Screening method for TGF-β signal inhibitor or Anti-fibrotic agent

Abstract

Provided is a method for evaluating or selecting a TGF-β signal inhibitor or an anti-fibrotic agent.　The method for evaluating or selecting a TGF-β signal inhibitor includes steps (1) to (3) below. (1) A step of bringing a test substance into contact with ST3GAL4, or with a tissue or cell capable of expressing ST3GAL4. (2) A step of measuring the expression or activity of ST3GAL4. (3) A step of evaluating or selecting, as a TGF-β signal inhibitor, a test substance that decreases the expression or activity of ST3GAL4 based on the results measured in (2).

Description

Screening method for TGF-β signal inhibitors or anti-fibrotic agents

[0001] The present invention relates to a method for evaluating or selecting a TGF-β signaling inhibitor or an anti-fibrotic agent.

[0002] Organ fibrosis is a disease in which chronic inflammation or other factors cause the accumulation of excess extracellular matrix in tissues, resulting in the replacement of normal tissue with the increased fibrous connective tissue, leading to organ dysfunction. A common cause of organ fibrosis is thought to be an excessive repair response associated with chronic inflammation and tissue damage. Specifically, immunocompetent cells mobilized to damaged tissues secrete various physiologically active substances, including TGF-β (Transforming Growth Factor-β), which promotes the proliferation of fibroblasts or hepatic stellate cells and their transformation into myofibroblast-like cells, as well as the production of extracellular matrix such as collagen, leading to the deposition of fibers. At the same time, the production of TIMPs, which are inhibitors of extracellular matrix-degrading proteases, inhibits fiber degradation, leading to the progression of fibrosis (Non-Patent Document 1). Furthermore, elevated TGF-β levels have been reported in patients with liver fibrosis, renal fibrosis, and pulmonary fibrosis compared to healthy individuals (Non-Patent Documents 2 to 4), and enhanced fibrosis has also been reported in animal models in which TGF-β has been artificially overexpressed (Non-Patent Document 5). Thus, enhanced TGF-β signaling is closely related to organ fibrosis.

[0003] TGF-β signal transduction is thought to occur via the following pathways 1) to 3). 1) After binding to type II and type I TGF-β receptors on the cell surface, TGF-β activates activin receptor-like kinase 5 (ALK5), a serine / threonine kinase domain of the type I receptor, and phosphorylates Smad2 / 3 in the cytoplasm. 2) The phosphorylated Smad2 / 3 forms a complex with Smad4 (phosphorylated Smad), translocates into the nucleus, and then binds to specific response elements on DNA to act as transcriptional activators. 3) As a result, physiological effects are exerted by inducing the expression of various response genes such as α-smooth muscle actin (α-SMA), collagen, tissue inhibitor of metalloprotease (TIMP), and connective tissue growth factor (CTGF) (Non-Patent Document 6). Here, nuclear localization of Smad2 / 3 is an essential process for TGF-β signaling, since TGF-β signaling is suppressed when a mutation is introduced into the nuclear localization signal of Smad3 (Non-Patent Document 7).

[0004] On the other hand, ST3GAL4, a β-galactoside α2-3 sialyltransferase involved in the terminal sialylation of glycoproteins and glycolipids, is reported to be involved in platelet coagulation through sialic acid addition to asialoglycoproteins, as well as in the adhesion, migration, and metastasis of cancer cells through the biosynthesis of tumor-associated sugar chains, and to be associated with epilepsy, etc. (Non-Patent Document 8). However, it is not known that ST3GAL4 is associated with the TGF-β signaling pathway or fibrosis.

[0005] Liver Int. 26(1), 8-22, 2006Hepatology 14, 269-273, 1991Curr. Opin. Nephrol. Hypertens. 3, 446-452, 1994Arch. Bronconeumol. 42, 380-383, 2006Am. J. Physiol. 276(4), G1059-68, 1999Cell 113(6), 685-700, 2003Proc. Natl. Acad. Sci. USA 97(14), 7853-7858, 2000Trends in Glycoscience and Glycotechnology 2011; 23(132) 178-193

[0006] The present invention relates to providing a method for evaluating or selecting a TGF-β signaling inhibitor or an anti-fibrotic agent.

[0007] While investigating molecules that act on the TGF-β signaling pathway, the present inventors found that ST3GAL4 is involved in glycosylation of the TGF-β type II receptor, and that suppression of ST3GAL4 expression suppresses the expression of mature TGF-β type II receptor and inhibits Smad2 / 3 phosphorylation by TGF-β1. Therefore, it is possible to search for or evaluate TGF-β signaling inhibitors or anti-fibrotic agents using ST3GAL4 expression as an indicator.

[0008] That is, the present invention relates to the following 1) to 2): 1) A method for evaluating or selecting a TGF-β signaling inhibitor, comprising the following steps (1) to (3): (1) contacting a test substance with tissues or cells capable of expressing ST3GAL4, or with ST3GAL4; (2) measuring the expression or activity of ST3GAL4; and (3) evaluating or selecting, as a TGF-β signaling inhibitor, a test substance that reduces the expression or activity of ST3GAL4 based on the results of the measurement in (2). 2) A method for evaluating or selecting an anti-fibrotic agent, comprising the following steps (4) to (6): (4) contacting a test substance with tissues or cells capable of expressing ST3GAL4, or with ST3GAL4; (5) measuring the expression or activity of ST3GAL4; and (6) evaluating or selecting, as an anti-fibrotic agent, a test substance that reduces the expression or activity of ST3GAL4 based on the results of the measurement in (5).

[0009] According to the present invention, drugs for preventing or treating diseases or pathological conditions caused by abnormalities in the TGF-β signaling pathway, such as organ fibrosis, obesity, diabetes, cancer metastasis, etc., can be easily screened or evaluated.

[0010] Expression analysis results of TGF-β type II receptor. Expression analysis results of Smad2 / 3. Phosphorylation ratio of Smad2 / 3.

[0011] As used herein, ST3GAL4 (ST3 Beta-Galactoside Alpha-2,3-Sialyltransferase 4) is a sialyltransferase belonging to the ST3gal family that transfers sialic acid to terminal galactose of sugar chains via α2,3-linkage using CMP-sialic acid as a donor substrate, and the acceptor sugar chain substrates are mainly Galβ1,3GlcNAc and Galβ1,4GlcNAc structures on glycoproteins and oligosaccharides (see Non-Patent Document 8).

[0012] N-glycans on the TGF-β type II receptor are essential for the receptor's functional expression, and it is known that inhibiting the binding of N-glycans to the receptor suppresses TGF-β-induced signaling and reduces the level of phosphorylated Smads (Biochem. J. (2012) 445, 403-411). As shown in the Examples below, suppressing the expression of ST3GAL4 suppresses the expression of mature TGF-β type II receptor and inhibits TGF-β1-induced Smad2 / 3 phosphorylation. Therefore, control of ST3GAL4 can be a target for the inhibition of TGF-β signaling or anti-fibrosis, and it becomes possible to evaluate or select TGF-β signal inhibitors or anti-fibrotic agents using the level of ST3GAL4 expression as an indicator.

[0013] The method for evaluating or selecting a TGF-β signaling inhibitor of the present invention comprises the following steps (1) to (3): (1) contacting a test substance with tissues or cells capable of expressing ST3GAL4 or with ST3GAL4, (2) measuring the expression or activity of ST3GAL4, and (3) evaluating or selecting, as a TGF-β signaling inhibitor, a test substance that reduces the expression or activity of ST3GAL4 based on the results of the measurement in (2). Furthermore, the method for evaluating or selecting an anti-fibrotic agent of the present invention comprises the following steps (4) to (6): (4) contacting a test substance with tissues or cells capable of expressing ST3GAL4 or with ST3GAL4, (5) measuring the expression or activity of ST3GAL4, and (6) evaluating or selecting, as an anti-fibrotic agent, a test substance that reduces the expression or activity of ST3GAL4 based on the results of the measurement in (5).

[0014] Examples of tissues or cells capable of expressing ST3GAL4 used in step (1) or (4) include tissues or cells that inherently have the ST3GAL4 gene and are capable of expressing it, and tissues or cells into which the ST3GAL4 gene has been exogenously introduced so as to be capable of expressing it. The tissues or cells may be tissues or cells collected from a living organism or cultured cells. Examples of tissues or cells that inherently have the ST3GAL4 gene and are capable of expressing it include any tissues or cells of a living organism, but are preferably cells collected from a mammal. Examples of mammals include humans, mice, rats, hamsters, rabbits, pigs, monkeys, etc. Humans are preferred. Examples of human-derived cells include cervical epithelioid carcinoma cells (e.g., HeLa cells, HeLa S3 cells), human malignant melanoma cells (e.g., A375 cells, A431 cells), human neuroblastoma cells (e.g., SH-SY5Y cells, IMR-32 cells), human fibroblasts (e.g., primary cultured lung fibroblasts, HFL-1 cells, IMR-90 cells, NIH-3T3 cells, WI-38 cells), human hepatic stellate cells (e.g., TWNT-4 cells, LI-90 cells, LX-2 cells), and human embryonic kidney cells (e.g., HEK293 cells).

[0015] Tissues or cells into which the ST3GAL4 gene has been exogenously introduced so as to be capable of expression can be obtained, for example, by introducing a gene encoding ST3GAL4 into any mammalian tissue or cell and transforming the tissue or cell so that the tissue or cell expresses ST3GAL4 or enhances ST3GAL4 expression. Cells into which a construct has been introduced in which a reporter gene such as luciferase is fused downstream of the promoter region of the ST3GAL4 gene can also be used. Methods for introducing genes or constructs into cells include, but are not limited to, vector introduction by electroporation, lipofection, etc.

[0016] Examples of the ST3GAL4 used in step (1) or (4) include ST3GAL4 isolated from a living organism, recombinant ST3GAL4 produced by genetic recombination, as well as cell membranes carrying the ST3GAL4, artificial lipid bilayer membranes, and the like.

[0017] The test substance used in step (1) or (4) is not particularly limited as long as it can be used as a TGF-β signal inhibitor or anti-fibrotic agent, and examples thereof include animals, plants, marine organisms, microorganisms, etc., and extracts thereof; natural components derived therefrom (e.g., nucleic acids, proteins, antibodies, etc.); synthetic compounds; and mixtures and compositions thereof.

[0018] In step (1) or (4), the means for contacting the tissue or cells, or ST3GAL4 with the test substance may be any means known in the art, including, for example, adding the test substance to tissue or cell culture medium, or directly adding the test substance to tissue or cells, or ST3GAL4 (e.g., dropping, applying, spraying, atomizing, patching, etc.). The concentration and contact amount of the test substance may be appropriately determined based on the form, chemical properties, cytotoxicity, etc. of the test substance. For example, a predetermined amount of the test substance diluted to an appropriate concentration may be exposed to HeLa cells or the like for 24 to 48 hours under conditions of 25 to 37°C and 5% CO2.

[0019] The expression or activity of ST3GAL4 measured in step (2) or (5) can be measured according to methods known in the art. Examples of ST3GAL4 expression measurement include measuring protein expression levels, measuring mRNA expression levels, or measuring promoter activity. Examples of methods for measuring protein expression include immunohistochemistry, ELISA, Western blotting, immunoprecipitation, mass spectrometry, and combinations thereof. Examples of methods for measuring mRNA expression include dot blotting, Northern blotting, RT-PCR, real-time RT-PCR, microarrays, RNA sequencing, and combinations thereof. Examples of methods for measuring promoter activity include fluorescent / optical measurement of promoter activity or transcriptional activity using a reporter gene (reporter assay).

[0020] ST3GAL4 activity can be measured, for example, by measuring sialic acid transfer from the donor substrate, cytidine monophosphate (CMP)-sialic acid, to the acceptor substrate, β1,3-galactosyl-N-acetylgalactosamine, using a recombinant ST3GAL4 protein. Sialic acid transfer can be quantified by mass spectrometry of the reaction product or by converting free CMP to ATP and measuring the luciferase activity.

[0021] Next, based on the results of the measurements as described above, test substances that reduce the expression or activity of ST3GAL4 are evaluated or selected as TGF-β signaling inhibitors or anti-fibrotic agents (step (3) or (6)). Such evaluation or selection is performed, for example, by comparing the results before and after the addition of the test substance, or by comparing a group to which the test substance has been added with a group to which the test substance has not been added or a group to which a control substance has been added. Alternatively, evaluation is performed by comparing the measurement results between test substances at various concentrations.

[0022] For example, if the expression or activity of ST3GAL4 in the test substance-added group is lower than that before the addition of the test substance, the test substance-free group, or the control substance-added group, the test substance is evaluated or selected as a TGF-β signaling inhibitor or anti-fibrotic agent. In this case, determination can be made based on whether the expression or activity of ST3GAL4 in the test substance-added group is statistically significantly lower than that before the addition of the test substance, the test substance-free group, or the control substance-added group. Alternatively, determination can be made based on whether the expression or activity of ST3GAL4 in the test substance-added group is below a certain level, for example, 90% or less, preferably 70% or less, and more preferably 50% or less, when the expression or activity of ST3GAL4 before the addition of the test substance, the test substance-free group, or the control substance-added group is set to 100%.

[0023] In the present invention, the TGF-β signal inhibitor is a drug that inhibits or suppresses the TGF-β signaling pathway to prevent, treat, or ameliorate diseases or pathological conditions caused by abnormalities in the TGF-β signaling pathway. Examples of target diseases or pathological conditions include fibrosis (organ fibrosis), obesity, diabetes, and cancer metastasis. The anti-fibrotic agent is a drug for preventing or treating fibrosis. Examples of fibrosis include organ fibrosis such as liver fibrosis, pulmonary fibrosis, renal fibrosis, dermal fibrosis, cardiac fibrosis, and pancreatic fibrosis. Furthermore, since fibrosis can cause fibrosis-associated symptoms such as inflammation and atrophy depending on the organ in which fibrosis has progressed and the degree of progression, the anti-fibrotic agent of the present invention also encompasses treatment of symptoms associated with fibrosis. Pulmonary fibrosis includes not only idiopathic pulmonary fibrosis but also pulmonary fibrotic conditions, including those that coexist with interstitial pneumonia. In other words, pulmonary fibrosis includes interstitial pneumonia, which may be accompanied by pulmonary fibrosis.

[0024] Test Example 1 Anti-fibrotic effect of ST3GAL4 siRNA <Experimental method> HeLa human-derived cervical cancer cell line was suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and 5 × 10 cells were placed in a 6-well plate. 4 After seeding at a density of 1000 cells / well, the cells were cultured at 37°C in the presence of 5% CO2 for 24 hours. Using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and Opti-MEM™ I Reduced Serum Media (Thermo Fisher Scientific), a complex of siRNA and lipids consisting of the following base sequence designed against ST3GAL4 was prepared, and the siRNA was diluted with antibiotic-free 10% FBS-DMEM to a final concentration of 37.5 nM. After removing the medium from the HeLa cells, these were added and cultured for 48 hours, after which 2 ng / ml TGF-β1 (Peprotech) was added and the cells were cultured for an additional 4 hours.

[0025]

[0026] The medium was then removed, the cells were washed with phosphate buffered saline (PBS), and the cells were harvested. After centrifugation, the cells were replaced with RIPA lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors (Roche) and allowed to stand on ice for 30 minutes to extract proteins. The supernatant was then harvested after centrifugation. Laemmli sample buffer was then added, and the cells were heated on a heat block at 95°C for 5 minutes to reduce the protein. This protein sample was used as a whole-cell extract. The sample was subjected to Western blotting or simple Western analysis to analyze the expression of TGF-β type II receptor and Smad2 / 3.

[0027] For Western blotting, whole cell extracts were separated by SDS-polyacrylamide gel electrophoresis at 20 mA for 90 minutes, then transferred to a PVDF membrane at 40 V, 144 mA for 40 minutes. Blocking was then performed for 30 minutes with Tris-buffered saline containing Tween-20 (TBS-T) containing 5% skim milk, and the membrane was incubated overnight at 4°C with anti-TGF-β type II receptor antibody (abcam) diluted 1 / 1000 with Can Get Signal Solution I (TOYOBO). After the incubation, the membrane was washed three times with TBS-T and incubated for 1 hour at room temperature with peroxidase-labeled anti-rabbit immunoglobulin antibody (Cell Signaling Technology) diluted 1 / 2000 with Can Get Signal Solution II (TOYOBO). After the reaction was completed, the plate was washed three times with TBS-T, reacted with Luminata Western HRP Substrate (Millipore), and then detected using a ChemiDoc imaging system (Bio-rad).

[0028] For simple Western analysis, whole cell extracts, anti-Smad2 / 3 antibody, and anti-phosphorylated Smad2 / 3 antibody (Cell Signaling Technology) were dispensed onto the plate provided with the 12-230 kD Separation module (Protein Simple) along with the included washing solution, blocking solution, and secondary antibody solution, and data were acquired using the fully automated Western blotting system Jess (Protein Simple). The included analysis software, Compass, was used to quantify the band intensities of total Smad2 / 3 and phosphorylated Smad2 / 3, and the phosphorylation ratio was calculated from the ratio.

[0029] <Discussion of Results> Western blotting revealed that the expression level of the mature TGF-β type II receptor with normal glycosylation, indicated by the arrow (next page), was reduced by ST3GAL4 siRNA treatment (Figure 1). Simple Western analysis showed that the same treatment significantly reduced the Smad2 / 3 phosphorylation level (Figures 2 and 3), suggesting that sialic acid addition by ST3GAL4 is essential for the expression of TGF-β type II receptor and its subsequent signal transduction function, and that suppression of ST3GAL4 expression has an anti-fibrotic effect via inhibition of TGF-β signaling, a major fibrotic signal.

Claims

1. A method for evaluating or selecting a TGF-β signaling inhibitor, comprising the following steps (1) to (3): (1) contacting a test substance with tissues or cells capable of expressing ST3GAL4, or with ST3GAL4; (2) measuring the expression or activity of ST3GAL4; and (3) evaluating or selecting a test substance that reduces the expression or activity of ST3GAL4 as a TGF-β signaling inhibitor based on the results of the measurement in (2).

2. A method for evaluating or selecting an antifibrotic agent, comprising the following steps (4) to (6): (4) contacting a test substance with tissues or cells capable of expressing ST3GAL4 or with ST3GAL4; (5) measuring the expression or activity of ST3GAL4; and (6) evaluating or selecting, as an antifibrotic agent, a test substance that reduces the expression or activity of ST3GAL4 based on the results measured in (5).

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