CN100484575C - Chromatin peptide medicinal molecule for sealing MYC cell proliferation path - Google Patents

Abstract

A class of chromatin peptide drug molecules that can specifically block the proliferation pathway of human and mammalian cancer cells MYC, the molecules are DNA affinity domain sequences or their biomimetic molecules, containing 3-100 amino acid residues, they can Specifically and firmly bind to the promoters of MYC family genes and the promoters of downstream genes driven by their expressed transcription factors, permanently shut down MYC family genes and related downstream genes, and terminate MYC caused by mutations in MYC family genes and related gene variations Overexpression of family genes, and/or termination of overexpression of downstream transcription factors and cell growth factor genes regulated by MYC family genes, thereby long-term closing of malignant cell proliferation pathways caused by escape of cell proliferation regulation by MYC family genes.

Description

A Class of Chromatin Peptide Drug Molecules Blocking MYC Cell Proliferation Pathway

technical field

The invention belongs to the field of human genomics and biological self-treatment approach/technology of malignant tumors. The invention relates to a class of biologically active chromatin peptide drug molecules that block the homeopathic regulatory element Cis-RE of the domino phenotype gene group on a specific chromatin template during the renewal process of regenerated body tissue cells. These drug molecules can specifically and firmly bind to the MYC proto-oncogene family related to most cancers and the enhancers/promoters/silencers of the chromatin templates of the downstream related genes driven by them, and permanently shut down the MYC Uncontrolled proliferation of cells caused by dominoes/Domino-style gene transcriptional regulation cascades/Transcriptional regulatory cascades caused by overexpression of similar genes. By reconstructing the orbit of the MYC gene family of cancer cells, the cancer cells can be transformed from a malignant phenotype to a normal phenotype, and the orderly proliferation regulation of cells can be restored. These molecules are curative drugs that can be used to treat the malignant uncontrolled proliferation of most cancer cells.

Background technique

Thousands of people are tortured to death by malignant tumors every day, while many more people survive with tumors for many years, and even recover completely; evidence shows that the human body produces 30-100 cancer cells every day. Cancer cells are constantly generated and eliminated in the body every day, but in most cases, people do not get sick because of the continuous generation of cancer cells. The human body itself shows a tenacious ability to suppress cancer, and even cure it. So how does the body cure many patients of cancer, and what mechanism does the cancer cell escape the control of the body, causing many patients to die? We found that there are some biologically active chromatin peptides in the human body, which are the structural domains/DNA-binding Domain fragments of certain transcription factors and DNA: during gene transcription, transcription factors specifically recognize and bind in the characteristic On the regulatory element/gene promoter, activates the transcription of the gene; then allosterically and disengages from the regulatory element; when the domino-like linkage of the downstream gene driven by the transcription factor is restricted by certain external conditions, such as anchorage and space constraints, the transcription of the expression When the factor protein accumulates excessively, it will trigger the protease complex/proteasome reaction and be degraded into these chromatin peptides, which retain the specific affinity of the original transcription factor for its DNA-substrate, and can bind to the transcription factor coding gene and be Binding sites of regulated downstream genes negatively feedback block the expression of these genes. The over-transcription of many proto-oncogenes that lead to tumorigenesis/deterioration is through the inherent "key off effect" of this living body to block the overexpression of itself and downstream regulated genes, thereby firmly shutting down the malignant proliferation of the cells; This may be the root cause of many cancer patients being cured or living with cancer for a long time; more patients died of excessive cell proliferation caused by the continuous overexpression of malignant genes, which may be due to their body not being able to trigger enough "break key off effect" ” chromatin peptides to shut down the overexpression of malignant genes. Therefore, synthesizing and supplementing these chromatin peptides to turn off the overexpression of malignant genes, and then turn off the malignant proliferation of tumor cells where they are located, is expected to be the best way to cure cancer. We identified chromatin peptides that shut down the expression of certain key malignant genes.

Every reaction in the organism is regulated by Cis and Trans. This positive and negative regulation maintains the balance and order of biological life activities. This regulation necessarily involves two interrelated regulatory molecules. Based on the principle of biological evolutionary thrift, after a molecule is regulated in one direction, its allosteric or primary structure changes, weakening or inhibiting the reaction it promotes, and becomes a regulating molecule in another direction. Trans chromatin peptides and their precursor transcription factors are functionally antagonistic to each other. In this way, the common source molecules complete the regulation of both Cis and Trans directions.

Chromatin peptide is an oligomeric peptide, which is the degradation product of some transcription factors and auxiliary proteins in organisms, generally containing 3-100aa. They are domains/DNA-binding Domain fragments of certain transcription factors and auxiliary proteins that bind to DNA. These chromatin peptides can recognize and bind to the promoter of the transcription factor gene itself and the promoters of the downstream related genes driven by it, block these promoters and shut down the uncontrolled expression of these genes.

The autonomous growth and proliferation of cells is essentially a domino linkage of gene transcription under the regulation of its chromatin template. When this linkage network is blocked by some intracellular and extracellular factors (such as contact inhibition, anchorage deficiency), the upstream of this gene expression cascade will accumulate too much transcription factor protein, and then stimulate the protease complex/proteasome/hydrolysis reaction to degrade these waste products, among which the recognition and affinity between those transcription factors and their substrate DNA The structural domain/DNA-binding Domain/fragment of and is retained, combined with the substrate DNA, blocking and closing this uncontrolled gene expression cascade and cell growth/proliferation activities, like a key with a broken handle, blocking The keyhole is like blocking other keys to open the door; therefore, the gene key/transcription factor breaking key response and chromatin peptide are physiological functions evolved by life to defend against uncontrolled gene expression caused by endogenous gene mutations. Chromatin peptides are a class of substances similar to antibodies. After excessive deposition of malignant transcription factors, the body will produce sufficient amounts of chromatin peptides. Under normal circumstances, it can monitor and remove the "foreign bodies" produced by the body's own mutations, such as, cancerous cells. When one's own defense ability is not enough to defeat this "foreign body" and maintain the permanent survival of life, by supplementing chromatin peptides to supplement and enhance the defense ability of life itself, it can maintain a longer continuation of life. By supplementing relevant chromatin peptides, we can control endogenous pathogen-malignant cell diseases.

The relationship between Trans transcription factors and their derived chromatin peptides is functionally antagonistic. The phenomenon of functional antagonism between this protein/derived peptide is that the promoters of the three cell proliferation initiation genes, HIF-1α/C-FOS/C-MYC, all contain Cis-transcriptional regulatory elements for histogenesis (i.e., tissue differentiation Trans-transcriptional regulatory element), the Cis-NFE2 element generated by erythrocytes on HIF-1α, the Cis-MITF element generated by B-cells on C-FOS and the Cis-NFAT element generated by T-cells on MYC, they It is clear that when the cell matures, the tissue differentiation functional gene group is activated, and the binding site of the embryonic stem cell transcription factor is blocked on the chromatin track of the HIF-1α/C-FOS/C-MYC gene transcription movement, thereby The chromatin phenotype of cells is reconstructed, and the progressive proliferation phenotype of embryonic stem cells is reconstructed into the dynamic equilibrium proliferation phenotype of mature differentiated cells.

The specific recognition and combination between protein/protein or protein/DNA is the basic way of life molecular communication, and the combination between the two is generally a mosaic/key-lock combination between rigid structures. The combination of the regulatory subunits/domains of the two full-sequence molecules will trigger the allosterism and function of the functional subunits, and then the two molecules will be separated from each other to achieve dynamic functions or trans-regulation of information transmission/Trans -regulating functional role; when the domain of one degrading molecule binds to the domain of another full-sequence molecule, a certain static and long-lasting cis-regulating/Cis-regulating phenotypic combination is formed. Therefore, the chromatin peptide/transcription factor is a stabilizing effect of the molecular network of life, and the key closing effect of the gene expression network is a stabilizing effect of the cell chromatin template and the growth phenotype, closing the cell to a stable phenotype. From dynamic fluctuation to static stability, this is the dynamic balance principle of cell/line proliferation.

The domain sequence of transcription factors, that is, the chromatin peptide sequence, when the sequence is long enough, contains more hydrophobic amino acids. When they are in the free state, they are difficult to be degraded by proteolytic enzymes because of their low water solubility; when the two When in the bound state between the two, this domain part cannot be degraded because of the inaccessibility of the protease molecule; due to these chemical characteristics of the domain, these structures often survive the reaction of the protease complex, and the hydrophobic derivatized peptide also Longer half-life than full-sequence proteins and much lower effective functional doses. In addition, the chromatin peptides we choose for drug therapy contain more basic amino acids, which is conducive to the concentration of drugs to the cancer mass in a slightly acidic environment.

To sum up, the autonomous proliferation of cells is confined to a limited space within the cell; the two domino-like intertwined directional movements of gene expression and expressed protein action are actually a kind of Multiple gene group switches are linked, one gene group is activated, and then it is closed after the activity, and then the activity of the downstream gene group is activated; after the positive activity of each protein is completed, it needs to degrade itself to make room for the subsequent activities; Sometimes it is necessary to shut down the expression or function of this protein in order to enter the linkage of the next protein; therefore, in the long-term evolution of living organisms, this kind of protein molecule has been cultivated to complete the activation of a functional activity and the closing of two switching actions. Effort-saving coordination effect.

Traditional killing therapy is aimed at proliferating cells. In fact, many important healthy cells, such as bone marrow and epithelial cells, proliferate faster than tumor cells. These treatments are all at the cost of sacrificing more healthy cells; this is the common drawback of traditional chemotherapy, radiotherapy and traditional Chinese medicine. Immunotherapy and gene therapy can be selective for malignant cells, especially anti-vascular therapy without even harming normal cells. Immunotherapy is ineffective because there is no tumor cell-specific immunogen. Existing studies have shown that compared with normal cells, the antigens of malignant tumor cells are only quantitatively different, but not qualitatively different; at present, gene therapy is ineffective because Choosing the wrong target of the drug: that is, positioning the drug target on the dynamic mRNA or protein that disappears instantly after the gene product is expressed instantaneously, which can only temporarily change and interfere with the malignant kinetic energy of the cell, but cannot permanently change and Block its malignant phenotype. Only by positioning the drug target on the stable and static canonical gene domino-like linked DNA track and the regulatory elements of gene expression can the malignant phenotype of cells be permanently changed and blocked to achieve a radical cure.

The MYC gene family is a class of genes encoding transcription factors related to cell contact communication. Expression and repression of MYC-like genes are associated with spatial and anchorage-dependent growth control of cells The MYC gene family genes currently found in the human body include three genes: MYC, NMYC, and LMYC. MYC family genes have similar structure and close homology. Studies have shown that they have similar functions in living organisms. They all contain 3 exons and 2 introns. Although their first exon showed no homology, this exon did not encode a protein. However, regions with homology in exons 2 and 3 amounted to five. The carboxy-terminal sequence homology exists in the 85 amino acids of the MYC family, one of which is an important basic region. The amino acid composition and sequence of the three MYC-encoded proteins are highly conserved and homologous, which is their binding to DNA. domain. In this way, it suggests a possibility to design a molecule or a class of molecules that can block all MYC family genes.

Under normal circumstances, the human MYC gene is located on 8q24; NMYC gene is located on 2p25; LMYC is located on 1p32.

Since MYC was cloned and identified, there have been more than 10,000 research papers on MYC family genes in medical research literature over the past 20 years. MYC has become an important target gene in the study of proto-oncogenes and cancer treatments.

The nuclear phosphoprotein Myc encoded by MYC forms heterodimers Myc/Max and Myc/Mad with the other two nuclear proteins Max and Mad respectively in the nucleus. Like most other transcription factors, Myc has two domains with different functions: the Binding Dormain that binds to other proteins and DNA, and the domain that regulates functional activity. The 85 amino acids at the C-terminus are necessary for the binding of Myc to Mad, Max and DNA. The N-terminus contains the transcriptional activation domain of Myc, as well as regions required for transcriptional repression, cell cycle regulation, transformation, apoptosis, etc. In addition to interacting with the RB family protein P107, the N-terminus also interacts with α-tubulin and some new adapter proteins BIN1, MM1, TAM, TRRAP and AMY1, etc., the structures of these proteins and their effects on the action of MYC It was shown that their interaction with Myc is related to the transcriptional regulatory system as well as cell cycle regulation, chromatin construction and apoptosis. In addition to binding to Max and Mad, the Myc C-terminus is also involved in the transcriptional repression of several growth-promoting genes and plays an important role in Myc-mediated cell transformation. And its C-terminus combines with Mad and Max to form heterodimer Myc/Mad and Myc/Max, which respectively complete the Trans and Cis regulation of the MYC gene and its regulated related genes, just like the two switches on and off. Like this structure, it dominates the transcriptional activity of MYC and related genes. In addition, the C-terminus also interacts with other cellular proteins YY1, AP2, BRCA1, TFIII and MIZ1; in addition, the expression activity of many genes in the human body is related to MYC, indicating the complexity of its regulatory function.

It is now known that Myc is a type of transcription factor. This transcription factor and the related genes it regulates have multiple functions. Cell cycle, cell differentiation, apoptosis, cell transformation, angiogenesis, gene stability, etc., are all related to MYC. Studies have shown that some other genes related to cancer, such as Ras, can only lead to the malignant proliferation of cancer cells through the activation of MYC.

Existing research data show that the malignant cells of most human cancer patients are accompanied by the overexpression of MYC, and the copy number of MYC in cancer cells of some patients is multiplied or even increased by hundreds of times. Studies have shown that MYC gene in Burkitt lymphoma, non-Burkitt lymphoma, breast cancer, cervical cancer, prostate cancer, gastrointestinal cancer, osteosarcoma, melanoma, teratoma, granulocyte and plasma cell and B lymphocyte leukemia, Malignant glioma, neuroblastoma and small cell lung cancer, overexpression in malignant cells of nasopharyngeal carcinoma; on the other hand, it is now recognized that the synergistic effect of activating proto-oncogenes is the key to malignant transformation of cells: most cases Under these conditions, the occurrence of cancer is related to two or more proto-oncogene mutations, for example, the occurrence of cervical cancer is related to MYC and HRAS; breast cancer is related to MYB, MYC, EGFR, HER2, HRAS and p53, and lung cancer is related to JUN, MYCN, MYCL, HRAS, RB1, p53, RAF1 are related, and this situation is widespread in human cancers. With the increase of data accumulated in the investigation of gene expression in human cancer, it is possible to find more abnormal expression of MYC in malignant cells. We speculate that MYC plays a key synergistic role in the malignant reproduction of cells caused by different gene mutations.

The growth of cells is regulated by the chromatin track in the nucleus, and the only way to prevent malignant growth is to close and configure its chromatin track. The W. Felsher research group of Stanford University in the United States simply turned off MYC through transgenic mouse animal experiments, and transformed/reconstructed the cell phenotype from malignant phenotype (uncontrolled proliferation) to benign (differentiation/apoptosis) The phenotypes achieved the atrophy and demise of liver cancer mass in vivo (Nature.2004 Oct 28; 431(7012): 1112-7. Epub2004 Oct 10.), and the atrophy and apoptosis of osteosarcoma in vivo (Bernard Weinstein : Science 297(5578) Issue of 5 Jul 2002, pp.63-64.)) and disappearance of hematological malignancies (Felsher, D.W. & Bishop, J.M. Reversible tumorigenesis by MYCin hematopoietic lineages. Mol. Cell 4, 199-207 (1999 ). These experimental results demonstrate the feasibility of remodeling chromatin phenotypes.

The method of blocking the MYC gene or clearing and inhibiting the activity of the expressed product can realize the reversal of cancer cells. However, the method of blocking MYC can cut off the source of MYC transcription factor from the source. Compared with the latter two methods, blocking the proto-oncogene has several advantages. First, it can prevent cancer cells with a benign phenotype from reverting to a malignant phenotype for a long time. Second, the method is simple. There is no side effect, while the drugs targeting DNA currently used clinically have strong side effects on normal cells.

Based on the above analysis, blocking the MYC gene and related genes, and reconstructing the chromatin orbit of MYC has broad significance for the treatment of various cancers.

Contents of the invention

The chromatin peptides involved in the present invention are essentially MYC family gene expression transcription factors and a class of oligomeric peptide molecules related to the transcription factors and auxiliary protein degradation, which participate in the regulation of cell proliferation in the human body itself. There are two types of transcription factors, Trans transcription factors and Cis transcription factors. Chromatin peptides derived from Trans transcription factors generally act opposite to their derived precursor transcription factors; chromatin peptides derived from Cis transcription factors act the same as their derived precursor transcription factors. Some biomimetic molecules of chromatin peptides have the same effect as natural chromatin peptides; the disordered overexpression of these transcription factors leads to the loss of control of the body's own malignant growth monitoring and clearance system of cancer cells, and initiates the malignant proliferation of tumor cells. The chromatin peptides derived/degraded from them shut down the disordered overexpression of MYC genes in these tumor cells, thus continuously realizing the "cure without treatment" of many cancer patients, and bionic molecules have similar effects; The technology includes several chromatin peptides screened from the human genome database and derivatives of these chromatin peptides, which can block the proto-oncogene MYC family and the downstream related genes regulated by it, and realize the reconstruction of the running track of MYC genes , to transform cancer cells from a malignant phenotype to a normal cell phenotype. For cancer cells with a reversed phenotype, if they are differentiated cells, they will gradually die or undergo apoptosis; if they are cells with differentiation ability, such as various stem cells, they will proliferate or differentiate according to normal cells. These chromatin peptides can be used for the treatment and prevention of various human malignant tumors, and are biologically active drugs and biomimetic drugs with curative effect and preventive effects.

Therefore, the purpose of the present invention is to provide:

1. A class of chromatin peptide drug molecules that can specifically block the proliferation pathway of human and mammalian cancer cells MYC, which are DNA affinity domain sequences or their biomimetic molecules screened from gene databases, through MYC The cistron analysis of protein transcription factor regulatory elements contains 3-100 amino acid residues, which can specifically and firmly bind to the promoters of MYC family genes and the downstream gene promoters driven by the transcription factors expressed by them, and last for a long time Close down MYC family genes and related downstream genes, terminate the overexpression of MYC family genes caused by MYC family gene mutations and related gene variations, and/or terminate the overexpression of downstream transcription factors and cell growth factor genes regulated by MYC family genes, Thus, the malignant proliferation pathway of cells caused by the escape of cell proliferation regulation by MYC family genes is closed for a long time.

2. The chromatin peptide drug molecule of item 1 above, which is any chromatin peptide within the range of 3-100 aa that can block all amino acid sequences of MYC family genes and genes related to the MYC proliferation regulation pathway.

3. The chromatin drug molecule of the above item 1, which is selected from one or more of the seven chromatin peptide drug molecules (Table 1, sequence list) in sequence 1-7 of the sequence listing, or has a relationship with these molecules A chromatin peptide that can block all amino acid sequences of MYC family genes or genes related to the MYC proliferation regulatory pathway with a minimum of 3 amino acid homologous series.

4. Any amino acid sequence that has 3 amino acid minimum homology series with the 7 drug molecules mentioned in item 2 above and can block MYC family gene promoters or downstream related genes regulated by MYC transcription factors.

5. Chemically modify the N-terminal and/or C-terminal and/or side chain groups of the chromatin peptide drug molecule described in item 1, 2, 3 or 4 above, or replace the obtained amino acid addition and deletion All chromatin peptide derivatives capable of blocking MYC family gene promoters or downstream genes regulated by MYC-like transcription factors.

6. A pharmaceutical composition, which comprises a pharmaceutically effective amount of the chromatin peptide drug molecule or derivative as described in any one of items 1-5 above and optionally a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to the above item 6, which is an injection preparation, an oral capsule and a sustained-release preparation.

8. Use of the chromatin peptide drug molecule or derivative described in any one of items 1-5 above in the preparation of a drug for preventing or treating malignant tumors in humans or mammals.

9. The chromatin peptide drug molecule or derivative described in any one of items 1-5 above, including chromatin peptides obtained by any method for blocking the MYC cell proliferation pathway.

Blockers that block MYC family genes were screened by cistron analysis of regulatory elements of MYC-like protein transcription factors.

MYC family gene blocker—chromatin peptide drug molecule can block the DNA-affinity domain of genes related to the cell MYC proliferation pathway.

MYC gene blocker - chromatin peptide drug molecules block MYC family genes and downstream related genes, with high specificity, except for blocking MYC family genes and downstream related genes, it has no effect on the activity of other genes. In addition, they have a much higher affinity to the DNA-affinity domain of MYC family genes and downstream related genes of tumor cells than to the allele DNA-affinity domain of normal cells.

These drug molecules can pass through the cell membrane and nuclear membrane to reach the target site. Therefore, they can be administered through intramuscular injection, intravenous injection, oral capsule, intravenous infusion route, in situ injection at tumor site and skin penetration. The dose is determined according to the specific conditions of the patient, such as body weight, age, specific disease and condition, and the doctor's clinical experience, but usually it can be 0.05-30 mg/day, and can be divided into one or more doses.

In addition, these drug molecules cannot resist the digestion and hydrolysis of gastric acid and pepsin, so they cannot be directly administered orally.

These drug molecules can make the malignant tumor cells related to MYC family genes change from malignant phenotype to normal cell phenotype by inhibiting the overexpression of MYC family genes and downstream related transcription factors and related growth factors. Treatment and prevention of related malignancies.

The current research data show that the occurrence of most malignant tumors is accompanied by the overexpression of MYC family genes, some of which are caused by mutations in the MYC family genes themselves, and some are caused by other gene mutations that lead to the activation of MYC genes; in addition, theoretically , the mutation of the downstream transcription factors and related growth factors regulated by MYC family genes can also cause the same malignant phenotype of cells as the activation of MYC family genes. Therefore, blocking MYC family genes and downstream related genes can prevent most malignant tumors And treatment, has broad implications.

These drug molecules are safe to the human body, do not affect the normal metabolism of organs and tissues, do not affect the renewal of tissues and cells, and do not affect human growth and development.

These drug molecules can block the expression of all members of the MYC gene family, including C-MYC, NMYC and LMYC and their regulated downstream related genes, which ensures the broad-spectrum efficacy of the drug.

Chemical processing and modification of these chromatin peptides may be able to improve the permeability of the cell membrane and nuclear membrane, prolong the half-life, increase the affinity for the target site, and thus improve the curative effect.

No matter what method is used to obtain these drug molecules, as long as they have the molecular characteristics designed by this patent, they will have the same effect.

Brief description of the drawings

Fig. 1 The inhibitory effects of seven chromatin peptide drugs on the growth of 4T1 breast cancer in mice. 6-thioguanine-resistant cell counts at the original tumor site.

In recent years, there are a lot of research materials on MYC, a proto-oncogene. Studies have found that the expression of MYC is very high in the malignant cells of most solid malignant tumor patients and fluid cancer patients, and the copy number of MYC gene in some cancer tissues is several times or even 100 times that of normal cells. In normal tissue cells of adults, the expression of MYC is very small. Even its expression cannot be detected, but in the fast-growing tissues and organs of fetuses and children, MYC has a relatively high expression level.

Some people simply block or delete the MYC gene, which can realize the conversion of cancer cell phenotype to normal cell phenotype; using monoclonal antibody to clear the expression product of MYC can also temporarily inhibit the malignant reproduction of cells. These studies show that the occurrence of many malignant tumors is related to the overexpression of MYC genes. Translocations and point mutations of MYC-like genes can activate their expression. In addition, there may be other gene mutations that lead to the activation of MYC genes, such as the inactivation of P53. In the current research, the methods for blocking the MYC gene are mainly antisense RNA and DNA, and no breakthrough has been made, so the fundamental problem has not been solved. First, the stability of RNA is relatively poor; second, in the human body, it is difficult for RNA and DNA to naturally pass through the cell membrane and nuclear membrane; third, the technology of transmitting antisense DNA and RNA through viral vectors is complicated and the risks are high. Difficult to judge. Blocking the expression product of MYC by monoclonal antibody cannot completely block the source of the malignant protein, but can only temporarily alleviate the malignant proliferation of the tumor. In addition, because the half-life of the protein in the body is relatively short, the effect of the monoclonal antibody against the MYC-encoded protein and The utility time is very limited.

It is currently known that the regulation of cell proliferation in the MYC pathway is controlled by the cell living space (direct contact communication between cells): after the body matures, when some tissue cells are injured and die, it is necessary to initiate the proliferation and supplementation of adjacent cells, This kind of cell proliferation is still limited by the limited space of the body, and normal solid tissue cells are controlled by contact inhibition and anchorage-dependent proliferation of adjacent cells. Solid tissue cells, including bone marrow cells and certain fluid malignancies (whose origin is in the bone marrow and other hematopoietic organs) escape these two controls, which can lead to in situ malignant proliferation of cells and ectopic metastasis in the blood circulation. It is now known that the escape from the body space control of cell proliferation is the famous proto-oncogene MYC, its upstream regulatory element MAD/MAX accepts transcriptional inhibition communication signals such as cell contact inhibition, and the regulatory element Myc/MAX accepts cell Anchorage-dependent and other transcriptional promotion communication signals, the transcription factor protein Myc expressed by this gene activates a large number of growth factors that promote cell proliferation, and directly activates the cell proliferation/Cell cycle gene group. The deletion or mutation of any gene encoding an inhibitory signaling protein on this cell communication network may lead to the cell entering the malignant/progressive proliferation/Progressive Proliferation pathway; thus, the Myc-driven Cell cycle gene group transcription waterfall breaks through this A line of defense for body space control. In fact, overexpression of MYC is indeed a common feature of many malignant cells, so effectively turning off the transcription of this MYC gene has become a common cancer treatment strategy in the research phase.

Turning off the MYC gene with chromatin peptide molecules has many advantages. The chromatin peptide that we designed to close the MYC gene is a natural or biomimetic molecule that the human body possesses, and has no harm to normal cells, tissues and organs. Chromatin peptides have a small molecular weight (the chromatin peptides we use are 3-100 peptides) and are easy to pass through the cell membrane and nuclear membrane. In addition, these chromatin peptides have more basic amino acids, and the entire molecule is basic, which is conducive to the concentration of drug molecules to the acidic nausea tumor site. In addition, these chromatin peptides have more hydrophobic amino acids when they have a larger molecular weight. These hydrophobic amino acids make the drug molecules form a special aqueous phase structure in aqueous solution, which can resist the hydrolysis of proteases. Therefore, these drug molecules It has good in vivo stability and long half-life.

Most importantly, the specificity of these drug molecules is relatively high. First, there is no other part of the human genome that can bind to these drug molecules except for the promoter region of the transcription factor MYC gene and the promoter region of related genes regulated downstream. Except for the relevant genes regulated, other genes will not be blocked, which is an important aspect of the safety of these drug molecules; second, the MYC and related genes of few normal cells are blocked, and the alleles of cancer cells can interact with these Chromatin peptides specifically bind, which may be that the DNA in the MYC gene coding region of normal cells is in a highly condensed state, which is not easy to combine with these chromatin peptides; while in malignant cells, the MYC coding gene is activated, in a stretched state, and easy to stain Mass peptide drug molecule binding.

However, studies have shown that, especially during fetal and child development, MYC can directly drive the transcription of the promoter molecule ClycD of the cycle gene group. If these chromatin peptides are used to block the cycle gene group, it actually turns off the blood that is necessary for many bodies. An important functional pathway for the proliferation and renewal of normal cells such as cells and epithelial cells. However, as with our analysis of the transcription factor HIF-1α, the transcriptional regulatory cascade is a complex network rather than a single pathway. In addition to the transcription factor MYC directly driving the transcription of the promoter molecule ClycD of the cycle gene group, there may be other transcription factors driving the transcription of the promoter molecule ClycD of the cycle gene group. Existing research results prove that the cell proliferation regulatory network related to MYC genes is not a single channel directly, but a network system.

In normal adults, the expression of MYC is very low or cannot be detected at all. However, from an evolutionary point of view, it is true that as everyone agrees, there is no reason for human evolution to retain a dedicated oncogene. We speculate that MYC may be activated during larval development and certain emergencies in adults to promote cell reproduction. In normal tissues of adults, the MYC gene is silent or relatively low in expression.

It can be seen from the above analysis that the chromatin peptide drug molecules designed to block the MYC gene and related genes are safe to the human body. However, many parts of the human gene regulatory network are still unknown, and the full role of Myc is not fully understood. The safety of any drug molecule targeting the MYC gene should be demonstrated through other tests that can directly prove its safety. Animal safety test results show that when the injection dose of rabbits and mice reaches 4mg/kg body weight (equivalent to 80 times the designed dosage for human body), long-term observation does not affect the health of the animals. The white rabbits with an average body weight of 1500g and the small white mice with 15g were selected for the test, and the results showed that the medicine had no effect on the weight gain of the white rabbits and small white mice (see examples).

Based on the above analysis and experiments, we designed chromatin peptide drug molecules that block MYC family genes and regulatory network-related genes for the treatment of human malignant tumors. Drug molecules and targeting positions are shown in Table 1 and the sequence listing.

Table 1 The drug molecule, the target site of the drug molecule on MYC, and the transcription factors and auxiliary proteins derived from the drug molecule

drug molecule Source of Drug Molecule Derivation target 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 THNELEK NRR 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, to5′-CACGCG-3′.

Description All listed chromatin peptide sequences can block MYC-HUMAN.

Table 2 Transcription factors and auxiliary proteins derived from chromatin peptides and their functional roles

Chromatin Peptide-Derived Precursors Drug Molecular Sequence Functional roles of transcription factors and accessory proteins MYC HUMAN VKRRTHNVLERQRR Overexpression of MYC is associated with different types of malignancies. MAD HUMAN RSTHNEMEKNRRA MAD forms a heterodimer with MAX and resists the expression promotion effect of MYC by competing with MYC for MAX. MAD4 HUMAN RSSHNELEKHRR It forms a heterodimer with MAX and resists the expression promotion effect of MYC by competing with MYC for MAX. MAX HUMAN KRAHHNA LERKRR Brain, heart, lung levels are higher. And liver, kidney, muscle and bone parts. MXI1 HUMAN RS THNELEK NRR MAD forms a heterodimer with MAX and resists MYC's expression-promoting effect by competing with MYC for MAX MLX HUMAN NSLDP GLFVEST TCFL4-MAD1, TCFL4-MAD4, TCFL4-MLX.complexes are expression suppressors MNT HUMAN REVHNKLEK NRR Forms a heterodimer with MAX to bind DNA and repress transcription

Example

The following examples illustrate the invention.

Example 1

Synthesize the polypeptides of sequences 1-7 in the sequence listing by solid-phase peptide synthesis (SPPS), including step-by-step addition of amino acids protected with protecting groups to growing oligopeptide chains anchored on chemically stable particles , so that the synthesized peptide can be separated from reagents and solvents by simple filtration. After the synthesis, the chain is cleaved from the carrier and purified.

Example 2

Pharmacodynamic tests of chromatin peptide drug molecules blocking MYC cell proliferation pathway in animals

Inhibition of MYC Cell Proliferation Pathway Blocker on Breast Cancer in Mice

Animal medication after inoculation

material method

Female nude mice HHc, 6-8 weeks old, were purchased from Qingdao Hailand Research and Development Center.

Mouse mammary gland tumor cell 4T1 comes from Qingdao Hailand Research and Development Center.

The chromatin peptide drug (sequence 1-7) used in the test is chemically synthesized. 98.5% purity.

Mammary glands were aseptically inoculated into the mammary glands of healthy female mice with 0.1 ml of PBS suspension containing cancer 4T1 cells (10 ⁵ ). The amount of inoculation is determined empirically, generally based on the growth of tumors with a diameter of 10 mm in mice that have not been given drugs 25 days after inoculation. On the tenth day after the inoculation, when the diameter of the tumor reached 4-5mm, it was given by intramuscular injection every 5 days, and the control group was injected with PBS at a dose of 0.08 mg/kg body weight. Doses were given every five days until day 25, for a total of 4 doses. 5 mice per group. On the 30th day, observe and measure the size of the mammary gland mass (results are shown in Table 2) and take the tissue from the original breast cancer site and use the colonogenic method to culture and differentiate the cells ⁽¹⁾ . Take a 5mm × 5mm × 5mm tissue block of the mouse’s original cancer mammary gland, and then take 0.1g of the central part, chop it up, and digest it with 5ml of enzyme mixture at 37°C for 2 hours with shaking (1XPBS, 1mg/ml type IV collagenase). After digestion, the sample Filter through a 70 µm diameter cell nylon filter and wash 2 times with PBS. Cells were suspended and serially diluted in RPM Medium 1640 containing 60 μM thioguanine in a cell culture incubator to detect malignant growth. Because 4T1 cells are resistant to thioguanine, after 14 days in culture, the disseminated cancer cells formed masses. Fix with methanol, stain with 0.03% methylene green, and count the cells. (See Figure 1 for the results)

Result analysis

Table 3 Effects of chromatin peptide drugs on the growth of 4T1 breast cancer in mice

control 1 2 3 4 5 6 7 diameter mm 9.8mm 0 0 0 0 0 0 0

Measured with a caliper on the 30th day after tumor inoculation. Because of the irregular shape of the tumor at the tumor site, the average value of the largest diameter and the smallest diameter was taken. The numbering of chromatin peptides is the same as that in the sequence listing.

Figure 1 shows the inhibitory effects of seven chromatin peptide drugs on the growth of 4T1 breast cancer in mice. 6-thioguanine-resistant cell counts at the original tumor site. The numbering of chromatin peptides is the same as that in the sequence listing.

Thirty days after the administration, the tumors of the experimental mice in the No. 1-7 chromatin peptide drug-treated groups disappeared completely. All the cancer lumps in the treatment groups began to shrink on the 5th day of administration, among which, the No. 1 and No. 2 polypeptide treatment groups disappeared the fastest, and the tumors disappeared completely on the 9th and 16th days after the first treatment, respectively.

The cell counts showed that the number of 6-thioguanine-resistant cancer cells at the original site of canceration in all drug treatment groups was reduced to less than 10% of that in the control group without drug treatment.

The above results show that chromatin peptides 1-7 have a strong inhibitory effect on breast cancer in mice. It should be noted that the MYC-like chromatin peptide blocking agent reversed the phenotype of cancer cells. However, there may be a small number of cells with reversed oncogene mutations that do not die or undergo apoptosis. Whether these cells will reverse and trigger malignant growth again will require further experiments to prove.

references

1 Pulaski, BA & Ostrand-Rosenberg, S. (1998) Cancer Res. 58, 1486-1493. [Abstract]

Example 3

Advanced breast cancer in dogs with extensive metastases. Firstly, the expression of C-MYC, LMYC and NMYC genes in dogs with breast cancer was determined, and the result was that the expression of NMYC was 128 times that of normal tissues. Treatment with chromatin peptide sealant of KRRTHNVLERQRR amino acid sequence, subcutaneous direct injection, 2 mg each time, once every 5 days. On the 5th day, the cancer began to shrink, and on the 30th day, the cancer disappeared completely, and there were scars on the breast.

Chromatin peptides using this sequence can block the disordered proliferation of malignant tumor cells caused by the dedifferentiation of differentiated cells caused by the overexpression of NMYC. It can be used directly in the early stage of tumor occurrence without surgery and radiation therapy. When the mass is relatively large, medication can be used before surgery, and the mass that has transformed into a benign cell phenotype and shrunk can be removed at an appropriate time. It can also be used after surgery to transform the phenotype of a small number of malignant cells that may not be completely removed by surgery.

Example 4

The first inventor of the patent, nasopharyngeal carcinoma, underwent chemotherapy twice. Three years after the second chemotherapy, relapse occurred. The copy number of C-MYC in the malignant cells of nasopharyngeal carcinoma patients was determined to be 55, and the expression was abnormal. After examination, the lung metastases were found, and there was no metastases in other parts. Use 0.05 mg of chromatin peptide whose amino acid sequence is RSTHNELEKNRR and 0.05 mg of chromatin peptide whose sequence is NSLDPGLFVEST to prepare 5 ml of normal saline injection, aim at the 4 corners of the tumor and the center of the tumor, and inject directly, once every 15 days; Intravenous administration, 10mg each time, once every 15 days; continuous medication for 3 months. The primary site and lung mass disappeared 3 months after the first medication, and the biopsy showed no cancer cells.

Example 5

The abnormal expression of C-MYC in malignant cells of gastric cancer patients was detected. The main tumor mass was 65mm×70mm×44mm, and it spread to the liver. The hypoxic sealing agent normal saline solution (5mg/250ml) (drug molecule invented and designed by us) was administered for the first time by intravenous injection, and the main tumor mass was spit out on the sixth day. After 10 days, the same method was given again (2mg/100ml), and after 20 days, it was checked that most of the cancerous tissues were necrotic, and the tumor mass was loosely vomited out. Chromatin peptide physiological saline solution whose amino acid sequence is VKRRTHNVLERQRR was intravenously injected, the first dose was 5mg/250ml, and the same method was injected every 15 days thereafter, each time 2mg/100ml, and the drug was used continuously for 4 months. Four months later, the biopsy showed no cancer cells, and the expression of C-Myc in the cells of the pathological site was small, and returned to the level of the normal site.

SEQUENCE LISTING

<110> Wang Chengqiu Bian Xiaozhuang He Mi

<120> A Class of Chromatin Peptide Drug Molecules Blocking MYC Cell Proliferation Pathway

<130>

<160>7

<170>Patent Inversion 3.1

<210>1

<211>13

<212>PRT

<213> Artificial design

<220>

<223> Promoter design for MYC gene

<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 design for MYC gene

<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 design for MYC gene

<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 design for MYC gene

<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 design for MYC gene

<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 design for MYC gene

<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 design for MYC gene

<400>7

Arg Glu Val His Asn Lys Leu Glu Lys Asn Arg Arg

1 5 10

Claims (3)

1. A class of chromatin peptide drug molecules capable of specifically blocking the proliferation pathway of human and mammalian cancer cells MYC, selected from one or more of the seven chromatin peptide drug molecules in sequences 1-7 of the sequence listing; It is characterized in that the chromatin peptide drug molecule can penetrate the cell membrane and the nuclear membrane, and specifically bind to the promoter of the MYC family gene and the target sequence 5'-CAC[GA ]TG-3' and 5'-CACGCG-3', turn off MYC family genes and related downstream genes, terminate the overexpression of MYC family genes caused by MYC family gene mutations and related gene variations, and/or terminate MYC family genes Regulates overexpression of downstream transcription factor and cell growth factor genes. 2. A pharmaceutical composition comprising a pharmaceutically effective dose of the chromatin peptide drug molecule according to claim 1 and optionally a pharmaceutically acceptable carrier. 3. The use of the chromatin peptide drug molecule of claim 1 in the preparation of a drug for preventing or treating human or mammalian malignant tumors.

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