KR20230009095A - A composition for treating drug resistance - Google Patents

Abstract

A composition according to the present invention can inhibit the growth of OXPHOS inhibitor-resistant cancers by overcoming OXPHOS inhibitor resistance or enhancing OXPHOS inhibitor sensitivity, and thus the composition can be very effectively used for preventing, alleviating, or treating various cancers, particularly OXPHOS inhibitor-resistant cancers.

Description

A composition for treating drug resistance {A composition for treating drug resistance}

The present invention relates to a composition capable of overcoming drug resistance or inhibiting the growth of resistant cancer by enhancing sensitivity to drugs.

Cancer is one of the incurable diseases that mankind must solve, and a huge amount of capital is being invested in development to cure it worldwide. Diagnosed, more than 60,000 people die. Although various anticancer therapies have been rapidly developed in the diagnosis and treatment of cancer in the past decade, the mortality rate due to cancer is still high. In addition, there are still side effects associated with trying various anticancer drugs and various anticancer therapies. Research to reduce these side effects is being actively conducted, and recently, a drug that treats cancer by controlling the cancer metabolic process as a new treatment method with less side effects has emerged.

As an effective target for anti-cancer therapy, mitochondria related to metabolic processes have emerged (Trends Mol Med. 2004 Aug;10(8):372-8.), and these mitochondria are a major component of cell survival through ATP generation. It is also known to act as an important factor in cell death because it determines cell fate by mitochondrial membrane-dependent cell death signals (Cell Death Differ. 2003 Aug;10(8):870-80.) . The process of synthesizing ATP through electron transfer and chemosmosis in mitochondria is called oxidative phosphorylation (OXPHOS), and almost all organisms that breathe oxygen can synthesize ATP more efficiently compared to anaerobic fermentation. Carries out oxidative phosphorylation. Drugs that inhibit such oxidative phosphorylation are called OXPHOS inhibitors, and the inhibitors include IM156, metformin, rotenone, methylmalonate, BAM 15, BAY 87-2243, nitropropionic acid (3 -Nitropropionic acid; 3NPA), Atovaquone, etc. exist. Among them, anticancer drugs that have been found to have anticancer effects include IM156, Metformin, Phenformin, and Rotenone, but there are also patients with anticancer resistance who do not respond to these drugs. do. Therefore, even if a patient with such resistance is treated with the drug, the effect of treatment cannot be guaranteed, and thus, there is a possibility that the time and cost of clinicians and patients are unnecessarily consumed. Therefore, when starting anticancer treatment, it is realistically necessary to have a scale that can estimate treatment efficiency in advance so that customized drugs suitable for individual characteristics can be selectively used.

As such, since there is almost no research related to drug resistance, the present inventors have developed a drug-resistant treatment that can overcome this by selecting patients resistant to OXPHOS inhibitors (oxidative phosphorylation inhibitor) in advance.

One object of the present invention is to provide a pharmaceutical composition for overcoming drug resistance.

Another object of the present invention is to provide a pharmaceutical composition for enhancing drug sensitivity.

Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating drug-resistant cancer.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.

Unless there is a specific definition within the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.

According to one embodiment of the present invention, it relates to a pharmaceutical composition for overcoming drug resistance or enhancing drug sensitivity.

In the present invention, the composition is an agent that reduces the activity or expression level of MEC17 (alpha Tubulin Acetyltransferase 1; aTAT1) protein; Alternatively, it may include an agent that reduces the expression level of the gene encoding the protein as an active ingredient.

In the present invention, the "MEC17 (alpha Tubulin Acetyltransferase 1; aTAT1)" is a protein encoded by the ATAT1 gene called alpha tubulin acetyltransferase, and is also called TAT, C6orf134, Nbla00487, alpha-TAT, or alpha-TAT1. It belongs to the transferase family, and more specifically to acyl transferases. The ATAT1 gene encodes a clathrin protein that plays an important role in vesicle formation and acetylates alpha tubulin on lysine 40 (K40). This process is known to be important for microtubule growth during chemotaxis and cilium formation.

In the present invention, the information of the "MEC17 protein or gene encoding it" is registered with NCBI (National Center for Biotechnology Information) (Gene ID: 79969, NM_001031722.4), and the MEC17 protein is an amino acid represented by SEQ ID NO: 1 sequence, and the gene encoding the MEC17 protein may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto. In non-limiting examples, at least 99% and less than 100%, at least 95% and less than 99%, at least 90% and less than 95%, at least 85% and less than 90%, or at least 80% and less than 85% the sequence of MEC17. It may be the case of having the homology of, and it may include all without limitation within the obvious range that it exerts the desired effect of the present invention to those skilled in the art.

The agent for reducing the activity or expression level of the protein of the present invention is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein or a part thereof. It may include, but is not limited to, a method known as a method commonly used in the art that corresponds to a means for deriving an effect of inhibiting its activity or expression by directly or indirectly acting on the target MEC17 protein. If it can be easily derived by technology, it is not limited thereto and may include all.

In the present invention, the "peptide mimetics" are peptides or non-peptides that inhibit the binding domain of the MEC17 protein leading to the inhibition of AcAT activity. Key residues of non-hydrolysable peptide analogs include the β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemiCal co. Rockland, IL, 1985), azepine (Huffman et al. in Peptides: chemistry and Biology, G.R. Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides; chemistry and Biology, G.R. Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohols (Gordon et al. Biochem Biophys Res. commun 126:419 1985) and substituted gamma-lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

In the present invention, the "Aptamer" is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. Since the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) was first developed (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), aptamers have been developed from low-molecular-weight organic substances to peptides and membrane proteins. Many aptamers capable of binding to various target molecules have been continuously discovered. Aptamers are comparable to single antibodies because of their inherent high affinity (usually pM level) and specificity of being able to bind to target molecules, and in particular, they have high potential as alternative antibodies to the extent that they are called "chemical antibodies."

In the present invention, the "antibodies" prepared by injecting the protein or purchased commercially can be used. In addition, the antibody includes polyclonal antibodies, monoclonal antibodies, fragments capable of binding to an epitope, and the like.

Here, the polyclonal antibody can be produced by a conventional method of injecting the protein into an animal and collecting blood from the animal to obtain antibody-containing serum. Such polyclonal antibodies can be purified by any method known in the art, and can be made from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc.

In addition, the monoclonal antibody can be produced using any technique that provides for the production of antibody molecules through the cultivation of continuous cell lines. Such technologies include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology.

In addition, antibody fragments containing specific binding sites for the above proteins can be prepared. For example, but not limited to, F(ab')2 fragments can be prepared by pepsin digestion of antibody molecules, and Fab fragments can be prepared by reducing disulfide bridges of F(ab')2 fragments. Alternatively, by miniaturizing the Fab expression library, monoclonal Fab fragments having the desired specificity can be quickly and conveniently identified.

In the present invention, the antibody may be bound to a solid substrate to facilitate subsequent steps such as washing or separation of complexes. Solid substrates include, for example, synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resin includes polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, and nylon.

In the present invention, the agent that reduces the activity or expression level of the protein may be one that specifically binds to the polypeptide represented by SEQ ID NO: 1 of the MEC17 protein, and preferably, the composition of the present invention is an antibody specific to the MEC17 protein. It may include, wherein the antibody may specifically bind to the polypeptide represented by SEQ ID NO: 1, but is not limited thereto.

The agent for reducing the expression level of the gene encoding the MEC17 protein of the present invention is a gene encoding the MEC17 protein, preferably an antisense nucleotide complementary to the gene or a part thereof, a small interfering RNA (short interfering RNA) RNA; siRNA), short hairpin RNA (short hairpin RNA) and ribozyme (ribozyme) may include any one or more selected from, but is not limited thereto, direct or indirect to the gene encoding the target MEC17 protein It acts as a means for deriving an effect of inhibiting its expression, and it is not limited thereto and may include all, as long as it can be easily derived by a known technique as a method commonly used in the art.

In the present invention, the "antisense nucleotide", as defined in Watson-Crick base pairing, binds (hybridizes) to a complementary nucleotide sequence of DNA, immature-mRNA or mature mRNA to interfere with the flow of genetic information from DNA to protein will be. The specific nature of antisense nucleotides for target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target RNA sequence. Recently, many studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins. Since many recent advances have been made in the field of oligonucleotide chemistry and the synthesis of nucleotides that exhibit improved cell line adsorption, target binding affinity, and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

In the present invention, the "shRNA" and "siRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and can suppress the expression of a target gene, thereby providing an efficient gene knockdown method or gene therapy used in a way shRNA is a hairpin structure formed by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by dicer to form small RNA fragments of 21 to 25 nucleotides in size siRNA, which is a double-stranded oligonucleotide, can specifically bind to mRNA with a complementary sequence and suppress its expression. In addition, siRNA refers to a small double-stranded RNA fragment of 21 to 25 nucleotides in size that induces RNA interference (RNAi) by modifying target mRNA by double-stranded RNA (dsRNA).

In the present invention, which means of the shRNA and siRNA to be used can be determined by a person skilled in the art, and a similar expression reduction effect can be expected if the mRNA sequences they target are the same. For the purpose of the present invention, it is possible to inhibit the expression of MEC17 by specifically acting on the gene encoding the MEC17 protein to cut the MEC17 gene (eg, mRNA molecule) to induce RNA interference (RNAi, RNA interference). can siRNA can be synthesized chemically or enzymatically. The method for preparing siRNA is not particularly limited, and methods known in the art may be used. For example, a method for chemically synthesizing siRNA directly, a method for synthesizing siRNA using in vitro transcription, a method for cutting long double-stranded RNA synthesized by in vitro transcription using an enzyme, An expression method through intracellular delivery of shRNA expression plasmids or viral vectors and an expression method through intracellular delivery of polymerase chain reaction (PCR)-induced siRNA expression cassettes, but are not limited thereto.

In the present invention, the "ribozyme" refers to an RNA molecule having a catalytic activity. Ribozymes with various activities are known, and ribozymes of the MEC17 gene include known or artificially produced ribozymes, and optionally ribozymes having target-specific RNA cleavage activity are prepared by known standard techniques It can be.

As an example of the present invention, as an agent for reducing the expression level of the gene encoding the MEC17 (aTAT1) protein, it may be siRNA represented by SEQ ID NO: 3 and siRNA represented by SEQ ID NO: 4, but the gene encoding the MEC17 protein As long as the target site corresponds to the case where the target site includes SEQ ID NO: 7, it is not limited thereto.

As another example of the present invention, as an agent for reducing the expression level of the gene encoding the MEC17 (aTAT1) protein, it may be siRNA represented by SEQ ID NO: 5 and siRNA represented by SEQ ID NO: 6, but encoding the MEC17 protein As long as the locus targeting the gene corresponds to the case of including SEQ ID NO: 7, it is not limited thereto.

In the present invention, the term "drug resistance" refers to a decrease in the effectiveness of a drug when the drug is used repeatedly in quantitative terms, and in order to obtain the same effect previously experienced by patients with drug resistance, the amount of use or the frequency of use must be increased. It is a condition in which the same effect cannot be obtained even if the substance needs to be increased or administered in the same amount as before.

In the present invention, the "resistance treatment" refers to an action in which the effect of a drug is reduced when a drug is used repeatedly in a quantitative manner, or a drug-resistant patient is restored to obtain the same effect previously experienced. More specifically, the same anticancer effect can be obtained even when the drug is applied less frequently or at a lower dose, or the same effect can be obtained even if the same dose or a lower dose is administered by returning the drug to the state before drug resistance occurred. It refers to the action of creating a state.

In the present invention, "drug" or "anti-cancer agent" and "anti-cancer agent" may be used interchangeably, and the agent corresponds to an anti-cancer agent having a novel mechanism of killing cancer cells by inhibiting mitochondrial oxidative phosphorylation. Since various types of immune cells use specific cellular metabolic processes to maintain cell survival, development and function, these drugs can be used to enhance the effectiveness of cancer treatment by inhibiting ATP production.

In the present invention, the anticancer agent is an OXPHOS inhibitor, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib nip, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxyl Carbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, nitric acid Holmium chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, 5-fluorouracil, fludagabine , enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, bin Cristine, Vinblastine, Teniposide, Doxorubicin, Idarubicin, Epirubicin, Mitoxantrone, Mitomycin, Bleromycin, Daunorubicin, Dactinomycin, Pirarubicin, Aclarubicin, Pepro Mycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, thre tonin, exmestane, aminoglutecimide, anagrelide, navelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine and carrion It may be one using a drug containing at least one selected from the group consisting of mustine, preferably an OXPHOS inhibitor, and more preferably an OXPHOS inhibitor as an example of a ubiquinone reduction activity inhibitor, mitochondrial electron transport chain I inhibitors transport chain I iinhibitor) or iron chelator (iron chelator) may correspond to one or more selected from the group consisting of.

In the present invention, the "OXPHOS inhibitor" is one selected from the group consisting of a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, or an iron chelator. Any drug that has a mechanism of killing cancer cells by inhibiting oxidative phosphorylation of mitochondria may be included, but is not limited thereto.

In the present invention, the ubiquinone reduction activity inhibitor may be IACS-010759, but is not limited thereto as long as it corresponds to a drug that affects the activity of ubiquinone, a fat-soluble electron transporter present in the mitochondrial inner membrane.

In the present invention, the mitochondrial electron transport chain I inhibitor may be at least one selected from the group consisting of IM156, Metformin, Phenformin, and Rotenone, and more preferably IM156. It is not limited thereto.

In the present invention, the iron chelator may be VLX 600, but is not limited thereto as long as it is a compound having a function of inhibiting mitochondrial respiration.

In the present invention, the "IM156 drug" has a CAS number of 1422365-94-3, and is a type of new biguanide derivative compound previously known as HL156A, similar to other biguanide drugs, which have mitochondrial complex I (mitochondrial It corresponds to a drug that acts by blocking complex I). It is a small-molecule oral drug derived from biguanides and is known to be a potent oxidative phosphorylation (OXPHOS) inhibitor. Recent studies have shown that after treatment of in vitro cultured rat peritoneal mesothelial cells and rat renal proximal tubular cells with IM156, AMPK activity is more effective than other AMP-activated protein kinases (AMPK), such as metformin ( Am J Physiol Renal Physiol. 2016 Mar 1;310(5):F342-50.). However, since the drug IM156 affects different cell types in different modes of action, studies on various mechanisms of action are ongoing.

In the present invention, the "rotenone drug" is a drug that functions to inhibit tumor growth dependent on oxidative metabolism by acting on mitochondrial respiratory chain complex I (Mitochondrial Complex I). It is a lipophilic, naturally occurring product obtained from the roots and stems of the leguminous plants Lonchocarpus and Derris species. It has been widely used as an insecticide, but its use is restricted in many countries due to its toxic effect. did In addition, rotenone is known to inhibit cell proliferation by causing apoptosis in various human cancer cell lines, and is under development to be used for cancer treatment. Examples of mitochondrial electron transport chain I inhibitors acting on mitochondrial respiratory chain complex I other than rotenone include IM156, metformin, and phenformin.

In addition, the composition of the present invention may further contain an anticancer agent to overcome drug resistance or increase the drug sensitivity enhancing effect, wherein the anticancer agent includes nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, four latinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, cabo Platin, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtusetan, heptaple Latin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin , Oteracil, azacitidine, methotrexate, uracil, cytarabine, 5-fluorouracil, fludabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor , raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mito Mycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, Altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exmestane, aminoglutesimide, anagrelide, navelbine, fadrazole, tamoxifen , toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine may be used, but is not limited thereto. .

In the present invention, "tumor" or "cancer" is a disease in which cell division continues due to unregulated cell cycle, and is divided into carcinoma and sarcoma according to the site of occurrence. Carcinoma refers to a malignant tumor arising from epithelial cells such as mucous membranes and skin, and sarcoma refers to a malignant tumor arising from non-epithelial cells such as muscle, connective tissue, bone, cartilage, and blood vessels. The cancers include thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and hematological cancer. , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, but If the progression of cancer, such as differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention, it is not limited thereto.

According to another embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating drug-resistant cancer.

The composition of the present invention is an agent that reduces the activity or expression level of the MEC17 (aTAT1) protein; Alternatively, it may include an agent that reduces the expression level of the gene encoding the protein as an active ingredient.

In the present invention, an agent that reduces the activity or expression of the MEC17 protein; Alternatively, an agent that reduces the expression level of the gene encoding the protein is an alpha-tubulin N-acetyltransferase (Alpha-tubulin N-acetyltransferase; aTAT1) protein activity or expression inhibitor; Alternatively, it may further include an inhibitor of the expression of the gene encoding the aTAT1 protein, but is not limited thereto and may include all, as long as it has a function of regulating acetylation of acetylated tubulin protein.

The composition of the present invention may further include an anticancer agent to increase the killing effect of drug-resistant cancer, wherein the anticancer agent includes nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, Gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, restautinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizu Mab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtucetan, heptaplatin, methylaminore fluoric acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, 5-fluorouracil, fludabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, Docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin , daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, altretmin, daka bazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exmestane, aminoglutesimide, anagrelide, navelbine, fadrazole, tamoxifen, toremifene, testo A drug containing at least one selected from the group consisting of lactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine may be used, but is not limited thereto.

In the pharmaceutical composition for preventing or treating drug-resistant cancer of the present invention, MEC17 protein or a gene encoding the same, an agent reducing the activity or expression level of the protein; Or, the description of an agent that reduces the expression level of the gene encoding the protein is the same as that described in the composition for overcoming drug resistance and enhancing drug sensitivity, so it is omitted in order to avoid excessive complexity in the present specification.

The "prevention" of the present invention can be included without limitation as long as it is an act of blocking, suppressing or delaying symptoms caused by uncontrolled growth of cancer cells due to drug resistance using the composition of the present invention. there is.

The "treatment" of the present invention may be included without limitation as long as symptoms caused by uncontrolled growth of cancer cells due to drug resistance are improved or beneficial by using the composition of the present invention.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.

The pharmaceutical compositions of the present invention are not limited thereto, but are formulated in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions, respectively, according to conventional methods. can be used The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration. In the case of injections, buffers, preservatives, painless A topical agent, a solubilizer, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, and the like may be used. The dosage form of the pharmaceutical composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.

The route of administration of the pharmaceutical composition of the present invention is not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Oral or parenteral administration is preferred.

The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration, but is not limited thereto.

The pharmaceutical composition of the present invention depends on various factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and is 0.0001 to 50 mg per day. /kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

The present invention can treat resistance to OXPHOS inhibitors among anticancer drugs, increase sensitivity to OXPHOS inhibitors, and furthermore effectively prevent, improve, or treat drug-resistant cancers.

1 is a diagram confirming the MEC17 (aTAT1) gene expression level measured in gastric cancer cell lines SK4, MKN45, and AGS according to an embodiment of the present invention through Western blot analysis.
Figure 2 shows the amount of change in MEC17 (aTAT1) gene expression level before and after drug treatment with the OXPHOS inhibitor drug rotenone or IM156 in a cell line susceptible to an OXPHOS inhibitor and a resistant cell line without sensitivity according to an embodiment of the present invention using ImageJ It is a figure that was quantified and compared and confirmed.
Figure 3 is a diagram confirming the protein expression level by Western blot analysis for the selection of a cell line in which the target gene is not expressed after aTAT1-crisper (CRSPR) knockout according to an embodiment of the present invention.
4 and 5 are oxygen in the cellular respiration process before and after IM156 drug treatment to control (PSK4, SSK4 cell lines), aTAT1-Crisper knocked out IM156 sensitive cell lines and IM156 resistant cell lines according to an embodiment of the present invention. It is a diagram that compares and confirms changes in drug sensitivity through Oxygen Consumption Rate (OCR) measurement.
Figure 6 shows cell viability before and after treatment with IM156 drug in control (PSK4, SSK4 cell lines) and aTAT1-CRISPR knocked out cell lines (aTAT1 KO PSK4, aTAT1 KO SSK4 cell lines) according to an embodiment of the present invention. ) is a diagram showing the results of measuring.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Cultivation of gastric cancer cell line

The inventors of the present invention conducted all experiments with the approval of the Yonsei University College of Medicine Evaluation Board (Institutional Review Board, IRB). SK4, MKN45, and AGS cell lines, which are human gastric cancer cell lines, were obtained from the Korean Cell Line Bank and cultured in RPMI 1640 medium containing 10% FBS with 1% penicillin-streptomycin (Gibco) according to the guide of the Korean cell line bank. was added and cultured in a 37° C., 5% CO ₂ incubator (HERAcell 150i, Thermo Scientific, Waltham, MA, USA) and used for experiments.

Example 2: Confirmation of MEC17 (aTAT1) expression in OXPHOS inhibitor drug resistant (drug resistant) cell lines

SK4 parental cells, which are the cell lines cultured in Example 1, are referred to as PSK4 cells (SK4 parent cells; PSK4 cells) in the following specification, and cells that survived through subculture over 30 days in a glucose-free environment from the PSK4 cells ( Only cancer stem cells (CSCs) are referred to as SK4 selected cells (SSK4 cells) in the specification below. In order to confirm the correlation between gene expression of MEC17 (aTAT1) related to acetylated protein and patient prognosis due to OXPHOS inhibitor Rotenone or IM156 drug, rotenone and each drug of IM156 were compared with a sensitive cell line (PSK4). A resistant cell line (SSK4) without sensitivity was treated, and changes in gene expression level were analyzed in the same amount of cells. The results are shown in FIG. 2, and in order to compare them more clearly, the results were quantified (fold change) based on the aTAT1 expression level before drug treatment using ImageJ, and are shown in FIG. 2 and Table 1.

aTAT1
expression change amount before drug treatment Rotenone drug IM156 drug PSK4 1.0 0.9375 1.0100 SSK4 1.0 1.5199 1.6454

As shown in Figure 2 and Table 1, the expression of the MEC17 (aTAT1) gene in the resistant cell line SSK4 cells was significantly increased by more than 50% after drug treatment, and the expression of the MEC17 (aTAT1) gene in the drug-sensitive cell line PSK4 cells. It was confirmed that this appears at a similar level or tends to decrease in the case of rotenone drugs. This means that when the expression of the MEC17 (aTAT1) gene is increased, the therapeutic response of the IM156 drug is low, and the patient's prognosis may be poor. As such, if the MEC17 (aTAT1) expression level after the drug treatment is higher than that of the control group, it can be seen that resistance to the drug has developed, which can predict a poor prognosis for the drug.

Example 3: Confirmation of the possibility of treating OXPHOS inhibitor drug resistance (drug resistance)

SK4, the above gastric cancer cell line, was divided into P cells (IM156 sensitive cell line) and S cells (IM156 resistant cell line) to determine whether the resistance of IM156 among OXPHOS inhibitors was overcome. For additional experiments using CRISPR (Clusters of Regularly Interspaced Palindromic Repeats) gene editing technology, a ToolGen company was commissioned to produce the sequence shown in Table 2 below, and experiments were performed.

Sequence (5'→3') note One ^st PCR F: GCCTTTTTGGTGACCTCTGA SEQ ID NO: 3 R: GGATGATCGTGAGGCTCATA SEQ ID NO: 4 Adapt PCR F: CCTAGGACCCGCTAATTTAG SEQ ID NO: 5 R: GATGGTATATAAGGGAGGCC SEQ ID NO: 6 MEC17 (aTAT1) target site TATGACCATTATAGATGAACTGG SEQ ID NO: 7

The pRGEN_Human_ATAT1_U6_SG2 vector containing the above target sequence and the pRGEN-Cas9-CMV/T7-Puro-RFP vector expressing Cas9 were transfected together for 48 hours, and then selected as a selection marker. (See FIG. 3), a cell line in which MEC17 (aTAT1), in which the target gene is not expressed, was knocked down and used in the experiment. 1) Control group (PSK4 and SSK4 cell lines) treated with IM156 and oxygen consumption rate (OCR) during cellular respiration, 2) MEC17 (aTAT1)-CRISPR knocked out PSK4 and SSK4 cell lines (IM156 sensitive cell line and IM156-resistant cell lines) in the process of cellular respiration before IM156 drug treatment, 3) MEC17(aTAT1)-CRISPR-knockout PSK4 and SSK4 cell lines (IM156 sensitive cell lines and IM156-resistant cell lines) after IM156 drug treatment The results of comparative measurement of consumption rate (OCR) are shown in FIGS. 4 and 5 .

As a result of the experiment, both PSK4 and SSK4 cell lines confirmed that mitochondrial function was reduced by reducing OCR by IM156 treatment after MEC17 (aTAT1) knockout, but in the case of SSK4 cell line (IM156-resistant cell line), mitochondrial function increased after IM156 treatment and previous results. Conversely, it was confirmed that OCR was greatly reduced when IM156 was treated after MEC17 (aTAT1) knockout (see FIG. 5). This means that mitochondrial function can be reduced through inhibition of MEC17 (aTAT1) when drug resistance to IM156, a mitochondrial complex I inhibitor, is acquired. In other words, the degree of reduction in oxygen consumption rate was the largest and significantly reduced when MEC17 (aTAT1) was knocked out in IM156-resistant cell lines. Through this, it can be seen that IM156 drug sensitivity is greatly increased when MEC17 (aTAT1) is knocked out. . In order to verify the above results, cell viability was measured in an additional experiment to confirm that the mitochondrial function had the same results as above. As a result, when MEC17 (aTAT1) was knocked out by CRSPR as shown in FIG. By doing so, it was confirmed again that the reactivity to the IM156 drug was improved.

Taking these results together, OXPHOS inhibitor-resistant cell lines were selected through the expression level of the MEC17 (aTAT1) gene, and furthermore, OXPHOS inhibitor-resistant cancer was prevented and improved by treating resistance to the drug or increasing sensitivity to the drug. or expected to be curable.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A composition for treating drug resistance <130> PDPB214114 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 421 <212> PRT <213> Homo sapiens <400> 1 Met Glu Phe Pro Phe Asp Val Asp Ala Leu Phe Pro Glu Arg Ile Thr 1 5 10 15 Val Leu Asp Gln His Leu Arg Pro Pro Ala Arg Arg Pro Gly Thr Thr Thr 20 25 30 Thr Pro Ala Arg Val Asp Leu Gln Gln Gln Ile Met Thr Ile Ile Asp 35 40 45 Glu Leu Gly Lys Ala Ser Ala Lys Ala Gln Asn Leu Ser Ala Pro Ile 50 55 60 Thr Ser Ala Ser Arg Met Gln Ser Asn Arg His Val Val Tyr Ile Leu 65 70 75 80 Lys Asp Ser Ser Ala Arg Pro Ala Gly Lys Gly Ala Ile Ile Gly Phe 85 90 95 Ile Lys Val Gly Tyr Lys Lys Leu Phe Val Leu Asp Asp Arg Glu Ala 100 105 110 His Asn Glu Val Glu Pro Leu Cys Ile Leu Asp Phe Tyr Ile His Glu 115 120 125 Ser Val Gln Arg His Gly His Gly Arg Glu Leu Phe Gln Tyr Met Leu 130 135 140 Gln Lys Glu Arg Val Glu Pro His Gln Leu Ala Ile Asp Arg Pro Ser 145 150 155 160 Gln Lys Leu Leu Lys Phe Leu Asn Lys His Tyr Asn Leu Glu Thr Thr Thr 165 170 175 Val Pro Gln Val Asn Asn Phe Val Ile Phe Glu Gly Phe Phe Ala His 180 185 190 Gln His Arg Pro Pro Ala Pro Ser Leu Arg Ala Thr Arg His Ser Arg 195 200 205 Ala Ala Ala Val Asp Pro Thr Pro Ala Ala Pro Ala Arg Lys Leu Pro 210 215 220 Pro Lys Arg Ala Glu Gly Asp Ile Lys Pro Tyr Ser Ser Ser Asp Arg 225 230 235 240 Glu Phe Leu Lys Val Ala Val Glu Pro Pro Trp Pro Leu Asn Arg Ala 245 250 255 Pro Arg Arg Ala Thr Pro Pro Ala His Pro Pro Pro Arg Ser Ser Ser 260 265 270 Leu Gly Asn Ser Pro Glu Arg Gly Pro Leu Arg Pro Phe Val Pro Glu 275 280 285 Gln Glu Leu Leu Arg Ser Leu Arg Leu Cys Pro Pro His Pro Thr Ala 290 295 300 Arg Leu Leu Leu Ala Ala Asp Pro Gly Gly Ser Pro Ala Gln Arg Arg 305 310 315 320 Arg Thr Arg Gly Thr Pro Pro Gly Leu Val Ala Gln Ser Cys Cys Tyr 325 330 335 Ser Arg His Gly Gly Val Asn Ser Ser Ser Pro Asn Thr Gly Asn Gln 340 345 350 Asp Ser Lys Gln Gly Glu Gln Glu Thr Lys Asn Arg Ser Ala Ser Glu 355 360 365 Glu Gln Ala Leu Ser Gln Asp Gly Ser Gly Glu Lys Pro Met His Thr 370 375 380 Ala Pro Pro Gln Ala Pro Ala Pro Pro Ala Gln Ser Trp Thr Val Gly 385 390 395 400 Gly Asp Ile Leu Asn Ala Arg Phe Ile Arg Asn Leu Gln Glu Arg Arg 405 410 415 Ser Thr Arg Pro Trp 420 <210> 2 <211> 21182 <212> DNA <213> Homo sapiens <400> 2 aaatctt gag gctgagggcg ggtggtacag atgtgtatgg gaaaccccaa cccctatata 60 ttgtaaatag atgggctggg ctaaacattg ttgccgtttc atacttctac caactcagct 120 tttacacaat aaagctctac tgtctctggt ttgctttggg ctgtttccga tgaatgccat 180 tagcgggggg tgggctgagt gatggtcttt tcatataagc aattgggtga tgctgtgggg 240 agataagtgg tcaggcttaa gccagccttg cctgtgacgc ctgggactag aagccgggga 300 tgggcagctg tgccactctg tcaagatgcc ttgtgggccc ccactccaca gcatggccca 360 ctgttcactg aggggataaa aggttggaca gtgagacact gggccaagga agactacgtt 420 gccatggcac tcactgccgt gggatgcagg gatggaaagg agtggcactg ctaggggcac 480 agctggtttg gcaagaaaaa cgggggccct gtcagttgcc aggacgctag ggggcaaggt 540 ctacaggcgg ggctcctgga aataaagact ccgagaggcg gtgcggcgag aggaggggcg 600 gaagtgacgt cgtgtggggc gggtccgacc gcgcacaatg ggccatggag ttcccgttcg 660 atgtggacgc gctgttcccg gagcggatca cggtgctgga ccagcacctg aggcccccag 720 cccgccgacc cggaaccaca acgccggccc ggtgacagct caaacccacc ctctggccct 780 tttctcccgg ttcctctcca aacctggtcc aggcaccacg cccccttctc actgactagt 840 gatcgcccct tttgatgtcc aggcct gcct ttttggtgac ctctgaccct gggcctagtg 900 ggattgatca gcgcttggat ctgtgacctt tcaccccggg cccaaaatgt cccaatcaaa 960 ggatgtggtt gacctggcct ttctgcttcc tcacaataac cttaagggag gagggagtgt 1020 gccaccttga aaggtgtgac agaagtttgg gtttcagaag ggtggggtgg gaaatcagat 1080 tggaagactc ccaggcaaag gcagggagcc ttcagtgtta aacctgggtt ggagttgtgg 1140 cccaggttcc caggactgac tgcctaggac ccgctaattt agtgagtatc tgactcttta 1200 tttcttctct ttctctagtg ttgatctaca gcagcaaatt atgaccatta tagatgaact 1260 gggcaaggct tctgccaagg tactggagag tttttagatg gagtaaaggg aggacctctg 1320 tggggatggt atataaggga ggcctgggtc cttcggagag acttgcagaa agtctgactt 1380 aatcttccct gcaggcccag aatctttccg ctcctatcac tagtgcatca aggatgcaga 1440 gtaaccgcca tgttgtttat attctcaaag acagttcagc ccgaccgtga gtgccacatg 1500 ctcttccatc ccatacttaa ttccttcctt cctcagccct tcccccatct ttgactatct 1560 cttgcagata gataccacta gcctgttcat tattttcccc gtcctacagg gctggaaaag 1620 gagccattat tggtttcatc aaagttggat acaagaagct ctttgtactg gtgagtgtta 1680 ttggatgcta ggagttcgta taccttggtt tct gagaaca aaagtgctgg aggttagggg 1740 gcagcagaga tgccggggtt cctaaaacat ttttattgtt tctctcttag gatgatcgtg 1800 aggctcataa tgaggtagaa ccactttgca tcctggactt ttacatccat gagtctgtgc 1860 aacgccatgg ccatgggcga gaactcttcc agtatatgtt gcaggtatca ctgacctctt 1920 cactggttca tccaaactag gggctccttt gccctgagcc cttccagaag ccctgcctcc 1980 caccccccat gttcccatgt cattctattc ccttcccagg cttctggctt cctgttggca 2040 tgctttcccc atacttcctc ctaccctgag tctccttttc cctgcagaag gagcgagtgg 2100 aaccgcacca actggcaatt gaccgaccct cacagaagct gctgaaattc ctgaataagc 2160 actacaatct ggagaccaca gtcccacagg ttagaggttt cagagaatag atccccactg 2220 agcattccca ttgaatttat ttgttattta tggcaaagaa gtagtgactt atttcctatc 2280 acataggttt cattttctac aaccaggctc tttctttctc ttgtggtacc atctctcatc 2340 ctgtagtgac ttctttctca tctattttga tttttttttt tgagatggag tctcgccatg 2400 ctgcccaggc tggagtacag tggcgcaatc tcagctcact gcaacctcca cttcctggtt 2460 tcaagcgatt ctcctgcttc agcctcctga gtagctggga ctacaggcac ccaccaccac 2520 acccagctaa tttttatatc tttagtggag acggagtta c accatactgg ccaggctggt 2580 ctcaaactcc tgaccttgtg atctgcccgc cttggcctcc caaaatgctg ggattacagg 2640 tgtgagccac cgcatctgac tttttttttt tttttttcaa agcagagtct cctgctgttg 2700 cccaagctgg agtgctatgg caggatcttg gctcactgca gcccaacctt ctgggctcaa 2760 gcgatactcc tcccttagcc tcctgagtag ctgagactac aggcatgcac caccatgcct 2820 ggctaatttt ttattttttg tagagatgag gtctcactat gttgcactgg gtggtcttga 2880 actcctggct caagagatcc acctgcctca gcctcccaaa gtgctgggat tataggcgtg 2940 agccactgta cccagactta ttttgattct ttaccacaag ttgtttccta cacctaattt 3000 ttcttttttt ttttttttgt gagatgtagc cttgctccat cgtccaggct ggattgcagt 3060 ggcacgatca cagctcactg caacctctgc ctccggggtt caagtgattc ttgtgcctca 3120 gcctcctgag tagtagggat tacaggcatg caccatcatg cccagctaat ttttgtattt 3180 ttagtagaga tggagtttca ccatgttgga cagactggtc ctgaactcat ggcctcaagt 3240 gatgtgccca cctcagcctc ccaaaagtgc tgggattaca ggtgtgagcc accgcacaca 3300 acccttatgc ctaatttttt tttgagacag agtcgctctg tcacccaggc tggagtgcag 3360 tggcacgatc tcagctcact gcaagctccg cctcccaggt tcac ggcatt ctcctgcctc 3420 agcctcccga gtagctggga ctacaggtgc ccaccaccat acccagctaa ttttttgtat 3480 ttttagtaga gatggggttt caccgtgtta gccaggatgg tctagatctc ctgaccttgt 3540 gatctgcccg cctcggcctc ccaaagtgct gggattacag gcgtgagcca ccgtgcccga 3600 ccccttatga ctaattttca acccaaacat agccagctca ttttcacctc cttgttttca 3660 catagttcat tactcatctg gtcagtcagt atttattaag ggtccagaat aatatgcatt 3720 ccctgtcctc atggagcttt ggcctaatat agggaaggaa gtcttgttta taactaagtg 3780 cagcaaaatg ttactaatgc tacccattca tccaataaac attgagtgcc tggcagtgtt 3840 ctgggcacta ggaatggttt actcaatgaa acagacaaca gcctgggcaa catagcgaaa 3900 ctctgtctct acaaaaaata caaaaaaaaa ttagccaggc gtggtggcac gagcctgtag 3960 tcccagctac ttgggaggct gaaatgggag aatcgcttga gcctgggagg cagaggttgc 4020 agtgagccaa gatcgcgcca ctgcattata gcctgggcaa cagagagaga ccctgtctcc 4080 aaaaatgaaa acaaaaacag aaaaaaaggc caggtgcggt gcggtggccc atgcccgtaa 4140 tcccagcact ttgggaggct gacgtgggcg aatcacttga ggtcaggagt ttgagaccag 4200 cctggtcaac atggtaaaac cccgtctcta ttaaaaatac aaaaattagc ggggcatgat 4260 ggtgggtacc tgtaatccca gctacccagg aggctgaggc aggagaatca cttgaacccg 4320 ggaggcagag gttgcagtga accaagattg caccactgca ctccagcctg agcgacagag 4380 tgaggactcc atctcaaaaa agaaaaagaa aaagggccag gcatggtggc tcatgcctgt 4440 aatccccaca ctttgggagg ccaaggcagg aggatcacct gatatcagga gttcgagatc 4500 agcatgtgga acatagtgaa accctgtctc tactaaaaat ataaaaatta actgggcatg 4560 atggcgtgcg cctgtaatcc cagctactcg ggaggctgag gcaggagaat tgcttgaacc 4620 ccggaggcag aggttacagt gagccgaggt cctgctacag cactccaccc tgggggacga 4680 agcgagactc ttgtctcgga acaaaaaaaa aaaacagaaa aagaagggaa cagacaaaag 4740 tccctgtctt agtggtggag cttatattct agctggagag acaaacaaac ataataaaca 4800 atatggttaa taagtgctct ggaaaaatga gagcaagtaa gggtttggga gtactcaagt 4860 aaggtgggga tgggagtatg tgggattgca ggttgaaagg ggatcatcac tgagaaagtg 4920 tcatttgagc aataactgaa aggaagtaag agtaaaaact ggccgggcac ggtggctcat 4980 gcctgtaatc ccagcacttt gggaggccga ggcgcgcgga tcacgaggtc aggagatcta 5040 gaccatcctg gctaacatgg tgaaaccctg tctccactaa aaaaaataca aaaaa attag 5100 ctgggtgcct gtagtcccag ctactcggga ggctgaggca ggagaatggc gtgaacctgg 5160 gaggcagagc ttgcagtgag ccgagatcgc gccactgcac tccagcctgg gtgacagagc 5220 gagactccat ctcaaaaaaa aagaataaaa accaaggctg ggcgtggtga cttacatctg 5280 caatcctagt acttagggag gccgaggtgg gtggatcact tgagcccagg agttcgagac 5340 tagcctaggc aacatggtga aaccccatct ctacaaaaaa cacaaaaatt agccaggtgt 5400 agtggcacgc acctgtggtc ccagctactt gggggtctga ggcaggagga ttgcttaagc 5460 ccaggaggtc gaagctgcag tgagccgaga tggtaccact gcactgcagc ctgggtaaca 5520 acgtgagact gtctcaaaac aaacaaacaa aaaaaaagag taagagccaa gaaatatctg 5580 gagagagagc atcccagaca gaaggtacca ccagtgtatg gccgtgaggt gggagtgtgc 5640 ctgaaagagc aaattggctg tgtccagagc agcatgagtc agcggaggag tatggtagga 5700 gatgagacca gagaggtaat gcggaggagg ggccttatag gctactgcaa agactggctt 5760 ttattttaag taaaaaataa gatcaggcca ggggtggcga ctcacacctg taatcccagc 5820 actttgggag gccgaggtag gtggatcacc tgaggtctct actgaaaata ccaaaattag 5880 ctgggtgtga tggcaggtgc ctgtaatccc agctgtttgg gagtctgagg caggagaatc 5940 actagaaccg ggaggcggag gttgcagtga gccgctgaaa ttgtaccact gcactcctgc 6000 ctgggcgaca gagcaagact ccttcttaaa aaaaaaaaaa aaaaaaaata gcgccaggtg 6060 tggtatctca ttcctgtaat cccagcattt tgggaggccc aggcaggtgg atcacaaggt 6120 caggagttcg agaccagcct ggccatatgg tgaaacccca tctctactaa aaatacaaaa 6180 attagccggg tgtggtggcg ggcacctgta gccccagcta cttgggaggc tgagatagaa 6240 gaatcgcttg aacctgggag gcagaggttg cagtgagctg agatcgcact actgcactcc 6300 agcctggata acagaacgag actccatcaa agaaaaaaga aaaagatcat tttggctgtg 6360 atcttgattt tttccttttt aacaagatca ctttggctgt taagaacagg caatagccgg 6420 gcacagtggc tcacacctgt aatcctagca ctttgggagg ccgaggcagg tggattgcct 6480 gaggacttca agaccagtct ggctaacatg gtgaaacccc atctctacta aaaatagaaa 6540 aaaaaattag ccaggtgtgg tggtgctcgc ctgtaatccc agctactcgg gagactgagg 6600 caggggaatt gcttgaatca gggaggtaga ggttgcagtg agctgagatt gtgccactgc 6660 actgcactct agcctggtga cagagtaaga ccccatatca aaaaaaaaaa aaaaatggaa 6720 acggcaataa gggagccaga gtagaagcaa gtagactaat taggcagcaa caatcctggc 6780 g aaaaatggt ggtggctcag accaaggtgg tagcagtagt gatggtaaag agtggtcaaa 6840 ttttaaatat tttgaaggta aagcaagtaa gatttcctga cagattgtat gtggagaaag 6900 aggactttag gacaatgcca aagcctgagc agctggaaga atgaagttgc tttaactgag 6960 atggtaggta gaccagcttt gggggaaata ctaggagtac atttttaata tgttaattgg 7020 agatgtctgt gatatgtcca ggtttgagta gacagttgga tacttccctg gagatcaggg 7080 aggaggtttg gggaggagag ttttcagcat acatctggta tctaaagcca acagacagga 7140 tgcgatcacc atagaaagat tatagataga gaagctgccc ctttgggccc tctttaagaa 7200 gtgaggaccc ccaactggct gctctgaaaa gccatctttg cattgttcct ggttcggtgt 7260 cctgctcacc acagccacct ccgccatgca cttcctctgc tgcctcagag tctggcagct 7320 taatcgacat agtccccaaa ctctcacttt cttcttaatc ccttgcatcg gatcaccgct 7380 gtgccccacc atgtcagagg cagttgtgga cacaagctcc gtgatcacca ccaaggactt 7440 caaggagaag ttgtggagga ggcagaaagt ggaagagacg cccatgctaa cgggaacgct 7500 aatgaggaaa atggggagca ggaggctgac aacgaggtag atgaagaaga ggaacagggt 7560 ggggagaaag aggagaagga agaggaaggt gatggtgaag aaaagaacgg agatgaaaac 7620 gaagcag ctg aggcggtatg gacaaatggg cagctgatga tgatgaagat gacgatgttg 7680 ataccaagca gcagaaggcc agtgaggatg attagacagc aaaaaaagaa aagttaaact 7740 ttaaattaag gccaccgtga cctattcacc ctccacttcc catctcagaa tctaaacatg 7800 gttgccctcg agaggcctgc ttgccctcca cagacagtgc cactgcagat gacaggcact 7860 caccaccacc caacccaaac cagagaattt gcaacagagg aggaaaaaag aaccaaaact 7920 tccaaggtct tgctctttta aaagtacttt aaaaaggaag tttgtttgta ttttttattt 7980 acattttata tttttgtaca tattgttagg gtcatttttt ttttctttga gacggagtct 8040 agctctgtcg ccaggctcaa gtgcagtggt gcgatcttgg ctcaccgcaa gctccacctc 8100 ctgggttcaa gtgattctcc tgcctcagcc tcctgagtag ctgggattac aggcgcccgc 8160 caccacaccc agctaatttt tgtattttta gcagagacag gctttcacca ggttggccag 8220 gatggtttct atctcctgac cttgtgatcc acctacctcg gcctcccaaa gtgctcgaat 8280 tacaggcgtg agccaccggc gcccagccag gttcagtcat ttttaatgat ctcagatgac 8340 caagccagcc tttggagggt tctctgtctt acttctgact ttacttgtgg tgtgaccata 8400 ttcattataa tctcaaagga ggaaaaaaaa aaaaaaaaaa aaccttgttt aaaaaaaaaa 8460 aaaaagcctg gg cgcggtgg ctcgcgcctg taatcccagc actttgggag gccgaggtgg 8520 gtggatcacg aggtcagaag atcgagacca tcctggctaa catggtgaaa ccccctgtct 8580 actaaaaata caaaaaatta gccaggcgtg gtggcgggag cctgtagtcc cagctacttg 8640 ggaggctgag gcaggagaat ggcgtgaacc cgggaggcag agcttgcagt gagccaagat 8700 tgtgccactg cactccagcc tgggcaacag agcgagacta catctcaaaa acaacaacaa 8760 caacaaaaag tctcgttctg agcattccag tagcttcttt agtgtatgta gttagttgta 8820 ccataagtag ttggtttgtg tgagatggtt aaaaaggcca aagataaaat gtttcattta 8880 tttgcctttt ttgtctatga aatggctgct tatttattta ggcctatttg atgtatgtgt 8940 gaaacaatat tgtgcaacaa taaacccaaa ttttattttg ctgagttgtt ctaacagcaa 9000 caaaaagaag ttaaggaaga gaagaagacc agcaaatgca accacagagt gactagtgaa 9060 gtagatgaaa actgaggccg ggtgtggtgg ctcacacctg taatcccagc actttgggag 9120 gccgagtcgg gtggatcacc tgaggtcagg agttcaagac caacatggtg aaaccccatc 9180 tctacaaaaa atacaaaatt agccaggcga ggtggctcat gcctgtaatc ccagctactt 9240 gggaggctga ggcaggacaa tcacttgaat ctgggaggtg gaggttgcag taagccgaga 9300 tcatgccatt gcactcca gc ctgggcaaca aagcgaaact ccatctcaaa aaaaaaaaaa 9360 aagaaaagaa aactgaaaag taaggtgacc tcaaaggcca ctgaagaaag tgtttccagg 9420 aggaaggaat ggtttacttg gtcaaatgct gctgatcaag gagcaaagag gtctgagaag 9480 ttaccattgg atttatctgc gttaggccat tggtgatctt aatgagcagt tttggtgcag 9540 cggtgtttgg aagcctggat gcagtgggtc ttgtagactg agaagctagg aacacagcaa 9600 gaataagcta ctcttttaaa tcctgcttta atgggaatag aaatagagca agagctggag 9660 agtgaagtgg atcaaaagag ttgatctttt gcagatggga gaacaaatag cattagaatg 9720 attcagtaga gagaaaatat tattatgtca gagaaagtgg ggagaactgt tgaagtgatg 9780 tcattgaatg ggcggcgggg gcgttgagat ttggttgaca agttagcctt ggataggaac 9840 atggacagtt aatccattgt aacatgattt gatagatgtg attacagagg aggcataagg 9900 acatggattt gagtgctatc ttgggctggg ggttgggtaa agaaagatga catgtctaat 9960 cttgaaaggc aagtgtttgt caggtggaca aaaggctaaa gtgcatttca tgtagaggaa 10020 caggcatgag caaaggcaga aaggtattaa accacctttc aggccaggcg tggtggctca 10080 cacctgtaat cccagcactt tgggaggcca aggtaggcgg atcacaaggt caggagatcg 10140 agaccatcct ggctaacacg gtaaaacccc gtctctacta aaaatacaaa aaaaattagc 10200 tgggcgtggt ggcaggcgcc tgtagtccca gctaatcagg aggctgaggc aggagaatgg 10260 cgtgaaccca ggaggcggag cttgcagtga gcccagatca tgccactgca ctccagcctg 10320 ggcgacagag caagacactg tctcaaaaaa aataaataaa taaataaaaa taaaccacct 10380 ttcaggacac tacaagcagt gtggtgtggt tggagtgctg ggcatgtgct tgttgggggg 10440 tgggggtgat gaggatgggc tggtagacat tacaacaagg tcaaggcaag ggataggcag 10500 ggtcttccta cagtatattt ttccattaag aggcaacaga gagcagtgga aggagcacag 10560 tttttttttg tttgtttgtt tgtattttga gatggagtct cagtctgtcg cccaggctgg 10620 agtgcagtgg cacaatctca gctcactgga acctctgcct cctgagtcca agcaattctc 10680 ttgcctcagc ctcctgagta gctgggatta gaggcgccca ccaccacacc tggctaattt 10740 ttgtgttgat gaggtttcac catgttggcc agacgtctcg aacttctgac ctcaagtgat 10800 ccgcccacct cggtctccca aagtgctacg attacagccg tgagccacca tacccggtcc 10860 tggagcacag tattcgatat gaaacacatt acccagttaa catgtaaggc cagagcagta 10920 tagagtgtaa ataataattc acatttcatg ggctctatgt ggtatctata tgcatcatct 10980 cagtggattc ttg cacatct ttttgaggta ggtactatta ttaaacctat tttgggttta 11040 tacaaattaa tgacttaacc aaattcacac agccagtaaa tagtagagtc cacatttgaa 11100 cccatagcca tttgcaccca gtgaactttt tttttttttt tctttttgag gcaaggtctt 11160 gctctgttgc ctaggctgga gtgcagtggc acgatcacgg ctcactgcag tctctacctc 11220 ctaggctcaa gagatcttcc ctaccagcct ggccaacatg gcgaaacccc atctctatta 11280 aaaatacaaa aataagccgg gcgtggtggc atgtgcctgt aatcccagct actcaggagg 11340 ctgagacagg agaagagctt gaacctggga ggtggaagtt gcagggagcc gagatgacac 11400 cattgcactc cagcatgggc aacagagtga gattccatgt taaaaaaaaa aaaaggccgg 11460 acgcattggc tcgcgcctgt aaccccagca ctttggaagg ccaaggcggg cggatcacga 11520 ggtcaagaga tcaagaccat cctggccaac atggtgaaac cctgtctcta ctgaaaatac 11580 aaaaattagc tgggcatggt ggcgcatgcc tgtagtccca gctgctccgg aggctgaggc 11640 aggagaatcg cttgaactca ggaggtggag gttgcagtga gctgagatct tgccactgaa 11700 gtccagcctg gcaacagagc gagactccat ctcaaaaaag atcttcccac ctcaacctcc 11760 caagtagttg ggactacagg cgcccaccac tatggctggc tgattttttg tatttttagt 11820 agagacgggg tttcaccgtg ttagccaggg tggtctcgat ctcctgacct cgtgatcggc 11880 ccgcctcggc ctcccaaagt gctgggatta caggcttgag ccactgggcc cggcccacgc 11940 ctggctaatt tttaaaaata tttttgtaga gatgaggtct tgctatattg cccaggctgg 12000 tcttgaactc ctgggctcaa gctatccaca tgagccacca tgcccagccc ccattaaac t 12060 tttttttttg agatggagtc tcactctgtc acccaggctg aagtacagtg gtgcaatctc 12120 agctcactac agcctctccc tcctggggtc aatggattct cctgcctcag cctcctgagt 12180 agctaggatt acaggcgcac gtcaccacac ccagctaatt tttgtatttt tagtagagac 12240 agggtctcga actcctgacc tcaagtgatc cacccgcctt ggcctcccaa attgttggga 12300 ttacaggcgt gatccaccac gcctggcccc cgagtttttt tttttttttt gagacggagt 12360 ctctctctgt cgcccaggct ggagtgcagt gttgccatct cggctcactg caagctctcc 12420 tcttgagtaa actcttaatg gctacactat tttcctggct aaaacactgc agctggaatc 12480 agaagtctga aagttgaggc ccagccctgc cacttgtagc tacttggcat tggccaagcg 12540 aagccatgtc tccaaggctg tatttccccc aaccttcttt caaatagtga cttccaggat 12600 tgtgaaggcc aaattaaatg tgaaaatata atgaagtaac tctaaaatta atagttacta 12660 gttatcaaag tagcatcctg gcctccagca tgtcttcccc tgactttccc caccccttgg 12720 aaccctgctg aattttttat ttatttattt atcctttgag acggagtctc attctcttgc 12780 ccaggctgga gtgcagtggc acgatctcag ctcactgcaa cctccgcctc ctgggttcaa 12840 gcgactctcc tgcctcagcc tcccaagtag ctaggattac aggtgcacac t gccatgcct 12900 ggctaatttt ttgtatttat aatagacaca gggtttcacc atcttggcca gaccggtctt 12960 gaactcctga cctcaagtga tgcctgccac agcctcccaa agtgctggga ttacaggtgt 13020 gagccactga acctggactt tagcaccttt ttatgtgctt attggccatt tgtgtatctt 13080 ctttagagaa aagtttatac aagtcctttg tctgttctta aattgtgttc ttttttgttc 13140 tgagagtttt tcatatattc tagatagaac gcacttatca gatgtatgac ttgcaaacat 13200 tttctcccat tctgtagatt gtcttttcac tttctttctt tttttttttt tttgagacgg 13260 agtcttgctc catcgcccag gctggagtgc agtggcacga tctcagctca ctgcaagctc 13320 tgcctcccgg gttcacgcca ttctgctgcc tcagcctccc gagtagctgg gactacaggc 13380 gcccgccacc acatccggct aatttttttg tatttttagt agagatgggg tttcaccatg 13440 ttagccagga tggtctcgat ctcctgacct catgatccgc ccgcctcggc ctcccaaagt 13500 gctgggatta caggcgtgag ccaccgcacc tgacctttac tgtacctttt ctgtgttgag 13560 gtatgtttag atacatcagc tgaaccatta tgttagaatt gcctacagct ggctgagcat 13620 ggtggctcac gtctataatc ccaggacttt tggaggctga ggcagaagga tcacatgagc 13680 cctggagttt gagactggcc tgggcatcat agtgagaccc ccat ctctac aaaaagttaa 13740 aaaaaaatta gtagccagat gtggtggcat gcacctgtgg tcctagctac ttgggaggct 13800 gaggtgggag gatcatttaa gcccaggttg atgctgcagt gagctgtgat ggcaccactg 13860 cactccagcc taggcaacag agcgagactc tgcctctcaa aaaaaaaaaa aaattgccta 13920 cagcattcag tacagtaaca tgctgtacag gtttgtagcc taggagcaat aggctgtatg 13980 atatagtctg ggtgtgttgt aggctatagt gtctaggttt gtgtaagtac actctgtgat 14040 gttcacacaa tgaaatcacc caatgacgta tttctcagaa tgtatcccca tcgttaagtg 14100 atgcatgatt gtattttgtt tgtttcatct tccaggtgaa caactttgtg atctttgaag 14160 gcttctttgc ccatcaacat cgtaagtttt tgcattttgt tggtcacgta gtcggggtga 14220 gggaaaggaa agagctggac tcttggtcct gccgacccct cactgagggg ccccgccgct 14280 tccttcctca cagggccccc tgctccctct ctgagggcaa ctcgacactc tcgtgctgct 14340 gcagtcgatc ccacgcccgc tggtaaagcc tgtattgaag gggtggaact gtagtgcagt 14400 gatggctact tactctagat gccacggggt acagtgccat ctgtgggcaa ttttggaaaa 14460 ttctaaagca acccaagtct ccagcagtca tgactgtttg cctttgccct catgggagct 14520 cagtgcattt tatatttggc aagactttta actaagc aag ctcattggga gcctgtttga 14580 cagctgatat caatggaccc tcttgccagt tcaggtccgt caacataggc cagagtcagg 14640 ctcctttgta aaccccaggc ttctgttagc cagtgaggga caggctggtg caaacagccc 14700 ttccatttgc agtcacagaa tagtgacaca aatggcccaa aatttaaatg ttacttttag 14760 aagataacac tcagagttta taacatttcc aaccagataa tgaaattgat atggagaaac 14820 caaacctcag aggcactaaa atgctgtcca gattccccat cccatataca cacatacaca 14880 cacacacaca cacacaaaca cacttactga cagtctgagc cccactcctt cctcttcctc 14940 accacctcca ccttaccaac ttctgacagc tgtacagtgc ttgcttgcac agaagagccc 15000 ccttcctgag ctggctctgt ggccaggaaa ggatgtaacc accatccaaa cagcagtctg 15060 taaccagcta tgagcatcac agtgtcaggc actgagaggc acctcaactc gctttggttt 15120 ccaaggcttc tcccatttag cttgttcaga accacaggct gtgagaggga ctgagggcca 15180 acaaggatgg tgaggtctca ggcctgcagg ggagggtgct gtggataaag cttaagtgaa 15240 tttgctgaga agtctttcat ttgccacaca tacatgatgg agaatctctt gagagggaaa 15300 gccgggagca agtagagaag tgaggagggg gaggctgaac tttggacatt acatcagcct 15360 cctgcttact ctgatagctc cctttcagat gcccatattt attttctttt ttttttttaa 15420 cctaataaaa cttcagtctc ttcccatttt cgtataggaa ggagagattg tgccctcctt 15480 ccaaacctcc cctgacctct ccagagcaat tcctgattaa ccaagggctt tgtccatctc 15540 atccagagga acccagggtc ctcgttggcc cggctgggac cattccactg ccccagaata 15600 ccagggggcc atgacagcac ccactgacag taagagctca cttcccttgg ctgcccttct 15660 cctgcatctc ccaggccccc agagtctccc cttcgatctt tctccctagc tctgtgtttg 15720 gcctactcct tctggctttc ctcaacagtg ttccacattc ccctcaaatt cccttttggt 15780 gtgctggcat tgccatggtg ctgctcctgc aagttctcag gaggaactgt ggtgtcaggg 15840 agcagaggtt tggggttggg gtatagtggc tgggaggagg ggtgcaaagt atgtctccta 15900 acgcttaccc tgcctatgtc ccctccactg ccagctccag caaggaagct gccacccaag 15960 agagcagagg gagacatcaa gccatactcc tctagtgacc gagaatgtaa gaggggcaag 16020 ggtcgggtgt ctgggcctgg ggtaccttaa cacaagggaa gagaatgctc aggggaccca 16080 gggaaaggat tcgttctctc taaagactca gatttcttgg gctgggcatg gtggctcatg 16140 cctgtaatcc cagcactttg agaggctaag gcaggcagat cgcctgagtc caggggttca 16200 agaccagcct ggccaacatg gt gaaacccc gtctctacta aaaatacaaa aattagctgg 16260 gcacggtggc acgtgcctgt aatcccagct acttgggagg ctgaggcagg agaatggctt 16320 gaacccagga ggcggaagtt gcagtgagcc aagatcgtgc cactgcactc cagcttgggt 16380 gacagagtga gactccgtct caaaaaagaa aaaaaaaaaa aagaaagact cagatttctc 16440 tttttttcta ccaaaacctt tgctgtcatg actctcttcc ttttttcttc tttttctgtc 16500 ttgctcttca ttctccctgt ccccagttct gaaggtagct gtggagcctc cttggcccct 16560 aaacagggcc cctcgccgcg ccacacctcc agcccaccca cccccccgct ccagcagcct 16620 gggaaactca ccagaacgag gtcccctccg cccctttgtg ccagagcagg agctgctgcg 16680 ttccttgcgc ctctgccccc cacaccctac cgcccgcctt ctgttggctg ctgaccctgg 16740 gggcagccca gctcaacgtc gtcgcaccag gtaataggag ttgaagggct aaggagcctc 16800 acagctataa aagaggatgt tagaaatggc aaagggcaat ttgaatccat cagagagatg 16860 gatcaataag atgggtggct tggggggggt cctgaaacct ttcaagaaaa atatttgtgc 16920 aagtgatctg ggaaaaaaat gcagtgaagg agcagaatag gaccttatat ggagcctagg 16980 gaccctggct ttaatgtgag agttatgtgg aatggtagga agaacaccga gatccatcga 17040 gttgggggaa cagag ccttc taagattggg aaaatcttcg cttaatactt gctggggaag 17100 gggcagtgtc tgacagagag tgggaagcca ctggcttgtg tgccaagagt ccatcgcagc 17160 aggcagggag tgggcatttc ctttatttct ctccctttct cttcacctct gacttctctg 17220 tttttctctc ccccgccccc cgccatttcc catctccctt cctcccatcc ataacatcct 17280 tccacagctc ccttccccgc tctgaggaga gtcgatacta acagctaccc tctccctgcc 17340 ctgggagacc tggggtgggc agggaacccc tccctgagaa cctcagaccc actcttccat 17400 tgcatcctgt aggacccagt ggaacctgac agagcccata ggattccctc ttctactttc 17460 ttagacagca gggatgtcag ggtctcaaac tgcctaacac tttgtagctt ttcttaacac 17520 aaaagcaccc cttctctcct aacttgggct ctgaatactt tcccaacagg aagtctgatc 17580 tgttgccaga cttcttggtt agatggctca tacatttatc tagagaagca cactcttgct 17640 tgctgtcaaa ctttagacca ccatggaagg tctaagggca tcctgtgcca gggaaacttt 17700 ttaaggaatt ttatctatgg gataaacccc atattccctc tagtgtctac tggtggctct 17760 aatactgctt tgtgctgcct gccacacttg ccctttgagc ctgcgaatgg ccgctagtga 17820 gcaagctctg cttcagagca gtctagttag gtagaacagg gacttaccag cttcccaaag 17880 ggatctac tc accattgcca aactcttcat ttccacattt tgtgtaggtg tcagggaacc 17940 ccaaactggt gttgctttgg ggtctctaaa ggagattggc tgacaccacc atttccccca 18000 gatccagatt ctctgaggga ggttgtttct tgagagtaga tccagagtgt caaggatctg 18060 ttagatcctg gaatcccttc ttgcatccat ccctccctgg tagctaggtc ccgatatact 18120 cctgtcttgt gagattgtcg agatgagatg ggggaccact cttcctctgt ccttcctctc 18180 tcctttcctc catagcaagg acgaccttcc ctgctccatg cccagagtat agctagatcc 18240 cttcccctcc ctaccctctg aatgtgtgct agatcaggtg ccccactgtg tttcctgaaa 18300 tccttgggag ccggatctcc ccatctcccc tactcactct tcccttttct tctctcagtg 18360 ttgtctgaat aaagtgtgaa atcttttgtg ttttctaaat tgacattttc aatgaaaaaa 18420 agaatcacaa aaaaaaaagt tgtcagcctc atttgtgcgt catcccttat tttcctggga 18480 tctcaggacc tctgtccctc tcatttctca cttctgagat ctgcacatct tttacccagg 18540 agcctcagag ctcctgagtc tggtgtctgc ctatccccat cttcactgtt agtcctcctg 18600 cagattctgt gtctcctttc atgtaggtgc tggatccctg tgtgtgggct tccgtatcta 18660 ctccctcatt ccctccagga acctccagct ctccccagtg acttctaccc tttactctgg 18720 gcgtgccttt gccaagatgt caaagcttac caacatctct ggatccacta attacctcct 18780 gcctcctgta ttcgtcttcc cactctgatt acctgacgtc tgctccacta aaccgctgga 18840 tctctctcaa gacaaaccct tacctccatt gagagtgcaa cacagtctgt caccctattt 18900 acagaggccc ccttcctttt cctcctaaat tcaaaattca gccttgtcac ttcctatttc 18960 cctctggtct aaggaatctt tttttttttt tttgagatgg agtcttgctc tgtcgccagg 19020 ctggagtgca gtggcacaat ctcagctcac tgcaacctcc gcctcctggg ttcaagcgat 19080 tctcctgcct tagcctccca agtagctggg attacagaag tgcaccaccg tgcccagcta 19140 gtttgtgtat ttttagtaga gacagggttt caccatgttg gccaggttgg tctcgatctc 19200 ctgatcacgt gatctgcccg tcttggcctc ccaaagtgct gggattacaa gcctgagcca 19260 ccgcgcccag cctggtctaa ggaatcttat agttaaggta accctgtttt ccaaaccaaa 19320 caccagagta cccgatccaa cacatttttg accacatgtg agtctgttct tctgacatga 19380 tttggatcac acctagccat agatttaaca cattacctca actagaaaga atagagcaat 19440 aaatcagaag cactccagaa aatcttgggt ataaaatgaa cttcccccgc ccttttctgg 19500 ggcacagctt tgattaaaac ctgttaggaa tgataattac ccccttctct ttgttcctg t 19560 gctattcctt ttactcctct cctctgattc ctccataccc acccatcttt catccagtag 19620 cctcctcccc atcatctccc atttcttcta cagggggact cccccaggtc tggtagccca 19680 aagctgctgc tacagccgcc atgggggggt gaattcctca tcccccaata caggtaagta 19740 ttcactcctc cctaccctca aatcaagtag gccacattca ctgtctactc ctgccttccc 19800 attcacatgc ctgatatttc cacaggcaac caagactcca agcagggaga acaggaaaca 19860 aagaataggt gaggtctaaa cccctcccct aacagcctcc caccaccatc tgactccctt 19920 cctaacatca ttctcagtca cttcctactc ttaaatctta ttgtatgaac tggacaccag 19980 ctcctcccac aattccttct accttacatc ctgcaagccc ctttccccca caggttcaac 20040 tctggtactt ccctttggaa tacggattct ctgagaggtt ttaaatttgg acatagcact 20100 aatggttcca gcttcatacc catcatgtgt cctacattaa aacctggccc gagaccttga 20160 agagtctgta atcttaattt cctctttagt attcctataa cccactctcc atctccccac 20220 ctaccaggtc tgccagtgag gagcaggcct tgtcacagga tgggtctggg gagaagccca 20280 tgcacacagc tcctccacag gccccggccc cgccagccca gtcctggaca gtgggtgggg 20340 acatactcaa cgccaggttc attcgaaacc tgcaggaacg tcgcagcacc a ggccttggt 20400 gaccgcagcc ccgtcaaaca tcttcaaagt attatttctc cctcactaca ggaaagagcc 20460 aaagcccaac cctcataata gatggataca ttcattcatt cattcattca gcaggcttat 20520 cagattcaag tcatttgtat cttttaacca gaccaataaa agtatttatt tttatcacaa 20580 gagctgttga aaaatttgac tcattatttc agccgcctca cccctcactg tcgttgcacc 20640 cattcagcct tcagccctgt ttttgctcag ctttttgctc aaaggcctca gctgtgaata 20700 cagcgcttgg gggggcgggg gaggctgtaa cttgcgcaag cgcactcagg cagtctccga 20760 gcccgcgggc gcaggcgcgc ttacagccga cagagcgctt cagccgcttc cctcgagcct 20820 gcagtgcgca agcgcgggac atctccgttt ccctccctca gccccttccc cccctacccc 20880 cccgccccgg cctcctttcc ccttcacgaa gccggctctg gggcgcgctc acccctgtga 20940 ggaggccgga ggtcggactc aggaggctcc ttctccactc ccggaagatc atgtaccagc 21000 ccagccgggg tgcggcccgg cgtctcggcc cttgcctgcg cgcctaccag gctcgacccc 21060 aggtgagcgg aggagaagag ggagggagga gagggggcgg ggagagaccc tcctcaaagc 21120 cggtgcgtgg ggcggagcgc gcgctgggtt ccgcgcaggc gcagagacac ccgccgcccc 21180 tt 21182 <210> 3 < 211> 20 <212> DNA <213> Artifi cial Sequence <220> <223> sense siMEC17 <400> 3 gcctttttgg tgacctctga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense siMEC17 <400> 4 ggatgatcgt gaggctcata 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense siMEC17 <400> 5 cctaggaccc gctaatttag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense siMEC17 <400> 6 gatggtatat aagggaggcc 20 <210> 7 <211> 23 <212> DNA <213> Homo sapiens<400> 7 tatgaccatt atagatgaac tgg 23

Claims (15)

Agents that reduce the activity or expression level of MEC17 (alpha Tubulin Acetyltransferase 1; aTAT1) protein; Or, a pharmaceutical composition for overcoming drug resistance comprising an agent that reduces the expression level of the gene encoding the protein as an active ingredient. According to claim 1,
The agent for reducing the activity or expression of the protein is any one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the MEC17 protein or a part thereof, composition. According to claim 1,
Agents that reduce the expression level of the gene include antisense nucleotides that complementarily bind to the MEC17 gene or a part thereof, short interfering RNA (siRNA), short hairpin RNA, and ribozyme Any one or more selected from the group consisting of, the composition. According to claim 1,
Wherein the drug is an oxidative phosphorylation (OXPHOS) inhibitor. According to claim 4,
Wherein the OXPHOS inhibitor comprises at least one selected from the group consisting of a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, and an iron chelator. According to claim 5,
The composition of claim 1, wherein the OXPHOS inhibitor is any one selected from the group consisting of IM156, metformin and rotenone. According to claim 1,
The above drugs are thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hematological cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof. Which is used for the treatment of cancer selected from the group, the composition. Agents that reduce the activity or expression level of MEC17 (alpha Tubulin Acetyltransferase 1; aTAT1) protein; Or, a pharmaceutical composition for enhancing drug sensitivity comprising an agent that reduces the expression level of the gene encoding the protein as an active ingredient. According to claim 8,
Wherein the drug is an oxidative phosphorylation (OXPHOS) inhibitor. According to claim 9,
Wherein the OXPHOS inhibitor comprises at least one selected from the group consisting of a ubiquinone reduction activity inhibitor, a mitochondrial electron transport chain I inhibitor, and an iron chelator. According to claim 10,
The composition of claim 1, wherein the OXPHOS inhibitor is any one selected from the group consisting of IM156, metformin and rotenone. According to claim 8,
The above drugs are thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hematological cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof. Which is used for the treatment of cancer selected from the group, the composition. Agents that reduce the activity or expression level of MEC17 (alpha Tubulin Acetyltransferase 1; aTAT1) protein; Or a pharmaceutical composition for the prevention or treatment of drug-resistant cancer comprising, as an active ingredient, an agent that reduces the expression level of a gene encoding the protein. According to claim 13,
Wherein the drug is an OXPHOS inhibitor selected from the group consisting of IM156, Metformin and Rotenone. According to claim 13,
The cancers include thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and hematological cancer. , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and combinations thereof. A composition, wherein the composition is a cancer selected from the group.

Images