WO2002006520A1 - Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose - Google Patents

Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose Download PDF

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WO2002006520A1
WO2002006520A1 PCT/JP2001/006171 JP0106171W WO0206520A1 WO 2002006520 A1 WO2002006520 A1 WO 2002006520A1 JP 0106171 W JP0106171 W JP 0106171W WO 0206520 A1 WO0206520 A1 WO 0206520A1
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compound
lkb1
mek
test sample
erk
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PCT/JP2001/006171
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Japanese (ja)
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Jun-Ichi Nezu
Takakazu Mizuno
Asuka Ose
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Chugai Seiyaku Kabushiki Kaisha
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • the present invention relates to a method for screening a compound that regulates MEK / ERK signaling, and a pharmaceutical use of the compound.
  • the present invention relates to screening for a compound that regulates intracellular signal transduction associated with Peutz-Jeghers syndrome and its pharmaceutical use.
  • Fenne, et al. Considered this to be a candidate for the causative gene, and performed mutation analysis of the LKB1 gene in PJS patients, and found that the LKB1 gene mutation was present in all patients examined (Jenne DE et al., Nat Genet 1998 Jan; 18 (l): 38-43).
  • LKB1 is very important in the regulation of cell proliferation and cell differentiation. It is suggested that it plays a role. Therefore, if the intracellular physiology involved in LKB1 can be clarified at the molecular level, drugs that target the molecular mechanism can be rationally designed, thereby artificially controlling these phenotypes. It is considered possible. Such drugs are also expected to be applied to the treatment and prevention of diseases such as S. However, at this stage, the molecular mechanism of LKB1-related intracellular physiology has not been elucidated at the molecular level. Disclosure of the invention
  • An object of the present invention is to elucidate the intracellular physiological mechanism involving LKB1, which is a causative gene, and to provide a compound that controls the intracellular mechanism and a screening method thereof.
  • Another object of the present invention is to provide a drug containing a compound that controls the intracellular physiological mechanism as an active ingredient.
  • the present inventors aimed to identify intracellular signaling pathways involving LKB1, and examined the involvement of LKB1 in various known signaling pathways. The results revealed that LKB1 strongly suppressed activation of the c-fos gene promoter by activated K_Ras in HeLa cells (Fig. 1).
  • This c-fos promoter has SRE (serum responsive element), CRE (cAMP responsive element), and TRE (TPA responsive element) as known transcription regulatory element binding elements. It is known that activation of the promoter occurs mainly by activation of SRE by a Ras / MEK / ERK-mediated pathway (Fukumoto Y et al., J Biol Chem 1990 Jan 15; 265 (2): 774-80).
  • this pathway activates c-Raf kinase by Ras, which in turn activates MEK1,2 kinase, then ERK1,2 kinase, and ultimately transcription factors.
  • Ras a pathway in which gene transcription is activated by phosphorylation of Elkl, which often plays a very important role in the regulation of cell growth as the so-called Ras / MEK / ERK cascade.
  • LKB1 was thought to act on any step of this signaling pathway activated by forced expression of activated K-Ras to suppress SRE activation. Then, the present inventors examined the steps involving LKB1, and found that LKB1 also strongly suppressed the activation of artificial SRE by MEK1, a downstream factor of Ras. ( Figure 3). This suggests that LKB1 acts at least downstream of MEK1.
  • the Ras / MEK / ERK signal transduction pathway is known to play an important role in normal cell proliferation control mechanism as a pathway for transmitting signals from growth factor receptor Yuichi through Ras.
  • LKB1 kinase is inactivated by gene mutation, and the suppression of MEK / ERK signal does not occur, resulting in excessive proliferation of cells, which is expected to appear as a trait such as polyposis or carcinogenesis. Therefore, inhibition of LKB1 kinase activity or reduction of LKB1 protein can activate MEK / ERK signaling, and conversely, activate LKB1 kinase or increase LKB1 protein Thus, it is thought that this signaling can be suppressed.
  • Drugs targeting the LKB1 gene and its product kinase are expected to be effective against various diseases caused by abnormal MEK / ERK signaling.
  • the present invention has been completed based on the above findings, and provides a compound that controls the MEK / ERK signal transduction mechanism, a method for screening the same, and a drug containing the compound that controls the signal transduction mechanism as an active ingredient. I do.
  • the present invention provides
  • a method for screening a compound that inhibits MEK / ERK signaling comprising:
  • step (c) selecting a compound that increases the kinase activity measured in step (b) as compared to the case where the test sample is not contacted;
  • step (c) selecting a compound that increases the expression level of the LKB1 gene measured in step (b) as compared to the case where the test sample is not contacted,
  • step (c) selecting a compound that reduces the kinase activity measured in step (b) as compared to the case where the test sample is not contacted,
  • step (c) selecting a compound that reduces the expression level of the LKB1 gene measured in step (b) as compared to the case where the test sample is not contacted,
  • step (c) selecting a compound that reduces the reporter activity measured in step (b) as compared to when the test sample is not contacted, (6) The method according to (5), wherein the protein constituting the Ras / MEK / ERK signaling pathway is selected from the group consisting of Ras, c-Raf. MEK1, and MEK2.
  • an agent that suppresses MEK / ERK signal transmission comprising a compound that promotes the kinase activity of LKB1 as an active ingredient
  • an agent that suppresses MEK / ERK signal transmission comprising a compound that promotes expression of the LKB1 gene as an active ingredient
  • an agent which suppresses MEK / ERK signal transduction comprising LKB1 as an active ingredient
  • a drug that promotes MEK / ERK signal transmission comprising a compound that inhibits the kinase activity of LKB1 as an active ingredient
  • an agent that promotes MEK / ERK signal transmission comprising a compound that suppresses LKB1 gene expression as an active ingredient
  • (21) a therapeutic agent for Peutz-Jeggars syndrome, comprising a c-fos promoter or a compound that inhibits the activity of SRE as an active ingredient;
  • MEK / ERK signal transduction means that c-Raf kinase is activated by Ras, one of low-molecular-weight GTP-binding proteins, followed by activation of MEK1,2 kinase, ERK1 and 2 kinases are activated, and finally transcription factors such as Elkl are phosphorylated and activated, and the target gene expression is induced by MEK (Ras / MEK / ERK signal transduction pathway). Means downstream signaling.
  • the present invention provides a method for screening a compound that regulates (promotes or suppresses) MEK / ERK signaling.
  • the LKB1 protein suppresses MEK / ERK signal transmission. It was suggested that this suppression was based on the kinase activity of the LKB1 protein.
  • One embodiment of this screening method relates to a method using the kinase activity of LKB1 protein as an index.
  • a test sample is brought into contact with the LKB1 protein (step (a)), and the kinase activity of the LKB1 protein is measured (step (a)).
  • b)) by selecting a compound that increases or decreases the kinase activity measured in step (b) compared to the case where the test sample is not contacted (step (c)). .
  • the test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, a fermented microbial product, a marine organism extract, a plant extract, a purified or crude protein, a peptide, and a non-peptide compound. , Synthetic low molecular weight compounds and natural compounds.
  • the protein of the present invention to which the material is brought into contact may be, for example, a purified protein or a form bound to a carrier.
  • the test sample may be brought into contact with cells expressing the LKB1 protein.
  • the kinase activity of LKB1 protein is determined by, for example, autophosphorylation by LKB1 protein.
  • the activity can be measured with a liquid scintillation counter or the like as the amount of 32 P transferred from [ ⁇ - 32 P] ATP to LKB1 protein.
  • plasmid DNA for LKB1 expression Approximately 10 ig of plasmid DNA for LKB1 expression (pcDNA3 / LKBlmyc), etc. was obtained by the method using SuperFect (Qiagen) by using C0S7 cells, HeLa cells (cells derived from cervical cancer), cells derived from HeLa cells ( HeLa S3, G361 cells (melanomas), human THP1, K562, SW480, HEK293, mouse NIH3 T3, HL60 (human leukemia-derived cells), normal fibroblasts, normal epithelial cells, normal vascular smooth muscle cells, Transfection into cells such as normal osteoblasts.
  • SuperFect Qiagen
  • NP40 kinase lysis buffer (aOmM Tris-HCl pH 7.8, 1% NP40, 0.15 M sodium chloride, lmM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrrolate, 10 g / ml aprotinin, lmM PMSF). Solubilize the evening protein by mixing at 4 ° C for 30 minutes. Protein A / G plus agarose (Santa Cruz) is added to the cell lysate thus obtained and mixed for 30 minutes to remove non-specifically adsorbed beads.
  • NP40 kinase lysis buffer aOmM Tris-HCl pH 7.8, 1% NP40, 0.15 M sodium chloride, lmM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrrolate, 10 g / ml aprotinin, lmM PMSF.
  • the test sample used is MEK / If the kinase activity is reduced, the test sample used will be a candidate for a compound that promotes MEK / ERK signaling, if the kinase activity is reduced.
  • Another embodiment of the method for screening a compound that regulates MEK / ERK signal transduction relates to a method using LKB1 gene expression as an index, comprising contacting a test sample with a cell that expresses LKB1 gene (step ( a))), measuring the expression level of the LKB1 gene in the cells (step (b)), and increasing or decreasing the expression level of the LKB1 gene measured in the step (b) as compared with the case where the test sample is not contacted. It can be carried out by selecting a compound to be reduced (step (c)).
  • the test sample is not particularly limited as in the above-mentioned screening.
  • Cells expressing the LKB1 gene include, for example, C0S7 cells, HeLa cells (cells derived from uterine facial cancer), HeLa cell-derived cells (HeLa S3, etc.), G361 cells (melanoma), human THP1, K562, SW480 Cells such as HEK293, mouse NIH3T3, HL60 (human leukemia-derived cells), normal fibroblasts, normal epithelial cells, normal vascular smooth muscle cells, and normal osteoblasts can be suitably used.
  • Detection of the expression level of the LKB1 gene can be performed using a method known to those skilled in the art, such as a Northern blotting method or an RT-PCR method.
  • the test sample used is MEK / If the expression level of the LKB1 gene is reduced, the test sample used is a candidate for a compound that promotes MEK / ERK signal transmission.
  • the present invention also provides a method for screening a candidate compound for a therapeutic agent for a therapeutic agent for Peutz-Jeghers syndrome.
  • LKB1 which is the causative gene of Peutz-Jeghers syndrome, suppresses MEK / ERK signaling.
  • candidate therapeutic compounds for Peutz-Jeghers syndrome can be screened using inhibition of MEK / ERK signaling as an index.
  • the method for screening a candidate compound for a therapeutic agent for Boyje-Garz syndrome according to the present invention is based on such findings.
  • a vector containing a structure in which a repo overnight gene is functionally linked downstream of the c-fos promoter or SRE, and a vector expressing a protein that constitutes miEK / ERK signaling were introduced.
  • Cells are provided (step (a)), a test sample is brought into contact with the cells, the reporter activity is measured (step (b)), and compared with the case where the test sample is not contacted, It can be carried out by selecting a compound that reduces the measured reporter activity (step (c)).
  • the promoter region of the c-fos gene can be obtained by screening a genomic DNA library by a conventional method using a c-fos cDNA or a synthetic oligo DNA containing the sequence of the c-fos gene as a probe. Alternatively, it can be obtained by PCR using genomic DNA as type III.
  • the C-fos promoter repo overnight gene can be prepared by incorporating an appropriate region of the DNA fragment thus obtained into an upstream regulatory region of an appropriate reporter gene.
  • pTAL_Luc CL0NTECH
  • pTA-Luc CL0NTECH
  • TAL-SEAP CL0NTECH
  • the SRE repo overnight gene is, for example, "AGGATGTCCATATT AGGACATCTZ SEQ ID NO: 7 "can be prepared by incorporating a sequence repeated several times into the upstream regulatory region of the appropriate repo overnight gene as described above.
  • the SRE reporter gene (pSRE-Luc) is commercially available (Stratagene) and can be used.
  • a vector that expresses a protein constituting the Ras / MEK / ERK signal transduction pathway can be prepared as follows. First, a cDNA having all open reading frames of Ras and MEK1, which are proteins constituting the Ras / MEK / ERK signal transduction pathway, is cloned by a conventional cDNA library-screening RT-PCR method. This is inserted into an appropriate animal cell expression vector, for example, pcDNA3 vector (Invitrogen) and used as an expression vector. At this time, an appropriate mutation can be added so that the protein is constantly activated. For example, in the case of Ras, replace the 12th codon with Val etc.
  • Mutagenesis can be introduced, for example, by a general site-specific mutagenesis method using GeneEditor TM (Promega) or the like.
  • proteins constituting the Ras / MEK / ERK signal transduction pathway to be expressed in a vector include Ras, c-Raf, MEK1, and MEK2.
  • a commercially available vector (Stratagene) can be used as a vector for expressing these proteins.
  • Examples of cells into which the vector is introduced include C0S7 cells, HeLa cells (cells derived from uterine face cancer), HeLa cell-derived cells (HeLa S3, etc.), G361 cells (melanomas), human TH Pl, K562, SW480 Cells such as HEK293, mouse NIH3T3, HL60 (human leukemia-derived cells), normal fibroblasts, normal epithelial cells, normal vascular smooth muscle cells, and normal osteoblasts can be used.
  • C0S7 cells HeLa cells (cells derived from uterine face cancer), HeLa cell-derived cells (HeLa S3, etc.), G361 cells (melanomas), human TH Pl, K562, SW480 Cells such as HEK293, mouse NIH3T3, HL60 (human leukemia-derived cells), normal fibroblasts, normal epithelial cells, normal vascular smooth muscle cells, and normal osteoblasts can be used.
  • Reporter genes that can be used for screening include, for example, SRE reporter gene (pSRE-Luc, Stratagene) and the like.
  • Method for introducing vectors into cells Examples are methods using phospholipids such as SuperFect (Qiagen) and Lipofect Amine (GIBCO BRL) (specifically, the method described in Example 2), calcium phosphate method, electoral poration method, DEAE- It can be performed by a method known to those skilled in the art, such as the dextran method.
  • test sample to be brought into contact with the cells includes, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide Sex compounds, synthetic low molecular compounds, natural compounds and the like can be used, but there is no particular limitation.
  • the reporter activity of the cells brought into contact with the test sample is measured by using a suitable substrate that emits chemiluminescence when decomposed by luciferase when luciferase is used as the reporter, and measuring the luminescence with a luminometer. Activity can be assessed.
  • a suitable substrate that emits chemiluminescence when decomposed by luciferase when luciferase is used as the reporter
  • Activity can be assessed.
  • firefly luciferase is used as a reporter
  • firefly luciferin can be used as a substrate.
  • coelenterazine can be used as a substrate.
  • kits and reagents such as Dua Luc if erase TM Reporter Assay System (Promega) can be used.
  • i3 galactosidase or alkaline phosphatase When using i3 galactosidase or alkaline phosphatase as a reporter, use an appropriate substrate that develops a color when decomposed by these enzymes, or an appropriate substrate that emits chemiluminescence, and spectrophotometrically measures the color or emission.
  • the activity can be evaluated by measuring with a luminometer.
  • chloramphenicol acetyltransferase (CAT) is used as the repo overnight
  • chloramphenicol acetyl produced by using chloramphenicol and acetyl CoA which are labeled with 14 C, as substrates, is used.
  • the activity can be evaluated by separately measuring the amount of chloramphenicol by thin layer mouth chromatography or the like.
  • the present invention also provides an agent that regulates (promotes or suppresses) MEK / ERK signaling.
  • LKB1 protein suppresses MEK / ERK signaling. It was suggested that this suppression was based on the kinase activity of LKB1 protein. This fact means that a compound that regulates the kinase activity of LKB1 protein or the expression of LKB1 protein can be a drug that regulates MEK / ERK signaling.
  • the agent for regulating MEK / ERK signaling of the present invention is based on such findings.
  • the agents of the present invention include both reagents used for test and research purposes and medicaments used for the treatment and prevention of diseases.
  • As the active ingredient of the drug for suppressing MEK / ERK signaling of the present invention a compound that promotes the kinase activity of LKB1 protein and a compound that promotes the expression of LKB1 gene can be used. These compounds can be isolated by the above-mentioned screening. It is also possible to use the LKB1 protein or a DNA encoding the protein.
  • a compound that promotes the kinase activity of LKB1 protein and a compound that suppresses the expression of LKB1 gene can be used as an active ingredient of the drug.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • human antibodies or humanized antibodies obtained by genetic recombination are preferred. These antibodies can be prepared by methods known to those skilled in the art.
  • the present invention also provides a therapeutic agent for Peutz-Jeghers syndrome.
  • LKB1 which is a causative gene of Peutz-Jeghers syndrome, suppresses MEK / ERK signal transduction. This fact implies that compounds that inhibit MEK / ERK signaling could be therapeutics for Peutz-Jeghers syndrome.
  • the screening method for a therapeutic agent for Boyle-Jaez syndrome of the present invention is based on such findings.
  • the active ingredient of the therapeutic agent for Peutz-Jeghers syndrome of the present invention includes MEK / ERK sig.
  • Compounds that suppress null transmission can be used.
  • the compound may be a compound that suppresses the activity of the c-fos promoter or SRE. Since SRE is an element specific to MEK / ERK signal transmission involving LKB1, a compound that inhibits the activity of SRE is suitable as an active ingredient of the therapeutic agent for Peutz-Jeggars syndrome of the present invention. is there. These compounds can be isolated by the above-described screening of the present invention.
  • the agent of the present invention is effective when used as a medicament for human animals such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bush dogs, sea lions, monkeys, baboons, and chimpanzees.
  • human animals such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bush dogs, sea lions, monkeys, baboons, and chimpanzees.
  • a known pharmaceutical method for example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as necessary, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids Can be used parenterally in the form of injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives It is possible to formulate a drug product by combining it with a drug, a binder and the like as appropriate and mixing it in a unit dosage form required for generally accepted pharmaceutical practice.
  • the amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid.
  • Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous injection solutions include, for example, saline, dextrose and other adjuvants Isotonic solution, eg!)-Sorbitol, D-mannose, D_manni! And sodium chloride, and suitable solubilizers, for example, alcohols, specifically, ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 (TM), HCO May be used with -50.
  • solubilizers for example, alcohols, specifically, ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 (TM), HCO May be used with -50.
  • the oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • the prepared injection solution is usually filled in an appropriate ampoule.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by methods known to those skilled in the art. Yes.
  • the dose varies depending on the weight and age of the patient, the method of administration, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art.
  • the dose of the drug of the present invention varies depending on the symptoms, but in the case of oral administration, generally, for an adult (assuming a body weight of 60 kg), it is about 0.1 to 100 mg, preferably about 1.0 to 100 mg per day. It is believed to be 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, target organ, symptoms, and administration method.
  • injection usually in adults (with a body weight of 60 kg)
  • the amount can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.
  • FIG. 1 shows the results of detecting the effect of LKB1 on activation of c-fos promoter by activated K-Ras (K-RasV12).
  • LKBl WT represents the wild type LKB1
  • LKBl K78I represents the K78I mutant. + Indicates that each protein was expressed, and-indicates that it was not expressed. The triangles indicate that the amount of transfected plasmid DNA increases as going to the right.
  • FIG. 2 is a schematic diagram of various intracellular signal transmissions involved in activation of various transcription regulatory factor binding elements on the c-fos promoter-reporter gene.
  • FIG. 3 shows the results of detecting the effect of LKB1 on SRE activation by MEK1.
  • FIG. 4 is a diagram showing the results of detecting the effect of LKB1 on activation of the c_fos promoter by MEKK1.
  • FIG. 5 is a view showing the results of detecting the effect of LKB1 on the activation of NFKB by MEKK1.
  • FIG. 6 is a graph showing the results of detecting the effect of LKB1 on activation of the C-fos promoter by PKA.
  • FIG. 7 is a diagram and a photograph showing the results of detecting the effects of various LKB1 mutants on SRE promoter activation by MEK1. The bottom shows the kinase activity of each mutant.
  • pcDNA3 / LKBlmyc was used as type III, and various mutants were produced by in vitro mutagenesis using GeneEditor TM (Promega). The following primers for mutagenesis were used.
  • K78I mutant 5 'agg agg gcc gtc ate ate e tc aag aag 3' / SEQ ID NO: 3
  • D176N mutant 5 'at t gig cac aag aac ate aag ccg ggg 3' Z distribution 'J number: 4 W308C Mutant: 5 'egg cag cac age tgc t tc egg aag aa 3' Z distribution system No .: 5 L67P mutant: 5 'gtg aag gag gtg ccg gac tcg gag acg 3' distribution!
  • Plasmid DNAs for expression of MEK1 and MEKKU PKA were purchased from Stratagene.
  • Activated K-Ras expression plasmid DNA (pCEV / k-RasV12) was kindly provided by Dr. Fukumo to Yauso of Kobe University School of Medicine. ⁇ Michitake luciferase expression plasmid (PRL / SV40) was purchased from Promega.
  • c_fosLV The c-fos promoter reporter gene (c_fosLV) was kindly provided by Dr. Fukumoto Yauso of Kobe University School of Medicine.
  • the SRE repo overnight gene (pSRE_Luc) was purchased from Stratagene.
  • the NFKB reporter gene (PNFKB_LUC) was purchased from Ciontech.
  • Transfection was performed using SuperFect (Qiagen) according to the method recommended by the manufacturer. That is, Hela cells were sown on a 12-well plate at 10 soils, cultured at room temperature, a mixture of DNA 1.5 and SuperFect 41 was added, and cultured for 3 hours. Thereafter, the medium was replaced, and after culturing for further 24 hours, luciferase activity was measured.
  • SuperFect Qiagen
  • Dual-Lucif eRase TM Reporter Assay System Promega
  • a dual assay method using Mycobacterium luciferase as an endogenous control was performed using the Dual-Lucif eRase TM Reporter Assay System (Promega). Measured. That is, the transfected cells were washed with PBS and then lysed with lXPassive Lysis Buffer of IOOI. Using 51 of these, LucifeRase Assay Reagent II (50 ⁇ 1) was used as a substrate for firefly luciferase, and Stop & Glo ( R ) Reagent (501) was used as a substrate for M. luciferase. Was measured.
  • the cfos promoter has SRE (serum responsive element), CRE (cAMP responsive element), and TRE (TPA responsive element) as known transcription regulatory element binding elements (Fig. 2). ),
  • SRE serum responsive element
  • CRE cAMP responsive element
  • TRE TAA responsive element
  • the activation of this promoter by Ras is known to occur mainly by the activation of SRE by the MEK / ERK-mediated pathway (Fukumoto Y et al., J Biol Chem. 1990 Jan 15; 265 (2): 774-80), LKBl was thought to act on any step in the MEK / ERK signaling pathway to suppress SRE activation.
  • LKBl was thought to act on any step in the MEK / ERK signaling pathway to suppress SRE activation.
  • LKB1 acts on pathways other than the MEK / ERK signaling pathway.
  • C-fosLV cfs promoter-reporter gene
  • pNF / cB-Luc NFKB reporter gene
  • the present invention provides a method for screening a compound that regulates MEK / ERK signaling targeting LKB1.
  • a method for screening PJS therapeutic zen compounds targeting MEK / ERK signaling was provided. This has enabled efficient screening of compounds that regulate MEK / ERK signaling and candidate compounds for PJS therapeutics.
  • the present invention provides pharmaceutical uses of these compounds.
  • the drug is expected to be used as a reagent for regulating MEK / ERK signal transduction or as a medicament for treating or preventing diseases including PJS.

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Abstract

Selon la présente invention, LKB1 inhibe spécifiquement la voie de transduction de signaux Ras/MEK (protéine kinase activée par mitogène)/ERK (kinase régulée par signaux extracellulaires). On suggère que cette inhibition est occasionnée par l'action de LKB1 sur la partie en aval de MEK1 dans la voie de transduction de signaux susmentionnée, et qu'elle dépend donc de l'activité de la kinase de LKB1. A partir de ces faits, on peut développer des médicaments qui régulent le signal MEK/ERK en utilisant LKB1 comme cible. On peut utiliser ces médicaments dans le traitement de diverses maladies, notamment PJS dans laquelle le signal ERK/MEK rentre en jeu.
PCT/JP2001/006171 2000-07-19 2001-07-17 Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose WO2002006520A1 (fr)

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AU2001271067A AU2001271067A1 (en) 2000-07-19 2001-07-17 Method of screening compound controlling mek/erk signal transduction and medicinal use of the compound

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EP2298768A1 (fr) 2004-06-11 2011-03-23 Japan Tobacco, Inc. Dérivés 5-amino-2,4,7-trioxo-3,4,7,8-tetrahydro-2H-pyrido[2,3-d]pyrimidine et composés similaires pour le traitement de cancer

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WO2005010174A3 (fr) * 2003-07-17 2005-06-30 Univ Dundee Procedes
JP2007530000A (ja) * 2003-07-17 2007-11-01 ユニヴァーシティー オブ ダンディー Ｌｋｂ１／ｓｔｒａｄ７ｍ０２５複合体の使用方法
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EP2298768A1 (fr) 2004-06-11 2011-03-23 Japan Tobacco, Inc. Dérivés 5-amino-2,4,7-trioxo-3,4,7,8-tetrahydro-2H-pyrido[2,3-d]pyrimidine et composés similaires pour le traitement de cancer
US8835443B2 (en) 2004-06-11 2014-09-16 Japan Tobacco Inc. Pyrimidine compound and medical use thereof

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