CN1785432A - Chromatin peptide medicinal molecule for sealing MYC cell proliferation path - Google Patents
Chromatin peptide medicinal molecule for sealing MYC cell proliferation path Download PDFInfo
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Abstract
A chromatin peptide medicine molecular able to specifically block the cell reproduction path of MYC cancer cell for people and mammal is the DNA affinity structure domain sequences or their bionic molecule containing 3-100 amino acid residues. The can be specifically and firmly combined with the promoter of MYC family gene or its downstream gene to stop the over-expression of MYC family gene and the downstream transcription factor and cell growth factor gene, so blocking the malignant reproduction path of cancer cells.
Description
Technical field
The invention belongs to human genome and malignant tumor biology self treatment approach/technical field.The present invention relates to a class in regeneration soma cell renewal process, seal the take advantage of a situation biological activity chromatin peptide drug molecule of controlling element Cis-RE of dominoes phenotype genes group on the specific chromatin masterplate.These drug molecules can be firmly bonded to specifically with most of cancer and take place on the enhancers/promoters/silencer of the downstream related gene chromatin masterplate that relevant MYC proto-oncogene family and they are driven, and close muchly because dominoes/Domino formula gene transcription regulation waterfall/Transcriptional regulatory cascades cell that causes out of control that the overexpression of MYC genoid causes does not have to control breeds.By rebuilding the orbit of cancerous cell MYC gene cluster, change cancerous cell into normal phenotype by the malignant phenotype, recover the orderly multiplication regulatory of cell.These molecules are the medicines that effect a permanent cure that can be used for the treatment of the pernicious nothing control of most of cancerous cell propagation.
Background technology
All have every day thousands of people to be tormented by malignant tumor and cause death, but have more people to be escaped death by a hair's breadth in addition, the band tumor is survived for many years, even is completely recovered; Evidence suggests that human body produces 30-100 cancerous cell every day.All have every day cancerous cell in health, constantly to take place and eliminate, but under most of situation, the people is but not sick because of the continuous generation of cancerous cell.Human health itself shows the inhibition ability to the tanacity of cancer, even the healing ability.Health is the cancer of what mode to have cured many patients by actually so, what mechanism cancerous cell is again by, the escaped control of health, do you cause many patients in the fatal position? we find to exist the chromatin peptide of some biologically actives in human body, they are the bonded domain of certain transcription factor and DNA/DNA-binding Domain fragments: in the genetic transcription process, transcription factor specific recognition and being combined on the controlling element/gene promoter of feature, the transcribing of activated gene; Allosteric and break away from controlling element then; The downstream gene dominoes formula interlock that drives when transcription factor is subjected to being limited by some external condition, such as anchorage and spatial constraints, the transcription factor protein excess accumulation of expressing gets up, will the elicitor protein combined enzyme agent/proteasome reaction and be degraded to these chromatin peptides, they remain with the specificity affinity of original transcription factor to its DNA-substrate, can be attached to transcription factor encoding gene and by the binding site of the downstream gene regulated and control and negative feedback ground seals these gene expressions.Many proto-oncogenes that cause tumor generation/deterioration excessively transcribe all be by this life entity inherent " a disconnected spoon blackout effect " sealed own and the downstream by the overexpression of controlling gene, thereby close the malignant proliferation of place cell securely; This may be the basic reason that many cancer patients are fully recovered or survive with tumor for a long time; The cell hyperproliferation that the lasting overexpression that more patient dies from pernicious gene causes may be that its health does not have to cause the chromatin peptide of enough " disconnected spoon blackout effect ", closes the overexpression of pernicious gene.Therefore, synthetic and additional these chromatin peptides are closed the overexpression of pernicious gene, and then close the malignization propagation of its place tumor cell, just promise to be the optimal path of curing cancer.We have found that some can close the chromatin peptide of some crucial pernicious gene expression.
Each reaction all is subjected to the adjusting of Cis and Trans both direction in vivo.The adjusting of this both forward and reverse directions, the balance of having kept biological vital movement is orderly.This adjusting must relate to two adjusting molecules that are mutually related.Based on the thrifty principle of biological evolution, a kind of molecule is after the adjusting of having finished a direction, and allosteric or primary structure change, and weaken or suppress the reaction that it promotes, and become the adjusting molecule of another one direction.The precursor transcription factor of Trans chromatin peptide and it is to closely question anti-ly mutually on function, and in this way, the molecule in common source is finished the adjusting of Cis and Trans both direction.
Chromatin peptide is a kind of oligomeric peptide, is the catabolite of interior transcription factor of organism and auxilin, generally contains 3-100aa.They are certain transcription factor and auxilin and the bonded domain of DNA/DNA-binding Domain fragment.These chromatin peptides can be discerned and be attached on the downstream related gene promoter that the promoter of this transcription factor gene own and it is driven, and seal these promoteres and close the expression out of control of these genes.
The autonomy growth and the propagation of cell come down to a kind of by the dominoes formula interlock of the genetic transcription under its chromatin masterplate standard, when this interlock network disturbed by some cell internal and external factors and when stopping (such as contact inhibition, the anchorage lacks), too much transcription factor protein will deposit in the upstream of this gene expression waterfall, and then elicitor protein combined enzyme agent/proteasome/ hydrolysis, decompose these junks, wherein identification and affine domain/DNA-binding Domain/ fragment are retained between those transcription factor and its effect substrate DNA, be combined on the substrate DNA, seal and closed this gene expression waterfall out of control and the activity of cell growth/propagation, key just as a disconnected handle, sealed lockhole, blocked other key and opened the door the same again; Therefore, the disconnected spoon reaction of gene key/transcription factor and chromatin peptide are the gene expression physiological functions out of control that defence endogenous gene that a kind of life evolves out suddenlys change and causes.Chromatin peptide is the material that a class is similar to antibody, in pernicious transcription factor excessively after the alluvial, health can produce q.s chromatin peptide, generally speaking, can guard and remove " foreign body " of health variation generation itself, as, cancerous tumor cell.When self protection capability is not enough to defeat this " foreign body ", when safeguarding the permanent existence of life,, replenish and strengthen the protection capability of life self by complementary stain matter peptide mode, just can safeguard the continuity that life is more permanent.We just can control endogenous pathogens-malignant cell disease with the method for replenishing relevant chromatin peptide.
Relation between Trans transcription factor and its deutero-chromatin peptide is the function antagonism.Between this protein/derived peptide there be the function antagonism, all include histogenetic Cis-transcriptional regulatory element (being the Trans-transcriptional regulatory element of histo-differentiation) on the promoter of these three cell proliferation promotor genes of HIF-1 α/C-FOS/C-MYC, erythrogenic Cis-NFE2 element on the HIF-1 α, the cytogenetic Cis-NFAT element of T-on cytogenetic Cis-MITF element of B-on the C-FOS and the MYC, they are clearly when cell development is ripe, when starting histo-differentiation functional gene group, on the chromatin track of HIF-1 α/C-FOS/C-MYC genetic transcription campaign, sealed the binding site of embryonic stem cell transcription factor, thereby reconstruct the chromatin phenotype of cell, period of embryo stem cell progressivity propagation phenotype is reconstructed into the dynamical balance feature propagation phenotype of period of maturation noble cells.
Specific recognition between protein/protein or the protein/DNA and combination are the basic modes of biomolecule communications activity, and the combination between the two generally is the combination of inlaying between the solid structure/key-lock mode.Combination between the adjusting subunit/domain of two kinds of complete sequence molecules can cause the enforcement of the allosteric and the function of function subunit, and two kinds of molecules then are separated from each other then, realizes trans adjusting/Trans-regulating function that dynamic function or information are transmitted; When between the domain of a kind of domain of the molecule of degrading and another kind of complete sequence molecule in conjunction with the time, will form certain static and permanent cis adjusting/Cis-regulating phenotype combination.Therefore, chromatin peptide/transcription factor is a kind of biomolecule network stabilization effect, and the stabilizing effect that the disconnected spoon blackout effect of gene expression network then is a kind of cyto-chromatin masterplate and growth phenotype is closed cell in stable phenotype.From the dynamic fluctuation to the steady stability, Here it is cell/be the principle of mobile equilibrium of propagation.
The domain sequence of transcription factor, promptly the chromatin peptide sequence when the sequence long enough, contains more hydrophobic amino acid, and when they were in free state, difficulty was degraded by proteolytic enzyme because low water solublity is arranged; When being in combined state between the two, this domain part also can not be degraded because protease molecule is difficult to approaching; Because these chemical features of domain cause these structures often to escape death by sheer luck in the reaction of protease complex, also than complete sequence albumen long half time, effective efficiency dosage is much lower for hydrophobic derived peptide.In addition, we select to be used for the chromatin peptide of Drug therapy, contain more basic amino acid, help medicine and concentrate to the cancer piece position of little acid environment.
In sum, the autonomy propagation activity of cell is limited in the interior limited space of cell; The directed movement of these two dominoes formula interlock weave ins of gene expression and the effect of representation protein is actually a kind of a plurality of gene group switch interlock, and a gene group starts, and promptly closes after the activity, then starts downstream gene group's activity; After each protein forward activity is finished, oneself need be decomposed, for activity space is abdicated in the activity of back; Sometimes also to close this protein expression or effect, so that enter next proteinic interlock; So life entity in evolution of long period of time, is practised austerities out a this protein molecule, finish the startup of a functional activity and close the laborsaving coordinating effect of two switch motions.
It is traditional that to kill and wound treatment be that in fact, many important healthy cells, bone marrow, epithelial cell or the like are all bred also sooner than tumor cell at the cell that is in vegetative state.These treatments all are to be cost to sacrifice more healthy cells; Traditional chemotherapy that Here it is, the common disadvantage of radiotherapy and tcm therapy.Immunization therapy and gene therapy are can accomplish to malignant cell optionally, and particularly anti-angiogenic treatment does not even injure normal cell.It is because there is not the immunogen of tumor cell specific that immunization therapy is not prove effective, existing studies show that, and antigen of malignant cell and normal cell comparison, the difference of the amount of having only, and do not have the difference of character; At present, gene therapy do not prove effective be because choosing wrong the target site of medicine: promptly drug target is positioned at gene prod, moment is expressed the back with regard to the dynamic mRNA of pop-off or above the protein, the pernicious kinetic energy with interference cell can only be temporarily changed, and its malignant phenotype can not be for good and all changed and seal.Have only drug target is positioned on the controlling element of the DNA track of stable static standard gene dominoes formula interlock and gene expression, could change muchly with closing cell's malignant phenotype and realize radical cure.
The MYC gene family is the class transcription factor encoding gene relevant with the cells contacting communication.The expression of MYC genoid is relevant with the anchorage dependence Growth Control with the space of cell with inhibition.The MYC gene family gene of finding at human body comprises MYC, NMYC, three kinds of genes of LMYC at present.The MYC family gene has similar structure and close homology.Studies show that they have similar function in vivo.They all contain 3 exons and 2 introns.Though their first exon does not show homology, this exon does not have coded protein.Yet the zone that has homology in exon 2 and 3 reaches 5.The c-terminus sequence homology is present in 85 aminoacid of MYC family, the alkalescence zone that one of them is important, and the aminoacid of three kinds of MYC encoding proteins is formed and sequence has high conservative and homology, and this is the bonded domain of they and DNA.Like this, just point out a kind of probability, design one or molecule, can seal the gene of all MYC families.
Under the normal condition, human MYC gene is positioned on the 8q24; NMYC gene mapping is in 2p25; LMYC is positioned on the 1p32.
Cloned and identify from MYC, the research paper of more than the 20 year relevant MYC family gene of medical research document is above ten thousand pieces.MYC becomes the important target position gene of the Therapeutic Method of research proto-oncogene and cancer.
Phosphoric acid albumen Myc in the nuclear of MYC coding forms heterodimer Myc/Max and Myc/Mad with other two kinds of nuclear protein Max and Mad respectively in nuclear.The same with other most of transcription factor, Myc has two domains that function is different: with other albumen and and bonded Binding Dormain of DNA and performance regulatory function active structures territory.85 aminoacid of C end are for Myc and Mad, and the combination of Max and DNA is essential.The N end contains the transcriptional activation domain of Myc, and transcribes inhibition, and Cycle Regulation transforms the zone that apoptosis etc. are required.The N end is except the albumen P107 with RB family interacts, also with α tubulin and some new adapter protein B IN1, MM1, TAM, interaction such as TRRAP and AMY1, these proteic structures and they show the effect of MYC effect, interaction and transcriptional regulatory system and the Cycle Regulation of they and Myc, and chromatin structure and apoptosis are related.Except with Max and Mad combine, Myc C end also participates in the inhibition of transcribing of some growth promoting genes, plays a significant role in the cell transformation of Myc mediation.And its C end combines formation and heterodimer Myc/Mad and Myc/Max with Mad and Max, the Trans and the Cis that finish respectively MYC gene and its regulation and control related gene regulate, just as two structures that the Push And Release of switch closes, the transcriptional activity of leading MYC and related gene.In addition, C end also with other cell protein YY1, AP2, BRCA1, TFIII and MIZ1 interact; In addition, a lot of expression of gene are active relevant with MYC in the human body, show the complexity of its regulatory function.
Know that now Myc is a class transcription factor.The related gene of this transcription factor and its regulation and control has many-sided function.Cell cycle, cell differentiation, apoptosis, cell transformation, angiogenesis, gene stability etc., all relevant with MYC.There are some researches show that in addition some with cancer some relevant genes take place, as, Ras, it has only the activation by MYC, just can cause the malignant proliferation of cancerous cell.
Existing data shows, all is attended by the overexpression of MYC in human most of cancer patient's malignant cells, also have the interior MYC copy number of some patient's cancerous cell to become multiple, even the Radix Achyranthis Bidentatae number increases.Studies show that the MYC gene is in the Burkitt lymphoma, non-Burkitt lymphoma, breast carcinoma, cervical cancer, carcinoma of prostate, human primary gastrointestinal cancers, osteosarcoma, melanoma, teratoma, granulocyte and plasma cell and bone-marrow-derived lymphocyte leukemia, glioblastoma, overexpression in neuroblastoma and the small cell lung cancer, nasopharyngeal carcinoma malignant cell; On the one hand, everybody recognizes now in addition, and the synergism that activates proto-oncogene is the key that makes malignant transformation of cells: in most cases, the generation of cancer is relevant with two or more proto-oncogene sudden changes, and for example the generation of cervical cancer is relevant with MYC and HRAS; Breast carcinoma and MYB, MYC, EGFR, HER2, HRAS is relevant with p53, pulmonary carcinoma and JUN, MYCN, MYCL, HRAS, RB1, p53, RAF1 is relevant, and such situation has popularity at human cancer.Along with increase, may find to have in the more malignant cell unconventionality expression of MYC to the data of the investigation accumulation of the expression conditions of human cancer.We infer that in the cell malignization breeding that the different genes variation causes, MYC plays crucial synergism.
The growth of cell is stoped malignancy to be had only from sealing and its chromatin track of configuration and does by chromatin track institute standard in its nucleus.The W.Felsher research group of U.S. Stanford University is by the transgenic mouse zoopery, MYC is closed simply, just transformation/reconstruct cell phenotype, become optimum (differentiation/apoptosis) phenotype from malignant phenotype's (uncontrolled proliferation) reconstruct, realized liver cancer piece atrophy and extinction (Nature.2004 Oct 28 in vivo respectively; 431 (7012): 1112-7.Epub2004 Oct 10.), osteosarcoma atrophy and apoptosis (Bernard Weinstein:Science 297 (5578) Issue of 5 Jul 2002 in vivo, and the disappearance of hematologic malignancies (Felsher, D.W.﹠amp pp.63-64.)); Bishop, J.M.Reversible tumorigenesis byMYCin hematopoietic lineages.Mol.Cell 4,199-207 (1999).These result of the tests have proved the feasibility of reconstruct chromatin phenotype.
Sealing MYC gene or removing and the active method of inhibition expression product can realize the reverse of cancerous cell.But, adopt the method for sealing MYC, can cut off from the source MYC transcription factor the source.With respect to back two kinds of methods, the sealing proto-oncogene has some advantage, first, the cancerous cell that can prevent optimumization of phenotype is muchly realized the reverse to the malignant phenotype once more, the second, and method is easy, the 3rd, human body is free from side effects, and the target position that uses clinically at present is the medicine of DNA normal cell is had very strong side effect.
Based on above analysis, sealing MYC gene and related gene, the chromatin orbit of reconstruct MYC to multiple treatment for cancer, has wide significance.
Summary of the invention
The chromatin peptide that the present invention relates to comes down to the oligomeric peptide molecule of the degraded of the transcription factor of MYC family gene expression and relative transcription factor of a class and auxilin, and their participate in the regulation and control of the cell proliferation of human body itself.Transcription factor is divided into two classes, Trans transcription factor and Cis transcription factor.The chromatin peptide in Trans transcription factor source, its effect is general opposite with their the precursor transcription factor of deriving; The chromatin peptide in Cis transcription factor source, its effect is identical with their the precursor transcription factor effect of deriving.The bionical molecule of some chromatin peptides has identical effect with natural chromatin peptide; The unordered overexpression that these transcription factor break through control causes human body self that the cancerous cell malignancy is monitored and scavenging system out of control, started the tumor cell malignant proliferation, and closed the unordered overexpression of the MYC genoid of these tumor cells from their derive/degrade chromatin peptides of forming, thereby constantly realized many cancer patients " recovery occurs without treatment ", bionical molecule also has similar effect; Present technique comprises, the youngster who filters out from the human genome data base plants the derivant of chromatin peptide and these chromatin peptides, they can seal the downstream related gene of proto-oncogene MYC family and its regulation and control, realize the reconstruct of MYC genoid orbit, cancerous cell is transformed to the normal cell phenotype by the malignant phenotype.For the cancerous cell that reverses phenotype, if the cell of differentiation, progressively death or apoptosis; If the cell of differentiation capability is arranged, as various stem cell, will be according to normal cell proliferation or differentiation.These chromatin peptides can be used for the treatment and the prevention of human various malignant tumor, are biologically active drug and the bionical medicines with the curative effect of effecting a permanent cure and preventive effect.
Therefore, the purpose of this invention is to provide:
1, one class can specificity the chromatin peptide drug molecule of the human and mammalian cancer cells MYC cell proliferation approach of sealing, this quasi-molecule is affine domain sequence of DNA or their the bionical molecule that screens from gene database, obtain by cistron analysis MYC protein transcription factor controlling element, comprise 3-100 amino acid residue, they can specificity be firmly bonded on the downstream gene promoter that the transcription factor of the promoter of MYC family gene and its expression drives, close MYC family gene and relevant downstream gene muchly, stop because the overexpression of the MYC family gene that sudden change of MYC family gene and related gene variation cause, and/or stop the downstream transcription factor of MYC family gene regulation and control and the overexpression of cell growth factor gene, thereby close for a long time by the MYC family gene escape the cell proliferation regulation and control and because of cell malignant proliferation approach.
2, above the 1st chromatin peptide drug molecule, its for 3-100aa scope any can seal the MYC family gene and with the chromatin peptide of all aminoacid sequences of MYC multiplication regulatory approach related gene.
3, above the 1st chromatin drug molecule, it is selected from as 7 chromatin peptide drug molecule (tables 1 among the sequence table sequence 1-7, sequence table) one or more in, perhaps with these molecules have minimum 3 amino acid homology series can seal the MYC family gene or with the chromatin peptide of all aminoacid sequences of MYC multiplication regulatory approach related gene.
4, have the minimum homologous series of 3 aminoacid and can seal MYC family gene promoter or any aminoacid sequence of the downstream related gene of MYC class transcription factor regulation and control with 7 drug molecules described in above the 2nd.
5, to the above the 1st, 2, the N end of 3 or 4 described chromatin peptide drug molecules and/or C end and/or side-chain radical carry out chemical modification, perhaps by the aminoacid additions and deletions, substitute resulting all can seal the chromatin peptide derivant of the downstream gene of MYC family gene promoter or the regulation and control of MYC class transcription factor.
6, a kind of pharmaceutical composition, it has comprised any one described chromatin peptide drug molecule or the derivant and the optional pharmaceutically acceptable carrier as above 1-5 item of pharmacy effective dose.
7, according to above the 6th pharmaceutical composition, it is ejection preparation, oral capsule and slow releasing agent.
8, described chromatin peptide drug molecule of any one of above 1-5 item or the derivant application in the medicine of preparation prevention or treatment people or mammal malignant tumor.
9, described chromatin peptide drug molecule of any one of above 1-5 item or derivant comprise the sealing MYC cell proliferation approach chromatin peptide that obtains with any method.
The sealer of sealing MYC family gene is by the cistron Analysis and Screening of MYC albuminoid transcription factor controlling element is come out.
MYC family gene sealer-chromatin peptide drug molecule can closing cell MYC proliferating way the affine domain of DNA-of related gene.
MYC gene sealer-chromatin peptide drug molecule sealing MYC family gene and downstream related gene has high degree of specificity, except sealing MYC family gene and downstream related gene thereof, to the not influence of activity of other gene.They have than normal cell allelic dna-much higher affinity of affine domain the MYC family gene and the downstream affine domain of related gene DNA-of tumor cell in addition.
These drug molecules can permeate through cell membranes, and nuclear membrane arrives target site, therefore, can be by intramuscular injection, intravenous injection, oral capsule, the method administration of venous transfusion approach and lump position in-situ injection and dermal osmosis.Dosage is according to patient's concrete condition, body weight for example, and the age, the concrete disease and the state of an illness, and doctor's clinical experience decides, but can be 0.05-30mg/ days usually, can be in single or divided doses.
In addition, these drug molecules can not be resisted gastric acid and pepsic digestion hydrolysis, therefore, and direct oral medication.
These drug molecules are by suppressing the overexpression of MYC family gene and downstream associated transcription factor and relevant somatomedin, the malignant cell relevant with the MYC family gene changed to the normal cell phenotype by the malignant phenotype, be used for the treatment and the prevention of the malignant tumor relevant with the MYC family gene.
Present research data shows, the generation of most of malignant tumor is accompanied by the overexpression of MYC family gene, and some is that some is the activation that other gene mutation has caused the MYC gene because the sudden change of MYC family gene itself causes; In addition theoretically, the sudden change of the downstream transcription factor of MYC family gene regulation and control and relevant somatomedin, also can cause and the identical cell malignant phenotype of activation MYC family gene, therefore, sealing MYC family gene and downstream related gene, prevention and treatment to most of malignant tumor have broad sense.
These drug molecules do not influence the homergy of organ and tissue to human body safety, do not influence tissue and cell and upgrade, and do not influence people's growth promoter.
These drug molecules can seal all members' of MYC gene family expression, comprise C-MYC, the downstream related gene of NMYC and LMYC and their regulation and control, and this has guaranteed the wide spectrum curative effect of medicine.
These chromatin peptides are carried out chemical process modify, may can improve cell membrane, the penetrating ability of nuclear membrane, prolong half-life strengthens the affinity to target position, therefore improves curative effect.
These drug molecules no matter how to obtain, as long as the characterization of molecules of this patent design is arranged, all have equal effect.
The accompanying drawing summary
Seven kinds of chromatin peptide medicines of Fig. 1 are to the inhibitory action of the growth of white mice 4T1 breast carcinoma.The cell counting of anti-6-thioguanine of original tumor locus.
In recent years, the research data to this proto-oncogene of MYC is a lot. Research finds that in most of solid malignant patient and fluid cancer patient's the malignant cell, the expression of MYC is very high, and the MYC gene has several times even 100 times to Normocellular copy number in some cancerous tissue. In adult's normal tissue cell, the MYC expression is very little. Even do not measure its expression, and in the histoorgan of fetus and children's Fast Growth, MYC has higher expression.
Someone is simple to seal or deletion MYC gene, just can realize that cancer cell phenotype is to the conversion of normal cell phenotype; Expression product with monoclonal antibody removing MYC also can temporarily suppress the pernicious breeding of cell. These study explanation, and the generation of many malignant tumours is relevant with the overexpression of MYC genoid. The transposition of MYC genoid and point mutation can activate their expression. In addition, may exist other gene mutation to cause the activation of MYC genoid, such as the inactivation of P53. Present research, the method for sealing MYC gene mainly is antisense RNA and DNA, does not obtain breakthrough, thereby does not solve root problem. The first, RNA's is stable poor; The second, in human body, RNA and DNA are difficult to nature permeate through cell membranes and nuclear membrane; The 3rd, by viral vectors transmission antisense DNA and RNA, technical sophistication, and also its risk is difficult to judge. And the expression product by monoclonal antibody sealing MYC, can not thoroughly block the source of pernicious albumen, malignant proliferation that can only the respite tumour, because the albumen half-life in vivo is shorter, the effect of the monoclonal antibody of anti-MYC encoding proteins and effectiveness time are very limited in addition.
Recognize at present, the regulation of cell proliferation of MYC approach is the control by cells survival space (directly contact communication between the cell): after physically mature, when some histocyte is injured and dead, just needing startup to face cytoproximal propagation replenishes, this cell proliferation still will be subject to the restriction of the health confined space, and normal solid tissue cell all is the propagation control that is subject to facing cytoproximal contact inhibition and anchorage dependence. The solid tissue cell comprises bone marrow cell and some fluid malignant tumour (its source is at marrow and other blood forming organ) these two kinds of controls of having escaped, and can cause dystopy transfer in cell in-situ malignant proliferation and the blood circulation. Know now, the outlet of body space control of this cell proliferation of escaping is exactly this famous proto-oncogene MYC, its upstream controlling element MAD/MAX accepts cells contacting inhibition etc. and transcribes the inhibition communication signal, controlling element Myc/MAX then accepts cell anchorage dependence etc. and transcribes the promotion communication signal, the transcription factor protein Myc of this gene expression starts the growth factor of a large amount of promotion cell proliferation, and direct active cell propagation/Cell cycle gene group. Disappearance or sudden change occur in the encoding gene of any one inhibition signal protein on this cell communication network, the pernicious/progressivity propagation/Progressive Proliferation approach that all may cause cell to enter; So the Cell cycle gene group that Myc drives is transcribed the defence line that waterfall has just been broken through this body space control. In fact, the overexpression of MYC is the common trait of many malignant cells really, therefore, effectively closes transcribing of this MYC gene, has become a kind of common strategy of cancer treatment in conceptual phase.
Close the MYC gene with the chromatin peptide molecule, have many good qualities. We close the chromatin peptide of MYC genoid at design, be human body self have natural molecule or bionical molecule, to normal cell and not injury of histoorgan. Chromatin peptide has less molecular weight (chromatin peptide that we adopt is the 3-100 peptide) and is easy to permeate through cell membranes and nuclear membrane. In addition, these chromatin peptides have more basic amino acid, and whole molecule is alkalescence, are conducive to drug molecule and concentrate to the nauseating lump position of slant acidity. These chromatin peptides are when having larger molecular weight in addition, with more hydrophobic amino acid, these hydrophobic amino acids make drug molecule in the aqueous solution, form special water inner structure, can resist the hydrolysis of protease, therefore, these drug molecules have preferably body internal stability and long half-life.
The most important thing is that the specificity of these drug molecule targets is higher. First, except with the promoter region of the related gene of the promoter region of transcription factor MYC gene and downstream regulation and control thereof in conjunction with, human genome do not have other position can with these drug molecule combinations, this species specificity has guaranteed except the related gene that MYC genoid and downstream are regulated and control, other gene can not be closed, and this is an importance of these drug molecule securities; Second, seldom Normocellular MYC and related gene are closed, and the allele of cancer cell can with these chromatin peptide specific bond, this may be that the DNA in Normocellular MYC gene code zone is in highly condensing state, is difficult for being combined with these chromatin peptides; And at malignant cell, the MYC encoding gene is activated, and is in to unfold state, is easy to and the combination of chromatin peptide drug molecule.
But, studies show that, especially in fetus and child development phase, MYC can directly drive the transcribing of startup molecule ClycD of cycle gene group, if with these chromatin peptide sealings cycle gene group, in fact just closed the critical function approach that the normal cell propagation such as the necessary blood cell of many healths and epithelial cell are upgraded. But just the same to the analysis of transcription factor HIF-1 α with us, transcriptional control waterfall is network rather than the single path of a complexity. Directly drive the transcribing of startup molecule ClycD of cycle gene group except transcription factor MYC, may have in addition other transcription factor to drive the transcribing of startup molecule ClycD of cycle gene group. Existing result of study proves that the regulation of cell proliferation network relevant with the MYC genoid is not direct single channel, but network system.
In normal adult, the MYC expression is very low or do not measure its expression at all, and still, from the evolution angle, really as everybody common recognition, human evolution's process is without any special oncogene of reason reservation. We infer, MYC may some in emergency circumstances starts at paedomorphosis and adult, promotes cell proliferation. In adult's normal structure, the MYC gene is in silence state or expression is relatively humble.
From above-mentioned analysis, the sealing MYC gene of our design and the chromatin peptide drug molecule of related gene are safe to human body. But about human gene regulated and control network details, a lot of parts also are in unknown state at present, and whole effects of Myc are familiar with fully. The security of the drug molecule that any target target is the MYC gene should can prove directly that the test of its security illustrates by other. The zoology safety testing is the result show, when rabbit and small white mouse reach the 4mg/kg body weight at ID (be equivalent to human design consumption 80 times), and long-term observation, the health of animal is not affected. Select average weight to test at the White Rabbit of 1500g and the small white mouse of 15g, the result shows that medicine is to the body weight gain of White Rabbit and small white mouse, without any impact (referring to embodiment).
Based on above analysis and test, we have designed the chromatin peptide drug molecule of sealing MYC family gene and regulated and control network related gene, are used for the treatment of human malignancies. Drug molecule and target location see Table 1 and sequence table.
Table 1 drug molecule, drug molecule on MYC target position and drug molecule derive the source transcription factor and auxilin
| Drug molecule | The drug molecule source of deriving | The target position sequence | |
| 1 | VKRRTHNVLERQRR | MYC HUMAN | CAC[GA]TG. |
| 2 | RSTHNEMEKNRRA | MAD HUMAN | 5′-CAC[GA]TG-3′. |
| 3 | RSSHNELEKHRR | MAD4 HUMAN | CAC[GA]TG. |
| 4 | KRAHHNA LERKRR | MAX HUMAN | 5′-CAC[GA]TG-3′ |
| 5 | RS THNELEKNRR | MXI1 HUMAN | 5′-CAC[GA]TG-3′. |
| 6 | NSLDP GLFVEST | MLX HUMAN | 5′-CACGTG-3′. |
| 7 | REVHNKLEK NRR | MNT HUMAN | E box sequence 5′-CACGTG-3′ and，with higher affinity，to 5′-CACGCG-3′. |
The chromatin peptide sequence that all are listed is described, can seals MYC-HUMAN.
Transcription factor and auxilin and their function that table 2 chromatin peptide is derived and originated
| The chromatin peptide precursor of deriving | The drug molecule sequence | The function of transcription factor and auxilin |
| MYC HUMAN | VKRRTHNVLERQRR | The overexpression of MYC is relevant with dissimilar malignant tumours. |
| MAD HUMAN | RSTHNEMEKNRRA | MAD and MAX form heterodimer, by competing MAX with MYC, the expression facilitation of opposing MYC. |
| MAD4 HUMAN | RSSHNELEKHRR | Form heterodimer with MAX, by competing MAX with MYC, the expression facilitation of opposing MYC. |
| MAX HUMAN | KRAHHNALERKRR | Brain, the heart, lung's level is higher. And liver, kidney, muscle and skeletal sites. |
| MXI1 HUMAN | RS THNELEKNRR | MAD and MAX form heterodimer, by competing MAX with MYC, the expression facilitation of opposing MYC |
| MLX HUMAN | NSLDP GLFVEST | TCFL4-MAD1, TCFL4-MAD4, TCFL4-MLX, compound are the expression inhibiting factors |
| MNT HUMAN | REVHNKLEK NRR | Form heterodimer with MAX and be combined DNA, suppress to transcribe |
Embodiment
Illustrate the present invention by the following examples.
Embodiment 1
Polypeptide by sequence 1-7 in solid-phase peptide synthetic technology (SPPS) the composition sequence table; comprise the aminoacid with the protecting group protection is added on the oligopeptide chain of growth step by step; this oligopeptide chain is anchored on the chemically stable granule; so that can be by simple filtration with synthetic peptide and reagent and separated from solvent; behind the end of synthesis; chain cracking from the carrier is got off, purify again.
Embodiment 2
The chromatin peptide drug molecule animal test of pesticide effectiveness of sealing MYC cell proliferation approach
Sealing MYC cell proliferation approach sealer is to the inhibitory action of white mice breast carcinoma
Inoculation back animal-use drug
The material method
Female nude mice HHc, age in 6-8 week is available from Qingdao Hailand research and development centre.
White mouse breast tumor cell 4T1 comes from Qingdao Hailand research and development centre.
Test chromatin peptide medicine (sequence 1-7), chemosynthesis.Purity 98.5%.
The mammary gland position contains cancer 4T1 cell (10 with 0.1ml
5) PBS suspension aseptic inoculation to the mammary gland of Healthy female Mus.Inoculum concentration rule of thumb determines, generally with in inoculation back 25 days, do not give the mouse of medicine, and the tumor that grows diameter 10mm is as the criterion.Inoculation back the tenth day when the lump diameter reaches 4-5mm, begins per 5 days by administered intramuscular once, matched group injection PBS, and dosage is the 0.08mg/kg body weight.Be administered once in per five days, up to the 25th day, administration was 4 times altogether.Every group of 5 Mus.The 30th day, observation was measured mammary gland tumor size (the results are shown in Table 2) and is got the former canceration of mammary gland position tissue and cultivate discrimination test with colongenic method cell
(1)Get the original cancer mammary gland of mouse position piece of tissue 5mm * 5mm * 5mm, get central part 0.1g again, chopping, with 37 ℃ of vibration digestion of 5ml enzymatic mixture (1XPBS, 1mg/ml four Collagen Type VI enzymes) 2 hours, after the digestion, sample filters with the cell nylon filter of 70 microns of diameters, with PBS washing 2 times.Suspension cell carries out serial dilution with containing 60 μ M thioguanine RPM culture medium 1640 in cell culture apparatus, detect pernicious growth.Because the 4T1 cell has resistance to thioguanine, to cultivate after 14 days, the cancerous cell of diffusion forms lump.Fix with methanol, with the green dyeing of 0.03% methylene, the pair cell counting.(the results are shown in Figure 1)
Interpretation of result
Table 3 chromatin peptide medicine is to the influence of white mice 4T1 growth of breast cancers
| Contrast | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| Diameter mm | 9.8mm | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Used kind of calliper after the attached kind of tumor on the 30th day.Because lump position lump out-of-shape is got maximum diameter and path meansigma methods.Chromatin peptide numbering and sequence table are together.
Fig. 1 shows the inhibitory action of seven kinds of chromatin peptide medicines to the growth of white mice 4T1 breast carcinoma.The cell counting of anti-6-thioguanine of original tumor locus.Chromatin peptide numbering and sequence table are together.
After the medication 30 days, the test Mus lump complete obiteration of 1-7 chromatin peptide drug treating group.All drug treatment group cancer pieces are in administration the 5th day beginning atrophy, and wherein, it is the fastest that No. 1 and No. 2 polypeptide processed group disappear, and after treatment for the first time the 9th day and the 16th day respectively, the lump complete obiteration.
Cell counting shows that the cancerous cell of the anti-6-thioguanine number at the original position of canceration of all drug treating groups all is reduced to not below 10% of treated with medicaments matched group.
The above results shows that the 1-7 chromatin peptide has stronger inhibitory action to white mice breast carcinoma.Need to prove that MYC class chromatin peptide sealer has reversed the phenotype of cancerous cell.But, may have the reverse that do not have death or apoptosis on a small quantity the cell that the oncogene sudden change is arranged.Whether these cells can reverse, and the re-initiation malignancy needs further test to be proved.
List of references
1?Pulaski，B.A.&?Ostrand-Rosenberg，S.(1998)Cancer?Res.58，1486-1493.
[Abstract]
Embodiment 3
The Canis familiaris L. advanced breast cancer shifts on a large scale.Measure earlier breast cancer disease Canis familiaris L. C-MYC, LMYC, the NMYC gene expression amount, the NMYC expression is 128 times of normal structure as a result.With the chromatin peptide sealer treatment of KRRTHNVLERQRR aminoacid sequence, subcutaneous direct injection, each 2mg, per 5 days are once.Cancerous protuberance began atrophy in the 5th day, checked that cancerous protuberance thoroughly disappeared on the 30th day, and there is cicatrix at the mammary gland position.
Utilize the chromatin peptide of this sequence can seal the dedifferenting of noble cells that causes because of the NMYC overexpression and the unordered propagation of the malignant cell that causes.Taking place in early days in tumor can direct drug injection and need not operation and radiotherapy.When lump is bigger, can medication before operation, when appropriate between excision changed the lump of benign cell phenotype and atrophy into.Also can the used after operation medicine, a small amount of malignant cell that may not remove fully operation carries out phenotype and transforms.
Embodiment 4
Patent first inventor, nasopharyngeal carcinoma, twice chemotherapy.After the chemotherapy 3 years for the second time, recurrence.Measuring nasopharyngeal cancer patient malignant cell C-MYC copy number is 55, and unconventionality expression.Through checking, pulmonary shifts, and does not have the transfer diffusion at other position.With the 0.05mg aminoacid sequence is that chromatin peptide and the 0.05mg sequence of RSTHNELEKNRR is the chromatin peptide of NSLDPGLFVEST, is made into the 5ml normal saline solution, aims at 4 corners, lump position and lump center, direct injection, and 15 days are once; By intravenous administration, each 10mg, per 15 days are once; Continuous use 3 months.Former position and pulmonary's lump disappeared vivisection, cancer-free cell in 3 months after the medication first time.
Embodiment 5
Measure Patients with Gastric Cancer malignant cell C-MYC unconventionality expression, main tumor piece 65mm * 70mm * 44mm, and be diffused into liver.With intravenous method administration anoxia sealer normal saline solution (5mg/250ml) (we invent the drug molecule of the design) first time, the 6th God's cancer piece spues.With quadrat method rechallenge (2mg/100ml), check after 20 days after 10 days, most of cancerous tissue necrosis, the cancer piece is loose to spue.With aminoacid sequence is the chromatin peptide normal saline solution intravenous injection of VKRRTHNVLERQRR, and for the first time dosage is 5mg/250ml, and later every interval 15 days is with the quadrat method injection, each 2mg/100ml, continuous use 4 months.Trace is expressed in biopsy after 4 months, cancer-free cell, pathogenic site cell C-Myc, returns to normal position level.
SEQUENCE?LISTING
＜110〉how quiet the Wang Chengqiu Bian Xiao village is
＜120〉the chromatin peptide drug molecule of class sealing MYC cell proliferation approach
<130>
<160>7
<170>PatentIn?version?3.1
<210>1
<211>13
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>1
Lys?Arg?Arg?Thr?His?Asn?Val?Leu?Glu?Arg?Gln?Arg?Arg
1 5 10
<210>2
<211>14
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>2
Val?Lys?Arg?Arg?Thr?His?Asn?Val?Leu?Glu?Arg?Gln?Arg?Arg
1 5 10
<210>3
<211>12
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>3
Arg?Ser?Ser?His?Asn?Glu?Leu?Glu?Lys?His?Arg?Arg
1 5 10
<210>4
<211>13
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>4
Lys?Arg?Ala?His?His?Asn?Ala?Leu?Glu?Arg?Lys?Arg?Arg
1 5 10
<210>5
<211>12
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>5
Arg?Ser?Thr?His?Asn?Glu?Leu?Glu?Lys?Asn?Arg?Arg
1 5 10
<210>6
<211>12
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>6
Asn?Ser?Leu?Asp?Pro?Gly?Leu?Phe?Val?Glu?Ser?Thr
1 5 10
<210>7
<211>12
<212>PRT
＜213〉artificial design
<220>
＜223〉promoter at the MYC gene designs
<400>7
Arg?Glu?Val?His?Asn?Lys?Leu?Glu?Lys?Asn?Arg?Arg
1 5 10
Claims (9)
1, one class can specificity the chromatin peptide drug molecule of the human and mammalian cancer cells MYC cell proliferation approach of sealing, this quasi-molecule is affine domain sequence of DNA or their bionical molecule, comprise 3-100 amino acid residue, they can specificity be firmly bonded on the downstream gene promoter that the transcription factor of the promoter of MYC family gene and its expression drives, close MYC family gene and relevant downstream gene muchly, stop because the overexpression of the MYC family gene that sudden change of MYC family gene and related gene variation cause, and/or stop the downstream transcription factor of MYC family gene regulation and control and the overexpression of cell growth factor gene, thereby close for a long time by the MYC family gene escape the cell proliferation regulation and control and because of cell malignant proliferation approach.
2, according to the chromatin peptide drug molecule of claim 1, its for 3-100aa scope any can seal the MYC family gene and with the chromatin peptide of all aminoacid sequences of MYC multiplication regulatory approach related gene.
3, according to the chromatin drug molecule of claim 1, it is selected from as in 7 chromatin peptide drug molecules among the sequence table sequence 1-7 one or more, perhaps with these molecules have minimum 3 amino acid homology series can seal the MYC family gene or with the chromatin peptide of all aminoacid sequences of MYC multiplication regulatory approach related gene.
4, have the minimum homologous serieses of 3 aminoacid and can seal MYC family gene promoter or any aminoacid sequence of the downstream related gene of MYC class transcription factor regulation and control with any one of 7 drug molecules described in the claim 3.
5, to claim 1, the N end of 2,3 or 4 described chromatin peptide drug molecules and/or C end and/or side-chain radical carry out chemical modification or by the aminoacid additions and deletions resulting all can seal the chromatin peptide derivant of the downstream related gene of MYC family gene promoter or the regulation and control of MYC class transcription factor.
6, a kind of pharmaceutical composition, it has comprised any one described chromatin peptide drug molecule or the derivant and the optional pharmaceutically acceptable carrier as claim 1-5 of pharmacy effective dose.
7, according to the pharmaceutical composition according to claim 6, it is ejection preparation, oral capsule or slow releasing agent.
8, described chromatin peptide drug molecule of any one of claim 1-5 or the derivant application in the medicine of preparation prevention or treatment people or mammal malignant tumor.
9, described chromatin peptide drug molecule of any one of claim 1-5 or derivant comprise the sealing MYC cell proliferation approach chromatin peptide that obtains with any method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106390096A (en) * | 2015-07-29 | 2017-02-15 | 北京京爱康科技有限公司 | Polypeptides for inhibiting proliferation of cancer cells, and uses thereof |
| CN106390095A (en) * | 2015-07-29 | 2017-02-15 | 北京京爱康科技有限公司 | Polypeptide for regulating cancer-related gene, and uses thereof |
| CN113499332A (en) * | 2021-08-24 | 2021-10-15 | 光明乳业股份有限公司 | Use of MYC agonists for the preparation of a medicament for promoting cell proliferation |
-
2005
- 2005-10-26 CN CNB2005101166166A patent/CN100484575C/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106390096A (en) * | 2015-07-29 | 2017-02-15 | 北京京爱康科技有限公司 | Polypeptides for inhibiting proliferation of cancer cells, and uses thereof |
| CN106390095A (en) * | 2015-07-29 | 2017-02-15 | 北京京爱康科技有限公司 | Polypeptide for regulating cancer-related gene, and uses thereof |
| CN113499332A (en) * | 2021-08-24 | 2021-10-15 | 光明乳业股份有限公司 | Use of MYC agonists for the preparation of a medicament for promoting cell proliferation |
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