WO2002006520A1

WO2002006520A1 - Method of screening compound controlling mek/erk signal transduction and medicinal use of the compound

Info

Publication number: WO2002006520A1
Application number: PCT/JP2001/006171
Authority: WO
Inventors: Jun-Ichi Nezu; Takakazu Mizuno; Asuka Ose
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2000-07-19
Filing date: 2001-07-17
Publication date: 2002-01-24
Also published as: AU2001271067A1

Abstract

It is found out that LKB1 specifically inhibits the Ras/MEK/ERK signal transduction pathway. It is suggested that this inhibition is caused by the action of LKB1 on the dowstream of MEK1 in the above signal transduction pathway and, therefore, depends on the kinase activity of LKB1. Based on these facts, it is possible to develop drugs control the MEK/ERK signal by using LKB1 as a target. These drugs are usable in treating various diseases including PJS in which the MEK/ERK signal participates.

Description

TECHNICAL FIELD 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. Background art

Peutz-Jeghers Syndrome (MIM 175200, PJS) is a genetic disease in humans whose main symptoms are polyposis in the gastrointestinal tract and pigmentation in mucous membranes and skin. PJS is inherited in an autosomal dominant manner, but in 1997 He et al. Inki et al. Showed that the causal gene was mapped to chromosome 19 pl3.3 by linkage analysis in families with PIS. (He J. inki A et al., Nat Genet 1997 Jan; 15 (1): 87-90). In this region, there is a novel serine-nothreonine kinase LKB1 (GenBank accession number U63333) which the present inventors have found in a novel protein kinase search project. 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). In addition, similar reports have been made by other groups one after another, and to date, at least 60 or more LKB1 mutations have been found in PJS patients (He marauder inki A et al., Nature 1998 Jan 8; 391 (6663): 184-7, Nakagawa H et al., Hum Genet 1998 Aug; 103 (2): 168-72, Resta N et al., Cancer Res 1998 Nov 1; 58 (21): 4799-801, Ylikorka la A et al., Hum Mol Genet 1999 Jan; 8 (l): 45-51). In addition, the present inventors, LKB (1) The gene product was a kinase with autophosphorylation ability, and it was demonstrated that the missense mutation found in PJS patients was a mutation that lost kinase activity (Mehenni H et al., Am J Hum Genet 1998 Dec. ; 63 (6): 1641-50). These facts have confirmed that loss of function due to LKB1 serine / threonine kinase gene mutation is the cause of HS development.

It is also known from epidemiological studies that the risk of developing various cancers in PJS patients is significantly higher than that in healthy subjects, indicating that the causative gene has tumor suppressor-like activity. Was speculated. In fact, it has been reported that sporadic cancers unrelated to PJS have mutations in the LKB1 gene, and it is clear that inactivation of the LKB1 gene is also involved in general sporadic cancers. (Dong SM et al., Cancer Res 1998 Sep 1; 58 (17): 3787-90, Rowan A et al., J Inves t Dermatol 1999 Apr; 112 (4): 509-11, Guldberg P et al., Oncogene 1999 Mar 4; 18 (9): 1777-80, Su GH et al., Am J Pathol. 1999 Jun; 154 (6): 1835-40, Wang ZJ et al., J Pathol. 1999 May; 188 (l): 9-13).

By the way, the fact that a single LKB1 gene abnormality causes phenotypic phenotypes seen in PJS patients such as polyposis and carcinogenesis suggests that 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

The present invention has been made in view of such a situation. 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). That is, as shown in Figure 2, this pathway activates c-Raf kinase by Ras, which in turn activates MEK1,2 kinase, then ERK1,2 kinase, and ultimately transcription factors. Is 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. Are known. 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.

On the other hand, activation of C-fos promoter by MEKK1 (Fig. 4), activation of NFκ (Nuclear Factor of κΒcels) binding element by MEKK1 (Fig. 5), and activation of PKA (cAMP-dependent kinase) Activation of C-fos promoter via CRE (Fig. 6) It turned out that LKB1 had no effect. This suggested that the action of LKB1 was specific to the Ras / MEK / ERK signaling pathway.

Next, the effects of mutants (L67P, D176N, W308C) found in PJS patients were examined (Fig. 7). As a result, it was clarified that the mutants in which these kinase activities were attenuated also had significantly reduced inhibitory effects.

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. In PJS patients, 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.

More specifically, the present invention provides

(1) A method for screening a compound that inhibits MEK / ERK signaling, comprising:

(a) contacting the test sample with the LKB1 protein,

(b) measuring the kinase activity of LKB1 protein; and

(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; (2) A method for screening a compound that inhibits MEK / ERK signaling, comprising:

(a) contacting a test sample with cells expressing the LKB1 gene,

(b) measuring the expression level of the LKB1 gene in the cells, and

(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,

(3) a method of screening for a compound that promotes MEK / ERK signaling,

(a) contacting the test sample with the LKB1 protein,

(b) measuring the kinase activity of the LKB1 protein, and

(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,

(4) a method of screening for a compound that promotes MEK / ERK signaling,

(a) contacting a test sample with cells expressing the LKB1 gene,

(b) measuring the expression level of the LKB1 gene in the cells, and

(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,

(5) A method for screening candidate compounds for the treatment of Peutz-Jet-Girz syndrome,

(a) Provide a vector containing a structure in which a reporter gene is functionally linked downstream of the c_fos promoter or SRE, and a cell into which a vector expressing a protein constituting the Ras / MEK / ERK signaling pathway has been introduced. Process,

(b) contacting a test sample with the cells, and measuring reporter activity; and

(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.

(7) a compound that inhibits MEK / ERK signal transmission, which can be isolated by the method according to (1) or (2),

(8) a compound that promotes MEK / ERK signal transmission, which can be isolated by the method according to (3) or (4),

(9) A candidate drug for a therapeutic agent for Peutz and Jögers syndrome, which can be isolated by the method according to (5) or (6).

(10) an agent that suppresses MEK / ERK signal transmission, comprising a compound that promotes the kinase activity of LKB1 as an active ingredient;

(11) The drug according to (10), wherein the compound is a compound that can be isolated by the method according to (1).

(12) an agent that suppresses MEK / ERK signal transmission, comprising a compound that promotes expression of the LKB1 gene as an active ingredient;

(13) The agent according to (12), wherein the compound is a compound that can be isolated by the method according to (2).

(14) an agent which suppresses MEK / ERK signal transduction, comprising LKB1 as an active ingredient,

(15) a drug that promotes MEK / ERK signal transmission, comprising a compound that inhibits the kinase activity of LKB1 as an active ingredient;

(16) the agent according to (15), wherein the compound is an antibody that binds to LKB1 protein;

(17) The agent according to (15), wherein the compound is a compound that can be isolated by the method according to (3).

(18) an agent that promotes MEK / ERK signal transmission, comprising a compound that suppresses LKB1 gene expression as an active ingredient;

(19) The agent according to (18), wherein the compound is a compound that can be isolated by the method according to (4). (20) An agent for treating Peutz-Egers syndrome, comprising a compound that inhibits MEK / ERK signal transduction 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; and

(22) The therapeutic agent according to (20) or (21), wherein the compound is a compound that can be isolated by the method according to (5) or (6).

In the present invention, “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. In this example, it was shown that the LKB1 protein suppresses MEK / ERK signal transmission. It was suggested that this suppression was based on the kinase activity of the LKB1 protein. These facts indicate that MEK / ERK signaling can be regulated by regulating the kinase activity of LKB1 protein or the expression of LKB1 protein. The method for screening a compound that regulates MEK / ERK signaling of the present invention is based on such findings.

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. Test 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.

(Autophosphorylation) activity. The activity can be measured with a liquid scintillation counter or the like as the amount of ³² P transferred from [α- ³² P] ATP to LKB1 protein.

An example of a specific method for detecting kinase activity is described below. 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. That is, about 10 ⁶ cells are seeded on a 10-cm dish, cultured for 1 hour, and then a mixture of 10 g of plasmid DNA and 601 SuperFect is added, followed by culturing for about 3 hours. After that, the culture solution is replaced with a new one, and the cells are further cultured for 2 days, and then the cells are removed with a trypsin-EDTA solution and collected. The cells were suspended in 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. Next, add anti-c_Myc antibody A (Santa Cruz), leave at 4 for 1 hour, add protein A / G plus agarose, and leave for another 1 hour. The immunocomplex is precipitated by centrifugation, washed several times with NP40 Quinase lysis buffer, a buffer containing 1 M sodium chloride, and 50 mM Tris-HCl (pH 7.8), and Serve to See. Kina one Zeatsusi is 50mM Tris-HCl (pH 7. 8), lmM DTT , ( such as M n) 10 mM divalent cation and 10 C i - carried out in [§ ^3Z P] total amount including ATP 5.0 1 of the reaction system. After incubation of the immunoprecipitate in this kinase assay solution at 37 ° C for 30 minutes, the reaction is stopped by adding SDS-PAGE sample buffer and boiling, and SDS-PAGE is performed. Fix the gel with a solution of methanol / acetic acid, dry it, and analyze the image with a BAS200 Image Analyzer-1 (Fuji Film). Kinase activity can also be measured by the method described in the literature (Am J Hum Genet (1988) Dec; 63 (6): 1641-50, Hum Mo 1 Genet (1999) Jan; 8 (l): 45-51). It can also be detected.

As a result of this measurement, if the kinase activity of the LKB1 protein measured when the test sample is brought into contact with the test sample is higher than when the test sample is not brought into contact, 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 according to the present invention 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. As a result of this measurement, if the expression level of the LKB1 gene measured when the test sample is brought into contact with the test sample is higher than when the test sample is not brought into contact, 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 Peutz-Jeghers syndrome. In this example, it was shown that LKB1, which is the causative gene of Peutz-Jeghers syndrome, suppresses MEK / ERK signaling. These facts indicate that 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.

In this method, 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. As the plasmid DNA containing the reporter gene, pTAL_Luc (CL0NTECH), pTA-Luc (CL0NTECH), TAL-SEAP (CL0NTECH) and the like can be used. 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. In the case of MEK1, it is known that the amino acid at positions 218 and 222, which is known to be phosphorylated by the upstream kinase, becomes an activated protein by substituting it with Glu or the like. . Mutagenesis can be introduced, for example, by a general site-specific mutagenesis method using GeneEditor ™ (Promega) or the like.

Examples of 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.

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.

The test sample to be brought into contact with the cells thus prepared 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. When firefly luciferase is used as a reporter, for example, firefly luciferin can be used as a substrate. In addition, when the Mycobacterium luciferase is used as a repo overnight, for example, coelenterazine can be used as a substrate. Commercially available kits and reagents such as Dua Luc if erase ™ Reporter Assay System (Promega) can be used. 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. When 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.

As a result of this measurement, if the reporter activity measured when the test sample is brought into contact is lower than when the test sample is not brought into contact, the compound contained in the test sample used Becomes a candidate for treatment of Peutz-Jeghers syndrome. The present invention also provides an agent that regulates (promotes or suppresses) MEK / ERK signaling. In this example, it was shown that 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.

On the other hand, as an active ingredient of the drug for promoting MEK / ERK signaling of the present invention, a compound that promotes the kinase activity of LKB1 protein and a compound that suppresses the expression of LKB1 gene can be used. These compounds can be isolated by the screening described above. Further, an antibody that binds to LKB1 protein can be used as an active ingredient of the drug. The antibody may be a polyclonal antibody or a monoclonal antibody. When administered to humans, 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. In this example, it was shown that 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. In addition to directly administering the compound itself as a component to a patient, it is also possible to formulate and administer the compound by 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. For example, 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. When 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.

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. 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. If 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.

In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, and administration method. For example, in the case of injection, usually in adults (with a body weight of 60 kg), It may be convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of detecting the effect of LKB1 on activation of c-fos promoter by activated K-Ras (K-RasV12). The relative activity was expressed as the ratio of the activity of firefly luciferase without K-Ras to the activity of Mycobacterium luciferase (mean soil standard deviation (n = 3) (hereinafter referred to as title). The law is the same))). LKBl WT represents the wild type LKB1 and 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. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of plasmid DNA (1) Expression plasmid DNA

LK El Fraimaichi (5 gat gaa tic ggg tec age atg gag gtg gtg gac 3 '/ configuration U number: 1) and E2 primer (5' gat gaa t tc t ta gag gtc t tc t tc tga gat g ag By performing PCR using ct t ctg e tc c tg c tg ctt gca ggc cga 3 ′ / SEQ ID NO: 2), a c-Myc epitope sequence (Glu Gin Lys Leu Lie Ser Glu Glu A sp Leu) was added to a DNA fragment encoding the entire human LKB1 amino acid sequence. This was cut with the EcoRI site added to the primer, and then incorporated into the EcoRI site of the PCDNA3 vector (Invitrogen), an expression vector for mammalian cells. A clone (pcDNA3 / LKBlmyc), which was confirmed to have no PCR error by sequencing, was selected and used for expression experiments.

In addition, pcDNA3 / LKBlmyc was used as type III, and various mutants were produced by in vitro mutagenesis using GeneEditor ™ (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 aaa 3' Z distribution system No .: 5 L67P mutant: 5 'gtg aag gag gtg ccg gac tcg gag acg 3' distribution! J number: 6 Mutagenesis The primers for selection and the primer for selection (for the bottom strand) attached to the kit were both annealed with the single-stranded plasmid DNA to synthesize a new DNA strand. This was introduced into Escherichia coli, and a clone carrying the plasmid having the mutation was selected as a GeneEditor ™ antibiotic-resistant clone. It was confirmed by sequencing that the mutation was present and used for expression experiments.

Plasmid DNAs for expression of MEK1 and MEKKU PKA (pFC-MEK1, pFC-MEKKK pFC-PKA, respectively) 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. ゥ Michitake luciferase expression plasmid (PRL / SV40) was purchased from Promega.

(2) Repo overnight gene

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.

[Example 2] Reporter gene Atsusei

(1) Transcription

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.

(2) Measurement of luciferase activity

Using the Dual-Lucif eRase ™ Reporter Assay System (Promega), a dual assay method using Mycobacterium luciferase as an endogenous control was performed using the Dual-Lucif eRase ™ 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.

(3) Result

Using the cells into which the cfs promoter-reporter gene (C-fosLV) was introduced, the effect of LKB1 on the activation of the c-fos promoter by Ras was examined. As a result, L KB1 markedly suppressed the activation of c-fos promoter by Ras (Fig. 1). On the other hand, when K78I, an LKB1 mutant that has lost autophosphorylation ability, was similarly examined, the activation of c-fos promoter Yuichi by Ras was not significantly suppressed (Fig. 1). ).

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). ), 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. Next, we examined the steps involving LKB1.

Using the SRE repo overnight gene (pSRE_Luc) -transfected cells, the effect of LKB1 on the activation of SRE by MEK1 was examined. As a result, LKB1 markedly suppressed the activation of SRE by MEK1 (Fig. 3). On the other hand, when K78I was similarly examined, MEK1 did not significantly suppress SRE activation (Fig. 3). This fact suggests that LKB1 acts at least downstream of MEK1 in the MEK / ERK signaling pathway.

Next, we examined whether LKB1 acts on pathways other than the MEK / ERK signaling pathway. Using cells transfected with the cfs promoter-reporter gene (C-fosLV) or NFKB reporter gene (pNF / cB-Luc) cells to activate the c-fos promoter or NFKB by MEKK1 or PKA The effects of LKBl were studied. As a result, LKB1 activates c-fos promoter overnight by MEKK1 (Fig. 4), activation of NFKB (NU clear Factor of κ) cells) binding element by MEKK1 (Fig. 5), PKA (cAMP-dependent kinase). ) Has no effect on the activation of the C-fos promoter (Fig. 6). This suggests that the action of LKB1 is specific to the MEK / ERK signaling pathway at least in the range examined.

Next, a mutant found in PJS patients (L67P; Leu at position 67 replaced Pro) Mutant, D176N; a mutant in which Asp at position 176 was replaced with Asn, a mutant in which W308C; a mutant in which Τι: ρ at position 308 was replaced with Cys) were examined. Using the cells into which the SRE reporter gene was introduced, the effects of these LKB1 mutants on the activation of the SRE promoter by MEK1 were examined. As a result, it was revealed that these mutants in which kinase activity was attenuated also significantly attenuated the MEK / ERK inhibitory effect (FIG. 7). Industrial applicability

The present invention provides a method for screening a compound that regulates MEK / ERK signaling targeting LKB1. In addition, 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. In addition, 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.

Claims

The scope of the claims

1. A method of screening for a compound that inhibits MEK / ERK signaling,

(a) contacting the test sample with the LKB1 protein,

(b) measuring the kinase activity of the LKB1 protein, and

(c) selecting a compound that increases the kinase activity measured in step (b) as compared with the case where the test sample is not contacted.

2. A method of screening for a compound that inhibits MEK / ERK signaling,

(a) contacting a test sample with cells expressing the LKB1 gene,

(b) measuring the expression level of the LKB1 gene in the cells, and

(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.

3. A method for screening for a compound that promotes MEK / ERK signaling,

(a) contacting the test sample with the LKB1 protein,

(b) measuring the kinase activity of the LKB1 protein, and

(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.

4. A method of screening for a compound that promotes MEK / ERK signaling,

(a) contacting a test sample with cells expressing the LKB1 gene,

(b) measuring the expression level of the LKB1 gene in the cells, and

(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.

5. A method for screening candidate therapeutic compounds for Peutz's Jaegers syndrome,

(a) A vector containing a structure in which a repo allele gene is functionally linked downstream of the c-fos promoter or SRE, and a protein constituting the Ras / MEK / ERK signaling pathway Providing a cell into which the expressing vector has been introduced,

(c) selecting a compound that reduces the reporter activity measured in step (b) as compared with the case where the test sample is not contacted.

6. The method according to claim 5, wherein the protein constituting the Ras / MEK / ERK signaling pathway is selected from the group consisting of Ras, c_Raf, MEKK and miEK2.

7. A compound that suppresses MEK / ERK signaling, which can be isolated by the method according to claim 1 or 2.

8. A compound that promotes MEK / ERK signaling, which can be isolated by the method according to claim 3 or 4.

9. A therapeutic candidate compound for the Peutz-Jet-Gaz syndrome group, which can be isolated by the method according to claim 5 or 6.

10. An agent that suppresses MEK / ERK signal transmission, comprising a compound that promotes the kinase activity of LKB1 as an active ingredient.

1 1. The agent according to claim 10, wherein the compound is a compound that can be isolated by the method according to claim 1.

12. An agent that suppresses MEK / ERK signal transduction, comprising a compound that promotes LKB1 gene expression as an active ingredient.

13. The agent according to claim 12, wherein the compound is a compound that can be isolated by the method according to claim 2.

14. An agent containing LKB1 as an active ingredient, which suppresses MEK / ERK signaling.

15. An agent that promotes MEK / ERK signal transmission, comprising as an active ingredient a compound that inhibits the kinase activity of LKB1.

16. The agent according to claim 15, wherein the compound is an antibody that binds to LKB1 protein.

17. The agent according to claim 15, wherein the compound is a compound that can be isolated by the method according to claim 3.

18 8. An agent that promotes MEK / ERK signal transduction, comprising a compound that suppresses LKB1 gene expression as an active ingredient.

19. The agent according to claim 18, wherein the compound is a compound that can be isolated by the method according to claim 4.

20. A therapeutic agent for Peutz-Jie-Garz syndrome, comprising a compound that inhibits MEK / ERK signaling as an active ingredient.

21. A therapeutic agent for Peutz-Jeghers syndrome, comprising as an active ingredient a compound that suppresses the activity of the c-fos promoter or SRE.

22. The therapeutic agent according to claim 20 or 21, wherein the compound is a compound that can be isolated by the method according to claim 5 or 6.