanized antibody, preferably the humanization natural antibody.

The method of making the antibody of known antigens has a variety of; This is that technology that those skilled in the art know is (referring to (the Cold Spring Harbor Laboratory Press of Cold Spring Harbor Laboratory publishing house; Cold Spring Harbor; NY) " the Antibodies:A Laboratory Manual " that publishes, Harlow and Lane, 1988; Or referring to No. the 01/25437th, international monopoly).

Can adopt immunization in the body, come immune people or other animal reservoirs, from the animal reservoir, extract antibody again with the immunogenic component that has that is derived from the target protein; Can obtain above-mentioned antibody through immune people or the animal except that the people, preferred mammal, for example rat, mouse, cavy, rabbit, goat, sheep, pig, perhaps birds (like chicken), further preferred mouse.

Also can adopt the mode of external immunity from immunocyte, to extract antibody.External immunization preferably adopts recombination and expression techniques to produce antibody, and the clone that for example DNA with the anti-PC4 antibody of suitable coding transforms, transfection, infection or transduction are fit to obtains recombination system and can express the anti-PC4 antibody of production; Also can adopt hybridoma technology known in the art to prepare antibody;

Or adopt biological mode of rebuilding, directly with the heavy chain and the light chain of purifying are built into antibody;

Particularly, can also select the method for chemosynthesis to obtain suitable antibody, perhaps adopt the method (for example the intracellular antibody technology is expressed anti-PC4 antibody in born of the same parents) of intracellular immunity.

In addition, antibody of the present invention has also comprised chimeric antibody and hybrid antibody.Technology (Morrison etc., Proc.Natl.Acad.Sci.USA, (1984) 81:6851-6855 of preparation chimeric antibody and hybrid antibody have been described in some documents; Neuberger etc., Nature, (1984) 312:604-608; Takeda etc., Nature (1985), 314:452-454).

Further, single-chain antibody also be applicable to the present invention (be two United States Patent (USP)s of Huston referring to the invention people: the 5th, 476, No. 786 and the 5th, 132, No. 405; Huston etc., Proc.Natl.Acad.Sci.USA, (1988) 85:5879-5883; The invention people is the 4th, 946, No. 778 United States Patent (USP)s of people such as Ladner; Bird, Science. (1988) 242:423-426; Ward etc., Nature. (1989) 334:544-546).Described single-chain antibody is a kind of small molecular antibody of being made up of single peptide chain; Synthetic method: between heavy chain of antibody and variable region of light chain (Fv) gene; With one section small peptide gene both are connected into the ScFv gene, this expression of gene product can be folded into the single chain polypeptide with antigen binding capacity.

The method of administration of antibody can adopt delivery of drug approach more well-known to those skilled in the art, and for example: directly injection possibly relatively be fit to antibody is discharged into the site that needs treatment.Also can adopt the liposome that is enclosed with antibody in its film, the liposome orientation is transported to target spot, thereby the expression of the target gene here or function are suppressed.Except monoclonal anti PC4 antibody, can also comprise some other medicaments in the described liposome, for example above-mentioned some can be released to the medicament (referring to document, Wolff etc., Biochem.et Biophys.Acta, (1984) 802:259) of treatment target spot.

The antisense nucleoside acid molecule

Also provide employing antisense oligonucleotide molecule to suppress the method that the PC4 gene perhaps suppresses the expression of PC4 gene and DNA topoisomerase I gene (these two genes are hereinafter to be referred as target gene) in the present invention.Well known in the art, the expression of the antisense RNA in the cell, particularly constructive expression, expression that can suppressor (possibly be or prevent the expression that the mode of montage is come suppressor) through the blocking-up translation.Based on this point; Disturb montage to make that those conservative propertys are relatively poor and produce stronger specific intron sequences and can play a role; Thereby can suppress the expression of corresponding gene product in the species, but not suppress the expression of the homologue of this gene outcome in other species.

Term " antisense component " is meant RNA or the dna molecular fully complementary with a specific mRNA molecule.Antisense rna molecule is special, and antisense RNA and mRNA molecular hyridization take place can cause the translation of mRNA to be suppressed.Described molecular hyridization environment in vivo takes place down.Antisense rna molecule of the present invention must have enough abilities complementary with target gene; About 15～30 nucleotide of length; So antisense rna molecule can suppress the expression of target gene with target gene (perhaps mRNA) hybridization, no matter hybridizes to occur in montage level, transcriptional level or translation skill.Antisense rna molecule of the present invention select can with the RNA molecule of any part hybridization that comprises coding region sequence, 3 ' or 5 ' non-coding area sequence or other intron sequences among the target cDNA, or can with the RNA molecule of target mRNA hybridization.As for how designing antisense nucleic acid molecule, on the basis of known mRNA sequence, according to prior art, the antisense nucleic acid molecule sequence can design at an easy rate.

Antisense rna molecule of the present invention; Can adopt the mode of conversion or transfection; Transport the entering host cell by carrier, described bearer type comprises retroviral vector and plasmid, connects the DNA of encoding antisense RNA and the suitable adjusting sequence that includes promoter; Together insert in the described carrier after the connection, thereby antisense RNA can be expressed in host cell.In one embodiment, the carrier that includes the cDNA fragment of encoding antisense RNA molecule has been realized stable transfection and constructive expression, and perhaps this expression possibly be under the control of the promoter of tissue specificity or development-specific, to realize.Also can use liposome that antisense rna molecule is transported the entering cell.

To interior therapeutic, present existing prefered method is directly antisense rna molecule to be transported to target spot, rather than has made up the expression vector of the cDNA fragment of encoding antisense RNA molecule on it through stable transfection.Antisense oligonucleotide molecule of the present invention has the length of 15 to 30 bases; Its sequence has determined whether this antisense oligonucleotide molecule can hybridize with any part that comprises coded sequence, 3 ' or 5 ' noncoding region or other intron sequences among the target cDNA; Perhaps preferred, whether can hybridize with target mRNA.Preferably select for use those to have the sequence of the strongest antisense ability with the sequence of the antisense oligonucleotide molecule of target gene hybridization.Antisense ability on the antisense oligonucleotide sequence molecule is decided by following factor: the length of antisense oligonucleotide, the accessibility of binding compatibility, target sequence.At the strong antisense oligonucleotide molecule of in-vitro screening antisense ability, can be through vitro detection its suppress the ability of the ability of target protein translation phenotype relevant with target gene with inhibition, for example, the ability of cell proliferation in the inhibition nutrient culture media.In general, most of zones of target cDNA (5 ' and 3 ' noncoding region, AUG sintering, code area, splice junction and intron sequences) can both utilize antisense oligonucleotide to locate.

Antisense oligonucleotide molecule of the present invention is preferred, and those are stable, that strong nuclease-resistant ability is arranged, have the oligonucleotide molecule that suitable pharmacokinetics performance makes it arrive destination organization and to have the ability to pass plasma membrane with non-toxic.

According to prior art, the chemical modification of antisense rna molecule mainly concentrates on phosphodiester backbone, base, sugar ring, and these modifications can improve the stability of antisense rna molecule or the ability of hybridizing with target cDNA or mRNA.The preferred phosphorothioate antisense oligonucleotide of antisense oligonucleotide molecule of the present invention molecule (phosphodiester bond of antisense oligonucleotide molecule by the phosphorothioate modification then obtain phosphorothioate antisense oligonucleotide molecule).The basic structure of phosphorothioate antisense oligonucleotide molecule also can further be optimized its antisense ability by modification.In addition, had been found that also N3 '-P5 ' phosphoramidate bond energy enough makes the oligonucleotide molecule stable and can improve the binding adhesion between antisense oligonucleotide molecule and the RNA in the presence of nuclease.Peptide nucleic acid key (PNA) has all replaced ribose and phosphodiester backbone can be stablized it in the presence of nuclease, can not cut by RNA H enzyme, and improve the affinity between antisense oligonucleotide molecule and target gene or the mRNA.About the modification of base, verified, after base is modified by some heterocycles, can strengthen the antisense ability of antisense oligonucleotide molecule and make that the antisense oligonucleotide molecule can be by RNA H degraded, for example, the C-5 thiazole is modified.At last, also can consider the modification of sugar ring, for example use 2 '-oxygen-propane base, 2 ' methoxyethoxy to replace ribose and make oligonucleotide can keep stability in the cell in vitro nutrient culture media or in the environment in the body nuclease.

The method of administration of antisense nucleic acid oligomer molecule of the present invention should select some that the approach of best antisense effect (standard according to above-mentioned is measured) can be provided.Show with cationic-liposome, retroviral vector to be that carrier carries the drug effect of antisense nucleic acid oligomer or direct administration better with the testing in vitro result in the body of antisense nucleic acid oligomer molecule.Another kind of possible mode of administration is to utilize the antibody that can combine with target cell surface specific mark with the molecular targeted purpose cell that is transported to of antisense nucleic acid oligomer.Antibody combine with target cell or with its on the purpose that combines to reach targeted of acceptor.

Small molecules interference RNA

In the present invention the small molecules interference RNA that can suppress target gene and express (small interfering RNAs is called for short siRNA, also claims RNAi, also claims the RNA RNA) is provided further also.Small molecules interference RNA is a kind of double stranded rna molecule, and typical length is 21 nucleotide (nt), if itself and target gene homology, then can the jamming target expression of gene.

The RNAi technology is relevant with the sequence-specific posttranscriptional gene expression process in the eukaryotic.In general, relate to the mRNA degraded of particular sequence in this process, this degraded is caused by the double-stranded RNA (dsRNA) with this section particular sequence homology.For example, can make the transmission of hereditary information become unstable with the expression of specific strand mRNA (ss mRNA) the corresponding long dsRNA of sequence, thereby disturb the expression of corresponding gene.Introducing all can suppress this expression of gene with the corresponding dsRNA of whole or most sequences of any selected genes mRNA.It seems that according to present research when a segment length dsRNA expressed, it at first was cut into the short dsRNA that length is merely 21～22 base-pairs by the rnase iii enzyme.Therefore, siRNA can introducing or the expression of the short dsRNA of homology receive direct influence with it because of these.

Have at least two approach to receive the influence of dsRNA in the mammalian cell.

In the sequence-specific approach, aforesaid, initial dsRNA is a plurality of small molecules interference RNAs of digested one-tenth (siRNA) earlier.These siRNA are made up of positive-sense strand and antisense strand that length is about 21 nucleotide, and every chain is made up of the nucleotide of 19 interference effects and two free nucleotides of 3 ' end.The sequence information that small molecules interference RNA is considered to provide certain makes the mRNA of respective specific sequence degraded by target.

On the contrary, and non-specific approach can be caused by the dsRNA of arbitrary sequence, as long as this dsRNA length is at least greater than 30 base-pairs.The initiation of non-special approach is because dsRNA has activated two enzymes: PKR enzyme and 2 ', 5 ' oligoadenylate synthetase (2 ', 5 '-AS).When the PKR enzyme is in activated state, can close the synthetic of all albumen through phosphorylation translation initiation factor eIF2; 2 ', 5 ' oligoadenylate synthetase can synthesize the molecule of a kind of activator RNA enzyme L (RNase L), and RNase L is the non-specific enzyme of a kind of all mRNA that can degrade.Non-specific approach is represented host's reaction of pressure and virus infections to external world usually, and in general, the effect of non-specific approach is preferably dropped to floor level.Clearly; Long dsRNA can only induce non-specific approach; Therefore, the dsRNA of 30 base-pairs of curtailment preferably through RNA disturb (RNAi) be the sequence-specific approach influence expression of gene (referring to Hunter etc., J.Biol.Chem. (1975) 250:409-417; Manche etc., Mol.Cell.Biol. (1992) 12:5239-5248; Minks etc., J.Biol.Chem. (1979) 254:10180-10183; Elbashir etc., Nature (2001) 411:494-498).

Verified, it is a kind of effective means that reduces gene expression dose that RNA disturbs, and the cell of most of types all is suitable for, for example HeLa cell, NIH/3T3 cell, COS cell, 293 cells and BHK-21 cell.Particularly compare with adopting antisensenucleic acids, siRNA can drop to destination gene expression more low-level, and in fact, siRNA often can make genes of interest not express (referring to Bass, Nature (2001) 411:428-429) fully.In addition, for reducing destination gene expression level in the mammalian cell, under the equal conditions, the effective dose of siRNA is than the low several magnitude (Elbashir etc., Nature (2001) 411:494-498) of effective dose of antisense RNA.

RNA disturbs the double-stranded oligonucleotide of preferred length less than 30 base-pairs, and further preferred length is the double-stranded RNA of 25,24,23,22,21,20,19,18,17 base-pairs.Length is that the siRNA effect of 21 base-pairs is better.It is better to include 3 ' free end siRNA effect.Typical free 3 ' end has two free nucleotide; These two nucleotide can be made up of the ribonucleotide residue of any kind; Preferred 2 '-deoxyribonucleotide, in cell culture medium with transfectional cell in, adopt 2 '-free 3 ' end that deoxyribonucleotide is formed not only reduced the synthetic cost of RNA; And can strengthen the ability that siRNA opposing nuclease enzyme cuts (referring to Elbashi etc., Nature (2001) 411:494-498).

Longer dsRNA molecule for example has the big molecule dsRNA of 50,75,100 or even 500 or more base-pair, also can in specific embodiment of the present invention, be applied.For the normal concentration that reaches the dsRNA that the RNA interference effect adopted is about 0.05nm, 0.1nm, 0.5nm, 1.0nm, 1.5nm, 25nm or 100nm; Though also can adopt other concentration, this depends on handled cell type, the target gene that is directed against and other correlative factors.

Synthesizing of longer dsRNA molecule: can be from the synthetic long-chain dsRNA of promoter transcription, described promoter such as t7 rna polymerase promoter well known in the art.Long dsRNA molecule synthetic that suppresses the single target gene: light from the target bit in the promoter downstream of cell-free transcription folder,, and be assembled into corresponding dsRNA immediately to two different directions RNA molecule of synthetic complementation simultaneously.Above-mentioned these dsRNA divide the period of the day from 11 p.m. to 1 a.m in design, must consider that each RNA molecule all should include the nucleotide sequence of a part and target gene mRNA homology.

The basic synthetic method of siRNA: adopt the synthetic suitable expression vector that perhaps makes up of chemical method in external or body, to synthesize.The synthetic standard rna of chemical method is made up of 21 ribonucleotides.Synthetic siRNA can adopt the gel separation method to carry out purifying.(Elbashir etc., Genes Dev (2001) 15:188-200).

When design siRNA sequence; Can select any one section nucleotide sequence adjacent as specific sequence with the coded sequence of target gene mRNA; That is to say, can select when designing siRNA of the present invention with PC4mRNA on the adjacent any one section nucleotide sequence of coded sequence as specific sequence.In addition, how from these nucleotide sequences, to have selected best specific sequence? General some existing sequences Design softwares that adopt.At first utilize the secondary structure of these software program target of prediction gene mRNAs, select the sequence that those appear at the exposed strand district on the folding mRNA probably again.Design method that is adopted and the material of suitable siRNA, can be with reference to United States Patent (USP) the 6th, 251, the corresponding contents of putting down in writing in No. 588 files.

Though mRNA is considered to linear molecule usually, comprised the information that directly instructs albumen synthetic in its nucleotide sequence, in fact, most mRNA demonstrates and includes a lot of secondarys and three grades of spatial structures.The last secondary building unit of RNA is to form through taking place between the zones of different in the same RNA molecule to interact, and described interaction mainly is the interaction of Watson-Crick type.Important secondary building unit comprises in double-stranded RNA (dsRNA) and the Nei Huan district by the double-spiral structure that forms in the molecule, hairpin structure and prominent ring structure.Formed more complicated three-dimensional structure is the tertiary structure unit when generation interaction or secondary building unit and strand district have an effect between the secondary building unit.Many researchers is measured the combination energy in a large amount of RNA duplex structures, thereby has drawn a series of rules that can be used for predicting the RNA secondary structure (for example referring to Jaeger etc., Proc.Natl.Acad.Sci.USA (1989) 86:7706; Turner etc., Annu.Rev.Biophys.Biophys.Chem. (1988) 17:167).These rules can help identification mRNA structural unit, and particularly for the exposed strand of identification zone, the exposed strand zone on these mRNA may become the best target fragment of siRNA, ribozyme or antisense RNA institute target.Accordingly, the best target fragment on the identification PC4mRNA can be used for designing based on the dsRNA oligonucleotide with RNA interference effect of the present invention's design, suitable ribozyme and tup forming core enzymatic compositions.

The above-mentioned dsRNA oligonucleotide with RNA interference effect can connect the target gene of same allos together through the mode transfered cell of support agent transfection, and described support agent such as liposome well known in the art be the lipofectamine Lipofectamine 2000 to attached cell system of Life Technologies company production for example.The lipofectamine Oligofectamine that the transfection carrier of the dsRNA oligonucleotide of target endogenous gene can select for use Life Technologies company to produce.Transfection efficiency can also detect through following mode: is after cotransfection is expressed with dsRNA oligonucleotide and hGFP-encoding pAD3 at mammalian cell; Utilize fluorescence microscopy to detect its transfection efficiency (referring to Kehlenback etc., J.Cell.Biol. (1998) 141:863-874).Behind the dsRNA transfered cell, its RNA jamming effectiveness can adopt a lot of methods to detect.These methods comprise western blot analysis (Western blot analysis), RT-polymerase chain reaction (RT-PCR) and Northern hybridization analysis (Northern blot analysis); Described western blot analysis is meant that waiting for that the endogenous gene storehouse is accomplished once has enough to meet the need metabolism; New round albumen is synthetic be suppressed after, the method that adopts antibody to come recognition objective gene outcome (PC4); RT-polymerase chain reaction and Northern hybridization analysis can be measured the level of target gene mRNA (PC4mRNA).

The reagent that is adopted about RNAi technology, methods and applications have further introduction in No. the 6278039th, 5723750 and 5244805, United States Patent (USP), being listed in here can be for referencial use.

Another object of the present invention provides the screening technique of cancer therapy drug, and this method is based on PC4 to be a kind of cancer protein and to can be used as this scientific discovery of cancer therapy drug target spot.

Screening technique based on promoter

The method of screening cancer therapy drug of the present invention, this method comprise following two steps that take place successively: (1) provides a kind of gene construct, and it comprises the promoter sequence of PC4 gene and the reporter gene that effectively is connected with this promoter sequence; (2) drug candidate and described gene construct are mixed the environment that places suitable described reporter gene expression, wherein, the drug candidate that suppresses reporter gene expression is a cancer therapy drug.

The PC4 gene promoter sequence is referring to Figure 1A.With the suitable reporter gene that this promoter sequence effectively is connected, can select like luciferase gene or green fluorescence protein gene.Those skilled in the art should be able to recognize; The exemplary sequence that in present specification, is provided, also have many suitable PC4 gene promoter sequences or other cis acting transcription factors of some PC4 genes also can be applied to method for screening anticancer medicine based on the present invention's design.

Except the above-mentioned screening technique based on promoter, method for screening anticancer medicine of the present invention can also adopt the detection method based on protein active.

Depend on the transcriptional activity detection method of PC4 albumen

The present invention also provides a kind of method of screening cancer therapy drug, and this method comprises following two steps that take place successively: (1) provides a kind of test of transcriptional level of the PC4 of detection albumen; (2) in above-mentioned test, add drug candidate, wherein, the drug candidate that reduces the PC4 transcriptional level is a kind of cancer therapy drug (referring to Ge and Roeder, Cell (1994) 78:513-523).

Detection method based on protein-protein or protein-DNA interaction

The present invention also provides the method for another kind of screening cancer therapy drug, and this method comprises following two steps that take place successively: (1) provides and detects between PC4 and the albumen interactional test between interaction or PC4 and the DNA; (2) in above-mentioned test, add drug candidate, wherein, reduce between PC4 and the albumen between interaction or PC4 and the DNA interactional candidate compound and be cancer therapy drug (referring to Ge, Nucleic Acids Res. (2000) 28, e3).

Preferred high throughput system (HTS) screening suppresses the compound of PC4 protein active.

In high flux screening detects; Usually (fluorescence resonance energy transfer FRET) comes molecular dynamics (for example protein-protein interaction, protein-dna interaction, protein conformation conversion etc.) to carry out quantitatively to use the FRET technology.General 96 orifice plates or the 384 orifice plate standards of adopting of high flux screening detection system based on the FRET technology.

Screen the compound of inhibition PC4 protein active of the present invention and use amphipathic helix (AH) the domain polypeptide that is marked with fluorescence as energy donor, the PC4 albumen of mark fluorescent is as energy acceptor.In case PC4 and amphipathic helix domain polypeptide form complex; Promptly owing to the interaction force between two molecules makes close between donor and the acceptor (apart from about 1～10nm); What observed this moment mainly is the emission light of PC4, because intermolecular fluorescence resonance energy is transferred to PC4 albumen from amphipathic helix domain polypeptide.The exciting light of amphipathic helix domain polypeptide and the emission light of PC4 receive the monitoring that fluorescence intensity is read the plate appearance.The effect of the inhibition PC4 protein active of any compound can both be measured through the variation of monitoring the fluorescence intensity that is read.General screening two compounds: 1) micromolecule complex; 2) native compound.Certainly, these compounds also need further be confirmed, prove that it can become candidate's anticarcinogen that a kind of potential treatment depends on the malignant tumour of PC4.

The method of cancer diagnosis of the present invention or screening cancer therapy drug is based on protein or based on the detection method of nucleic acid, these methods all are conventional methods well-known to those skilled in the art.Anti-PC4 antibody and anti-topoisomerase I antibody all are antibody that some are known and that can on market, buy.For example, with the antibody of nano Au particle covalent cross-linking, can buy from BioassayWorks company (Ijamsville, Maryland), the enough level of PC4 albumen or the levels of topoisomerase I of monitoring quantitatively apace of this antibody capable.Same, reverse transcriptional PCR (RT-PCR) can be monitored the activation levels of PC4 and topoisomerase I at rna level.

In sum; The inventor has found that PC4 is relevant with cancer; Opened up the anticancer research frontier; And invented a series of Method for cancer diagnostics and reagent (based on the expression that detects PC4) thus, and the method for treatment and prophylaxis of cancer with medicine (based on function and the expression of inhibition PC4), screen the method for cancer therapy drug etc.

Description of drawings

Figure 1A, Figure 1B, Fig. 1 C provide the sequence information of people PC4 gene; Wherein, Figure 1A provides people PC4 cDNA sequence and promoter region, and promoter region has marked underscore (referring to the GenBank sequence number: NM_006713); Figure 1B provides the dna sequence dna of people PC4 code area; Fig. 1 C provides the amino acid sequence of people PC4 albumen (127 amino acid), and the 77th amino acid (F: phenylalanine has been marked underscore) site can make PC4 lose the ability that combines with single stranded DNA if the single amino acids sudden change takes place;

Fig. 2 shows PC4N-petiolarea mutation analysis collection of illustrative plates, and said N-petiolarea comprises base region and the 77th amino acid; Wherein, WT representes wild type, and mt representes saltant, and mt-1～7 have been represented 7 different saltant PC4 albumen of the present invention respectively;

Fig. 3 A, Fig. 3 B, Fig. 3 C show the functional analysis collection of illustrative plates of PC4 mutant (mt-1～7); Wherein, Fig. 3 A shows the standardized testing of various purified recombinant PC4 albumen; Fig. 3 B shows electrophoretic mobility shift assay (EMSA) experimental result of the various albumen that Fig. 3 A detected, promptly detect each albumen and dsRNA combine active), the PC4+DNA among the figure is meant the complex of PC4 and dsDNA; Fig. 3 C shows the in-vitro transcription test experience result of the various albumen that Fig. 3 A detected, and wherein pG5HM is the template of transcribing that depends on PC4, and pML Δ 53 is the templates of transcribing that do not rely on PC4;

Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D, Fig. 4 E, Fig. 4 F show the protein arrays analysis result of wild type PC4 albumen and various saltant PC4 albumen; Wherein 5 and 40 of the collection of illustrative plates left side represented two different protein concentrations (pmol of unit), Fig. 4 C shows the interaction result between each albumen and the transcription factor TFIIA (p55); Fig. 4 E shows the interaction result between each albumen and the transcription factor Spl; Fig. 4 F shows the interaction result between each albumen and the ssDNA; Fig. 4 A shows Ponceaux (Ponceau S) coloration result, and whether this experiment is to detect each albumen successfully to forward on the nitrocellulose membrane; The interaction result that Fig. 4 B shows between anti--PC4 antibody (anti-PC4) and each albumen, this experiment be for the amount that detects each albumen that forwards on the film whether consistent; Fig. 4 D is the blank result, detects between each albumen and the Gal4 (1-94) whether non-specific binding is arranged;

The presentation of results of Fig. 5 A, Fig. 5 B, Fig. 5 C expression and the relation of Africa xenopus development by metamorphosis of PC4; Every different development by metamorphosis stage of row gel band representative; Wherein, Fig. 5 A and Fig. 5 B show the expression of the PC4 in 2～58 and 56～64 each stage respectively, and " O " represented egg mother cell (oocytes) among Fig. 5 A, and " E " represented embryo (embryos), and PC4-P representes the PC4 albumen (inactivation) of phosphorylation; Fig. 5 C shows the relation of PC4 activation and Africa xenopus differentiation and development, and expression curve and the PC4 expression curve of expression curve and Thyroid Hormone Receptors (TR α and TR β) that wherein shows thyroid hormone (T3 and T4) is as contrast;

Fig. 6 A is control group (the HeLa cell of untransfected) fluoroscopic examination result; The fluoroscopic examination result of the HeLa cell of Fig. 6 B is transfection wild type PC4; The fluoroscopic examination result of serine region mutation type PC4 (PC4-Δ S) that Fig. 6 C has been transfection; Illustrated when PC4 by transfection in the Hela cell time, PC4 is arranged in nucleus and can causes chromosome condensation and cell death.

Fig. 7 shows the testing result of PC4 mRNA; Selected test sample is following: transformation cell lines (cos, HeLa and 293), three breast cancer cell lines ((MCF7, MD231 and MDA468), primary cell line (NIH3T3), rat normal tissues (brain m-brain and testis tissue m-Testis); Fig. 7 A representes the abundance measurement result of total RNA in each sample; Fig. 7 B shows the expression (being the express spectra of p52) of p52 mRNA as contrast; Fig. 7 C shows the expression of PC4 mRNA;

Fig. 8 A and Fig. 8 B show the western blot analysis result of DNA topoisomerase I and PC4 in various tumor tissues and the corresponding normal tissues respectively; Wherein, Kidn (nephridial tissue), Lung (lung tissue), Bladd (bladder body), Colon (colon), Prost (prostata tissue), Breast (breast tissue), Pancrs (pancreatic tissue), Endomt (endometrial tissue), Thyro (parathyroid tissue), S-Bow (small intestine); N representes normal tissues; T representes malignant tissue's (tumour), and PC4a representes to have active wild type, and PC4b representes to have active inferior wild type;

Fig. 9 A and Fig. 9 B show the immunohistochemical analysis result of PC4 expression in human body normal lung tissue and the cancerous lung tissue respectively;

Figure 10 shows the multiple function of PC4; These all functions especially mark the relevant with cancer of underscore or relevant with the pathogenesis of cancer, so PC4 may become the treatment target spot of a lot of cancer therapy drugs.

Embodiment

Come further to set forth the present invention below in conjunction with embodiment and accompanying drawing.

Embodiment 1

Saltant PC4 albumen

The inventor has located some important amino acids that concerning the activity of PC4 albumen, are absolutely necessary through making a series of single amino acids sudden change.Thus; The inventor filters out 7 kinds of saltant PC4 albumen that can be used as the PC4 antagonist; Collection of illustrative plates as shown in Figure 2, mt-1 (K23I/K29A), mt-2 (K35I/K41A), mt-3 (R27A/K28I/K29A), mt-4 (R47N/K35I/K41A), mt-5 (F77P), mt-6 (K29A), mt-7 (K41A).

The inventor has also carried out recombination to construct, expression, purifying, evaluation with above-mentioned 7 kinds of saltant PC4 albumen, and is specific as follows.

The recombination to construct method of mutant: produce various sudden change construct (the double-stranded oligonucleotide that includes the specified point sudden change can commercial synthetic obtaining) thereby the PC4 wild type district in the bacterial expression vector is replaced as the double-stranded oligonucleotide that includes specified point sudden change.

Each sudden change construct all will carry out sequencing analysis and in E coli, just can be identified after the expression.Expression PC4 albumen (comprising wild type and saltant) in Ecoli can obtain through 2 following step method of purification: the first step: P11 cellulose phosphate ion-exchange chromatography; Second step: heparin affinity chromatography.The concentration of albumen can be measured through the Bradford method behind the purifying, and purity can be passed through polyacrylamide gel electrophoresis (SDS-PAGE) and measure.PC4 albumen behind the purifying (WT, mt-1～7) is gone up SDS-PAGE, and electrophoresis result is shown in Fig. 3 A.

Adopt following method to detect the activity of PC4 albumen behind the purifying: 1) electrophoretic mobility shift assay experiment (result sees Fig. 3 B); 2) in-vitro transcription test experience (result sees Fig. 3 C); 3) protein arrays analysis (result sees Fig. 4), these three kinds of method concrete operations are following:

1) (Electrophoresis mobility shift assay EMSA) detects the ability that PC4 albumen is bound dsDNA to adopt the electrophoretic mobility shift assay method

The operation of EMSA is following: configuration 20ul reaction system (wherein including the 10ng various PC4 protein samples of purifying) and the length that is marked with 32P are the dsDNA probe (deriving from the adenovirus major late promoter district) of 64bp; The reaction mixture that both is mixed the back gained places 4 ℃ of incubations after 1 hour; Last 8% polyacrylamide gel electrophoresis can detect mobile PC4-dsDNA complex through radioautograph.Shown in Fig. 3 B, mt-1, mt-2, mt-3, mt-5 have lost the ability of binding with dsDNA.

2) in-vitro transcription test experience

Utilization from the HeLa cell or from a reorganization system general transcription factor of purifying, like TFIIA, TFIIB, TFIID, TFIIF, TFIIH, rna plymerase ii wait to transcribe test experience at reconstruction in vitro.The activation of this in-vitro transcription system depends on PC4 and activates son (referring to Ge etc., 1996) with other.Except these above-mentioned general transcription factors, this system of transcribing also comprise template (pG5HM is the template of transcribing that depends on PC4) that a response activates son, one do not rely on the basic templates (pML Δ 53 is the templates of transcribing that do not rely on PC4) that activates son and ³²P-CTP.This is transcribed, and 30 ℃ of incubations are after 1 hour in the system, and the new synthetic RNAs of extraction carries out SDS-PAGE with it and analyzes radioautograph again.The result is shown in Fig. 3 C, and mt-1, mt-2, mt-3, mt-5 have lost PC4 albumen basically as transcribing the auxilliary function that activates son.

3) protein arrays analysis experiment

Protein arrays analysis experiment is based on " general protein arrays " method of (also claiming UPA) (referring to document Ge 2000).Will be in a large number purified proteins be fixed on the nitrocellulose membrane by certain arrangement regulation, form intensive lattice array, the about 1mm of every spot diameter includes the albumen (size that depends on albumen) of 10～100ng.With PC4 albumen on the lattice array interactional target protein takes place and can utilize specific antibody (antibody of for example anti-transcription factor TFIIA, Gal4, Sp1) to detect, as a result Fig. 4 C, Fig. 4 D, Fig. 4 E.

If detect the interaction of albumen and DNA, utilize the autoradiographic technique monitoring mark to have ³²The dna probe of P-dCTP and the DNA that has been bound.The result is shown in Fig. 4 F.

Can find out that by Fig. 4 the phenylalanine in the 77th site is absolutely necessary for PC4 combines ssDNA.In addition, the interaction of F77P saltant PC4 and transcription factor TFIIA and Sp1 significantly strengthens.

In sum; The PC4 that single amino acids sudden change (F77P) taken place except the 77th site has lost ability and the transcriptional activity that combines DNA; The The above results proof also greatly reduces in the activity that the PC4 albumen of dual sudden change (K23I/K29A and K35I/K41A) and triple mutant (R27A/K28I/K29A) takes place N-end group local area; Therefore PC4 mutant: F77P, K23I/K29A, K35I/K41A, R27A/K28I/K29A can be used as cancer therapy drug, reach the effect that treatment depends on the cancer of PC4 through competing with endogenous wild type PC4.

Embodiment 2

Short polypeptide with the amino acid sequence homologous in the activation structure territory of PC4

The inventor designs and has synthesized two peptide species; Their sequence is consistent with two PC4 activation structure domain amino acid sequences respectively; Proved that through above-mentioned protein arrays analysis (test experience interacts between the protein-protein) and in-vitro transcription test experience this two peptide species can combine other to activate son with wild type PC4 competition through the inventor, thereby reached the active effect of PC4 that suppresses.

PC4-AD15, sequence KRKKQVAPEKPVKKQ sees SEQ ID No.1;

And the short polypeptide that is connected with the migration domain accordingly, like TLD-AD15, sequence: PLSSIFSRIGDPKRKKQVAPEKPVKKQ sees SEQ ID No.2;

PC4-S15, sequence D SDSEVDKKLKRKKQ sees SEQ ID No.3;

And the short polypeptide that is connected with the migration domain accordingly, like TLD-S15, sequence: PLSSIFSRIGDPDSDSEVDKKLKRKKQ sees SEQ ID No.4.

Embodiment 3

The simulation activation structure domain polypeptide that combines with PC4

(sequence is the amphipathic helix polypeptide: ELQELQELQALLQQQ; See SEQ ID No.5) can simulate a kind of activation structure territory and PC4 generation strong interaction; Sealed the activation structure territory of PC4, suppress PC4 and endogenous activation and interact, thereby it has been active to suppress PC4;

And the short polypeptide that is connected with the migration domain accordingly, like TLD-AH15, sequence: PLSSIFSRIGDPELQELQELQALLQQQ sees SEQ ID No.6.

Embodiment 4

Utilization suppresses the compound of PC4 protein active based on the high flux screening detection system screening of FRET technology.

Screen the compound of inhibition PC4 protein active of the present invention and use amphipathic helix (AH) the domain polypeptide that is marked with fluorescence as energy donor, the PC4 albumen of mark fluorescent is as energy acceptor.In case PC4 and amphipathic helix domain polypeptide form complex; Promptly owing to the interaction force between two molecules makes close between donor and the acceptor (apart from about 1～10nm); What observed this moment mainly is the emission light of PC4, because intermolecular fluorescence resonance energy is transferred to PC4 albumen from amphipathic helix domain polypeptide.The exciting light of amphipathic helix domain polypeptide and the emission light of PC4 receive the monitoring that fluorescence intensity is read the plate appearance.The effect of the inhibition PC4 protein active of any compound can both be measured through the variation of monitoring the fluorescence intensity that is read.General screening two compounds: 1) micromolecule complex; 2) native compound.Certainly, these compounds also need further be confirmed, prove that it can become candidate's anticarcinogen that a kind of potential treatment depends on the malignant tumour of PC4.

The micromolecule complex comprises commercial the developing with some scientific research institution inside of obtaining of synthesizing.Selected compound is kept in 96 orifice plates or 384 orifice plates, and sets up their a detailed data storehouse.Each hole on the reaction plate all has 10～50ul to comprise the compound to be detected that concentration is 1mM (384 orifice plates are used 10ul, and 96 orifice plates are used 50ul) reaction mixture, and has also comprised PC4 albumen and the amphipathic helix domain polypeptide that is marked with fluorescence in the reaction mixture.4 positive control holes (use concentration as the unlabelled amphipathic helix domain of 10mM polypeptide surrogate markers fluorescence) and 4 negative controls (water substitutes fluorescently-labeled amphipathic helix domain polypeptide as energy donor) are all arranged on each plate.Read the OD value that plate appearance (Molecular Dynamics company produces) reads OD535 with fluorescence intensity.The anticancer effect of the positive molecule that screens need adopt the different detection method to come further to confirm, described different detection method comprises that external protein-protein interaction detects, transcribes the drug effect that adopts cell model and animal model to carry out before detection and the clinical testing and detects.Most prescription medicine all is a native compound, and anticarcinogen more than 50% and the anti-infectious agent that can buy on the market all come from natural products.

For identifying that can native compound suppress the activity of PC4 albumen; Can choose the compound that some derive from soil microorganism, marine microorganism, plant or other nature materials, utilize high throughput system that their screenings are detected based on the FRET technology.The starting material that screen can be further purified again, identify and in some above-mentioned different detection systems, further confirm again.

Above-mentioned summary of the invention and embodiment only for illustrating inventive concept of the present invention and main points, can not limit protection scope of the present invention with this.All according to embodiments of the invention and combine equivalence that spirit of the present invention does to change or modify, these all are that those skilled in the art can expect, all should be encompassed in protection scope of the present invention.Moreover, relevant position mark in the text all such as the teaching material of all references or other document publications here.

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Claims (9)

1. method of screening the treatment or the medicine of prophylaxis of cancer; Described medicine has comprised the PC4 antagonist of effective dose; It is characterized in that: described PC4 antagonist is for modify or do not have function PC4 polypeptide or albumen, and this method comprises following two steps that take place successively: (1) provides a kind of test of transcriptional level of the PC4 of detection albumen; (2) in above-mentioned test, add drug candidate, wherein, reduce the medicine of the drug candidate of PC4 transcriptional level for treatment or prophylaxis of cancer.

2. the method for the medicine of screening treatment according to claim 1 or prophylaxis of cancer is characterized in that: described to have modified or do not had function PC4 albumen be saltant PC4 albumen.

3. the method for the medicine of screening treatment according to claim 2 or prophylaxis of cancer is characterized in that: described saltant PC4 albumen is F77P, K23I/K29A, K35I/K41A, R27A/K28I/K29A saltant PC4 albumen.

4. according to the method for the medicine of claim 1 or 2 or 3 described screening treatments or prophylaxis of cancer, it is characterized in that: describedly modified or do not have the PC4 albumen that function PC4 albumen is phosphorylation.

5. the method for the medicine of screening treatment according to claim 1 or prophylaxis of cancer is characterized in that: described modified or do not have function PC4 polypeptide for the short polypeptide of the amino acid sequence homologous in PC4 activation structure territory.

6. the method for the medicine of screening treatment according to claim 1 or prophylaxis of cancer is characterized in that: described modified or do not had function PC4 polypeptide be the simulation activation structure domain polypeptide that combines with PC4.

7. according to the method for the medicine of claim 1,2,5 or 6 described screening treatments or prophylaxis of cancer, it is characterized in that: described PC4 antagonist is polypeptide or the protide PC4 antagonist that is connected with migration domain polypeptide.

8. the method for the medicine of screening treatment according to claim 7 or prophylaxis of cancer is characterized in that: described migration domain peptide sequence is shown in SEQ ID No.7.

9. the method for the medicine of screening treatment according to claim 8 or prophylaxis of cancer is characterized in that: the amino acid sequence of described polypeptide class PC4 antagonist is shown in SEQ ID No.2 or SEQ ID No.4.

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