JP2011215091A - Method for screening substance inhibiting c-met enzyme activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve a simple screening of a substance (for example, an anticancer drug) that can inhibit a c-MET enzyme activity.SOLUTION: The method for screening a substance which can inhibit a c-MET enzyme activity includes evaluating whether a target substance causes an increase in the amount of a c-MET extracellular domain fragment. For example, the method includes the steps of (a) causing the target substance to be in contact with a c-MET expression cell in a culture medium; and (b) measuring the amount of a c-MET extracellular domain fragment in the culture medium; and may further include the step of (c) selecting a target substance that causes an increase in the amount of the c-MET extracellular domain fragment. The method may further includes the steps of: (a') measuring the amount of the c-MET extracellular domain fragment in a biological sample collected from a mammal into which the target substance has been administrated; and (b') selecting a target substance that causes an increase in the amount of the c-MET extracellular domain fragment.

Description

  The present invention relates to a screening method for substances (eg, anticancer agents) that can inhibit c-MET enzyme activity.

  Development of molecular targeted drugs that act specifically on target molecules is important for treating patients suffering from cancer caused by abnormalities in the target molecules. Therefore, search for a target molecule and development of a molecular target drug for the target molecule are being actively conducted. As a target molecule of a molecular target drug desired to be developed, for example, c-MET (receptor tyrosine kinase) which is one of oncogenes can be mentioned. It has been shown that it is possible to inhibit the growth of a predetermined cancer by inhibiting the c-MET enzyme activity (Non-patent Document 1), and the development of c-MET inhibitors as anticancer agents has been promoted. Yes.

  As for the c-MET gene, ectodomain shedding, which is a phenomenon of releasing a c-MET extracellular domain fragment (secreted c-MET or soluble c-MET) has been reported (Patent Literature). 1). In addition, it has been reported that c-MET extracellular domain fragment can be used as a biomarker for cancer (Patent Document 2) and that ectodomain excretion can be associated with cancer malignancy (Non-Patent Document 2). .

US 2007037209 (A1) WO2007 / 056523

Cancer Res 2007; 67: 2081-2088. Clin Cancer Res 2006; 12: 4154-4162.

  An object of the present invention is to realize a simple screening for substances (eg, anticancer agents) capable of inhibiting c-MET enzyme activity.

  As a result of intensive studies, the present inventors have found that inhibition of c-MET enzyme activity can be evaluated using an increase in the amount of c-MET extracellular domain fragment as an index. Therefore, the present inventors have conceived that a substance capable of inhibiting c-MET enzyme activity can be easily screened by evaluating whether or not a test substance increases the amount of c-MET extracellular domain fragment. The present invention has been completed. The prior art described above does not describe or suggest a relationship between the amount of c-MET extracellular domain fragment and c-MET enzyme activity.

That is, the present invention is as follows.
[1] A screening method for a substance capable of inhibiting c-MET enzyme activity, comprising evaluating whether or not a test substance increases the amount of c-MET extracellular domain fragment.
[2] The screening method of [1] above, wherein the substance capable of inhibiting c-MET enzyme activity is an anticancer agent.
[3] The screening method of the above [1] comprising the following steps (a) to (c):
(A) contacting the test substance with c-MET-expressing cells in a culture medium;
(B) measuring the amount of c-MET extracellular domain fragment in the culture medium; and (c) selecting a test substance that increases the amount of c-MET extracellular domain fragment.
[4] The screening method according to [3] above, wherein the c-MET expressing cell is a cancer cell.
[5] The screening method of the above [1], which comprises the following steps (a ′) and (b ′):
(A ′) measuring the amount of c-MET extracellular domain fragment in a biological sample collected from a mammal administered with the test substance; and (b ′) increasing the amount of c-MET extracellular domain fragment; A step of selecting a test substance to be made.
[6] The screening method according to [5] above, wherein the mammal is a mammal suffering from cancer.

According to the present invention, a substance that can inhibit c-MET enzyme activity can be easily screened using the amount of c-MET extracellular domain fragment as an index. In the conventional method, when c-MET enzyme activity is evaluated, after preparing an extract of cells treated with a test substance, it is necessary to evaluate the activation of c-MET in the extract using phosphorylation or the like as an index was there. However, such a method takes time and effort. On the other hand, according to the present invention, c-MET enzyme activity can be evaluated by measuring the amount of c-MET extracellular domain fragment in the cell culture supernatant, which is convenient.
Moreover, according to the present invention, the inhibitory activity against c-MET can be evaluated using a biological sample (eg, blood, urine, saliva) with low invasiveness. This is because the inhibitory activity against c-MET, which is a protein present in cells, can be evaluated using the amount of c-MET extracellular domain fragment present outside cells as an index. Thus, the present invention enables the evaluation of inhibitory activity against c-MET in clinical trials, for example.
Furthermore, according to the present invention, there is a significant difference in the amount of c-MET extracellular domain fragment measured between the inhibitory and non-inhibitory conditions for c-MET enzyme activity, as well as Since the inhibition of the c-MET enzyme activity can be clearly grasped due to the difference, it is highly reliable in evaluating whether or not the test substance can inhibit the c-MET enzyme activity.
Furthermore, according to the present invention, a large amount of c-MET extracellular domain fragment is observed under the condition of inhibiting c-MET enzyme activity, and measurement of such a large amount of c-MET extracellular domain fragment is easy. Therefore, inhibition of c-MET enzyme activity can be easily evaluated.

FIG. 1 is a graph showing an increase in the amount of c-MET extracellular domain fragment (soluble c-MET) in the culture supernatant of cells treated with a c-MET inhibitor. Abbreviations: IP (immunoprecipitation); pY Blot (Western blotting with anti-phosphotyrosine antibody); EGFR Blot (Western blotting with anti-EGFR antibody); c-MET Blot (Western blotting with anti-c-METβ chain antibody (14G9)) Soluble c-MET (SDS-PAGE under non-reducing and reducing conditions followed by Western blotting with anti-c-MET alpha chain antibody (EP1454Y))

  The present invention provides a screening method for substances that can inhibit c-MET enzyme activity. The screening method of the present invention may comprise evaluating whether the test substance increases the amount of c-MET extracellular domain fragment.

  c-MET is a receptor tyrosine kinase of hepatocyte growth factor (HGF), and activates intracellular signal transduction by binding of ligand HGF. c-MET is composed of an α chain and a β chain generated by cleaving a precursor of c-MET (pro-c-MET), and forms a dimer by a disulfide bond. c-MET is a receptor that penetrates the cell membrane, and the entire α chain and a part of the β chain exist outside the cell. c-MET is known to phosphorylate its own tyrosine residues (eg, Y1234, Y1235, Y1349, Y1356) upon activation thereof (eg, Mark et al., The Journal of Biological Chemistry, 1992, Vol. 267, No. 36, pp. 26166-26171; History of Medicine, 2008, Vol. 224, No. 1, pp. 51-55). For human c-MET and its α and β chains, refer to GenBank Accession Number: NP — 000026.2.

  As used herein, the term “c-MET extracellular domain fragment” refers to a c-MET extracellular domain polypeptide or its degradation product that is excreted by ectodomain shedding. Say. c-MET extracellular domain fragment is synonymous with soluble c-MET or secreted c-MET.

  The test substance to be subjected to the screening method may be any known compound or novel compound, such as a nucleic acid (eg, nucleoside, oligonucleotide, polynucleotide), carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide). , Polysaccharides), lipids (eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides), organic low molecular weight compounds, using combinatorial chemistry technology Compound libraries prepared in this manner, random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. The test substance may also be an expression vector containing a polynucleotide encoding a predetermined gene.

  The screening method of the present invention can be performed in any form as long as it can be evaluated whether the test substance increases the amount of c-MET extracellular domain fragment. For example, the screening method of the present invention can be performed using c-MET-expressing cells or biological samples collected from mammals.

The screening method using c-MET expressing cells can include, for example, the following steps (a) to (c):
(A) contacting the test substance with c-MET-expressing cells in a culture medium;
(B) measuring the amount of c-MET extracellular domain fragment in the culture medium; and (c) selecting a test substance that increases the amount of c-MET extracellular domain fragment.

  In the step (a), the test substance is brought into contact with c-MET expressing cells. Contact of the test substance with c-MET-expressing cells can be performed in a culture medium.

  A c-MET-expressing cell refers to a cell that expresses c-MET and produces a c-MET extracellular domain fragment by ectodomain excretion. Examples of c-MET-expressing cells include primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, and cell lines available from cell banks. The c-MET expressing cell can also be a cell derived from a mammal. Examples of mammals include primates, pets, and working animals. Specific examples of mammals include humans, chimpanzees, dogs, cats, cows, sheep, horses, pigs, rabbits, mice, and rats, with humans being preferred.

  In one embodiment, the c-MET expressing cell is a cancer cell. The cancer cell may be accompanied by amplification of the c-MET gene. Therefore, the cancer cell may be a cell showing sensitivity to a c-MET inhibitor. Examples of such cancer cells include primary cultured cancer cells isolated from mammals suffering from cancer, and cancer cell lines. Cancer cells can be derived from the mammals described above.

  The cancer cell may also be a cell exhibiting resistance to an anticancer agent other than a c-MET inhibitor. Examples of such cancer cells include primary cultured cancer cells isolated from mammals treated with anticancer agents other than c-MET inhibitors, and cancer cells treated with anticancer agents other than c-MET inhibitors. Strains. Examples of anticancer agents other than c-MET inhibitors include EGFR inhibitors, ALK inhibitors, PDGFR inhibitors, and c-KIT inhibitors. EGFR inhibitors include, for example, gefitinib, erlotinib, cetuximab, lapatinib, ZD6474, CL-387785, HKI-272, XL647, PD64730B, E6473 2992, EKB-569, PF-299804. Examples of the ALK inhibitor include WHI-P154, TAE684, and PF-234066. Examples of PDGFR inhibitors include Gleevec, Desatinib, Valatinib, and Pazopanib. Examples of c-KIT inhibitors include imatinib, sunitinib, varatinib, desatinib, masatinib, and motesanib, and zotanib.

  As used herein, the term “cancer” (eg, “cancer” in cancer cells, “cancer” in anticancer agents) refers to any cancer, such as lung cancer (eg, squamous cell carcinoma, glandular gland). Non-small cell cancer such as cancer and large cell cancer, and small cell cancer), digestive system cancer (eg, stomach cancer, small intestine cancer, colon cancer, rectal cancer), pancreatic cancer, kidney cancer, liver cancer, thymic cancer , Spleen cancer, thyroid cancer, adrenal cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer (eg, endometrial cancer, cervical cancer), bone cancer, skin cancer, brain tumor, sarcoma, melanoma, blastoma (Eg, neuroblastoma), adenocarcinoma, squamous cell carcinoma, solid cancer, epithelial cancer, mesothelioma.

  The medium in which the test substance is contacted with the c-MET-expressing cells is appropriately selected depending on the type of cells used, and for example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum. Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 150 hours.

  In step (b), the amount of c-MET extracellular domain fragment in the culture supernatant of the cells contacted with the test substance is measured. The amount of c-MET extracellular domain fragment can be measured by a method known per se. For example, the measurement can be performed using a substance having affinity for the c-MET extracellular domain fragment. Measurement may also be performed by immunological techniques. Examples of such immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples include immunochromatography, luminescence immunoassay, spin immunoassay, western blot, and latex agglutination. Examples of methods other than the above that enable measurement of the amount of c-MET extracellular domain fragment include LC-MS.

  As used herein, the term “substance with affinity for a c-MET extracellular domain fragment” refers to a substance that has the ability to bind to a c-MET extracellular domain fragment. The substance having affinity for the c-MET extracellular domain fragment consists of a substance having affinity for the polypeptide consisting of the entire α chain of c-MET or a partial peptide thereof, or the extracellular region of the β chain of c-MET. It may be a substance having affinity for a polypeptide or a partial peptide thereof. Examples of substances having affinity for c-MET extracellular domain fragment include antibodies to c-MET extracellular domain fragment, aptamers to c-MET extracellular domain fragment (eg, see WO01 / 009159), and hepatocytes. HGF variants that retain the ability to bind growth factor (HGF) and c-MET.

  In step (c), a test substance that increases the amount of c-MET extracellular domain fragment is selected. Whether or not the test substance increases the amount of the c-MET extracellular domain fragment is determined by, for example, not contacting the test substance with the amount of the c-MET extracellular domain fragment in the culture supernatant of the cell contacted with the test substance. It can be determined by comparing the amount of c-MET extracellular domain fragment in the culture supernatant of control cells. The amount of the c-MET extracellular domain fragment in the culture supernatant of the control cell not contacted with the test substance is determined in advance with respect to the measurement of the amount of the c-MET extracellular domain fragment in the culture supernatant of the cell contacted with the test substance. Although it may be measured at the same time or at the same time, it is preferably an amount measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment. Since a test substance that increases the amount of c-MET extracellular domain fragment can inhibit c-MET enzyme activity, it is useful as a medicine such as an anticancer agent or a reagent. As an anticancer agent, the anticancer agent with respect to the cancer mentioned above is mentioned, for example.

The screening method of the present invention using a biological sample collected from a mammal can include, for example, the following steps (a ′) and (b ′):
(A ′) measuring the amount of c-MET extracellular domain fragment in a biological sample collected from a mammal administered with the test substance; and (b ′) increasing the amount of c-MET extracellular domain fragment; A step of selecting a test substance to be made.

  In the step (a ′), the mammal described above can be used, but a mammal suffering from cancer is preferable. The test substance is the same as described above. The biological sample is not particularly limited as long as the c-MET extracellular domain fragment is present, and examples thereof include blood, urine, saliva, ascites, tissue sample, cell sample, tissue extract, and cell extract. Of the above, blood, urine, and saliva are preferable from the viewpoint of low invasiveness. If necessary, the biological sample may be processed in advance prior to measurement. Examples of such treatment include centrifugation, extraction, concentration, fractionation, cell fixation, tissue fixation, tissue freezing, and tissue thinning.

  One of the advantages of the present invention is that the inhibitory activity against c-MET, which is a protein present in cells, can be measured using the amount of c-MET extracellular domain fragment present outside cells as an indicator. Since c-MET is a protein present in cells, it is necessary to obtain a cell sample in order to directly evaluate the inhibitory activity against c-MET. However, obtaining cell samples is highly invasive to mammals. Therefore, it is particularly difficult to obtain cell samples from humans as mammals. On the other hand, according to the present invention, inhibitory activity against c-MET can be indirectly evaluated using a biological sample (eg, blood, urine, saliva) with low invasiveness. Therefore, the present invention is useful, for example, in that it makes it possible to evaluate the inhibitory activity against c-MET in clinical trials.

  In the screening method of the present invention, instead of step (a ′), (a1 ′) a step of administering a test substance to a mammal, (a2 ′) a step of collecting a biological sample from the mammal administered with the test substance, and (A3 ′) One, two or all of the steps of measuring the amount of c-MET extracellular domain fragment in the collected biological sample may be included. Administration of a test substance to a mammal and collection of a biological sample from the mammal can be performed by a method known per se.

  In step (b '), a test substance that increases the amount of c-MET extracellular domain fragment is selected. Whether or not the test substance increases the amount of c-MET extracellular domain fragment is determined by, for example, examining the amount of c-MET extracellular domain fragment in a biological sample collected from a mammal administered the test substance. It can be determined by comparing the amount of c-MET extracellular domain fragment in a biological sample taken from a control mammal that has not been administered the substance. The amount of c-MET extracellular domain fragment in a biological sample collected from a control mammal not administered with the test substance is the amount of c-MET extracellular domain in the biological sample collected from the mammal administered with the test substance. For the measurement of the amount of domain fragment, it may be measured in advance or at the same time, but it is preferably an amount measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment. . Since a test substance that increases the amount of c-MET extracellular domain fragment can inhibit c-MET enzyme activity, it is useful as a medicine such as an anticancer agent or a reagent. As an anticancer agent, the anticancer agent with respect to the cancer mentioned above is mentioned, for example.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

A gefitinib resistant cell line (showing c-MET gene amplification) was prepared according to the method disclosed in Science 2007; 316: 1039-1043, a gefitinib sensitive cell line HCC827 (derived from human non-small cell lung cancer, Clin Cancer Res 2006; 12: 7117-7125.) Was subcultured in a medium containing gefitinib for 3 months. Gefitinib-resistant cells were isolated from EGFR inhibitor gefitinib or c-MET inhibitor PHA-666552 or SU11274 (eg, Science, 2007, vol. 316, p1039-1043, Cancer Research, 2005, vol. 65, p1479-). 1488) was cultured for 5 days in RPMI-1640 medium containing 10% fetal bovine serum containing 1 μM, and then the medium was replaced with serum-free RPMI-1640 medium containing the same concentration of inhibitor, and the culture was continued for 24 hours. For the cells, a cell extract was prepared with an NP-40-containing buffer, and the culture supernatant was concentrated with an ultrafiltration membrane. The cell extract was immunoprecipitated with an anti-EGFR antibody or anti-c-MET β chain antibody (14G9, obtained from Santa Cruz), and Western blotting with the same antibody or blotting with an anti-phosphotyrosine antibody was performed. The concentrated culture supernatant was transferred to a PVDF membrane by developing with reduced SDS-PAGE or non-reduced SDS-PAGE, and Western blotted with an anti-c-METα chain antibody (EP1454Y, obtained from abcam). The anti-c-MET beta chain antibody (14G9) recognizes the beta chains of immature c-MET (pro-c-MET) and mature c-MET (uncleaved c-MET). On the other hand, an anti-c-MET α-chain antibody (EP1454Y) is prepared using a partial peptide of an α-chain (linked to the extracellular domain in β-chain via a disulfide bond and present outside the cell) as an antigen. And thus recognizes the α chain of immature c-MET (pro-c-MET) and mature c-MET (uncleaved c-MET) and also a fragment of the c-MET extracellular domain ( That is, the α chain of secreted c-MET) can be recognized.
As a result, phosphorylation of c-MET detected in the cell extract of the untreated sample (DMSO) or the EGFR inhibitor-treated sample (gefitinib) was treated with c-MET inhibitors PHA-665752 or SU11274. In some cases (see FIG. 1, cMET IP: pYBlot). Along with this, the amount of c-MET extracellular domain fragment (soluble c-MET) detected in the concentrated culture supernatant was significantly increased with c-MET inhibitors (FIG. 1, soluble c-MET). See non-reducing and reducing). This indicates that inhibition of c-MET enzyme activity can be evaluated using the increase in the amount of c-MET extracellular domain fragment (soluble c-MET) as an index.
From the above, it was shown that a substance capable of inhibiting c-MET enzyme activity can be screened by evaluating whether or not the test substance increases the amount of c-MET extracellular domain fragment.

  The present invention is useful, for example, for the development of substances that can inhibit c-MET enzyme activity (eg, drugs such as anticancer agents or reagents).

Claims (6)

  A screening method for a substance capable of inhibiting c-MET enzyme activity, comprising evaluating whether a test substance increases the amount of c-MET extracellular domain fragment.   The screening method according to claim 1, wherein the substance capable of inhibiting c-MET enzyme activity is an anticancer agent. The screening method according to claim 1, comprising the following steps (a) to (c):
(A) contacting the test substance with c-MET-expressing cells in a culture medium;
(B) measuring the amount of c-MET extracellular domain fragment in the culture medium; and (c) selecting a test substance that increases the amount of c-MET extracellular domain fragment.   The screening method according to claim 3, wherein the c-MET-expressing cell is a cancer cell. The screening method according to claim 1, comprising the following steps (a ') and (b'):
(A ′) measuring the amount of c-MET extracellular domain fragment in a biological sample collected from a mammal administered with the test substance; and (b ′) increasing the amount of c-MET extracellular domain fragment; A step of selecting a test substance to be made.   The screening method according to claim 5, wherein the mammal is a mammal suffering from cancer.