KR101275777B1 - Cell line which is transfected with Wnt active measurement vector and High-Throughput Screening method of selective ligands by using it - Google Patents

Abstract

The present invention relates to a cell line transformed with a recombinant vector for measuring Wnt activity and a high efficiency screening method using the same, and more particularly, a recombinant vector for measuring Wnt activity, a cell line transformed with the vector and a Wnt protein activity using the same. As a method for searching related substances, the recombinant vector of the present invention and the transformed cell line including the same may be effectively used for developing osteoporosis treatment, osteoporosis treatment aid, and bone metabolic disease treatment by efficiently searching for substances related to the activity of Wnt protein. will be.

Description

Cell line which is transfected with Wnt active measurement vector and High-Throughput Screening method of selective ligands by using it}

The present invention relates to a cell line transformed with a recombinant vector for measuring Wnt activity and a high efficiency screening method using the same, and more particularly, a recombinant vector for measuring Wnt activity, a cell line transformed with the vector and a Wnt protein activity using the same. It relates to a method of searching for related substances.

The incidence of osteoporosis is increasing in modern people due to socio-environmental and genetic influences. This phenomenon is gradually increasing with an increase in the elderly population, and osteoporosis is a reality that is rapidly deteriorated with aging because it is started / developed without any special symptoms. Many drugs have been developed for the treatment of osteoporosis, but these drugs are mainly aimed at relieving pain according to the symptoms of the disease, or are effective only in reducing the bone density, and thus have no effect on the bone density increase in patients with advanced osteoporosis. In addition, there are limitations that are used only as injections, or they have problems such as many side effects when taken for a long time. Therefore, there is a demand for developing new drugs to solve them.

In order to overcome the limitations of the absorption inhibitors that have been used in the past for the treatment of osteoporosis, researches on how to fundamentally overcome the osteoporosis by promoting the production of new bone have recently emerged. In other words, the paradigm of the treatment of osteoporosis has changed from the purpose of inhibiting bone resorption by osteoclasts to the direct promotion of bone formation by osteoblasts.

Factors that promote osteoblast formation by controlling osteoblast differentiation include transcription factors Runx2 and osterix, and growth factors such as IGF-1, TGF-β, and BMP-2. In addition, in 2001 and 2002, it was known that Winding (hereinafter, “Wnt”) signaling is an important factor for promoting osteoblast differentiation.

The Wnt protein is a cysteine-rich secreted glycoprotein of about 40 kDa, and is known to be involved in various developmental processes including cell proliferation and differentiation and cell polarity (Moon RT et al., Science , 2002; Reya T and Clevers H, Nature , 2005). In humans 19 wnt proteins have been reported, 10 frizzled proteins and 2 co-receptors (LRP5 and 6) that act as wnt receptors (He XC et al., Nat Genet, 2004; Tamai K et al, Mol Cell , 2004; Tamai K et al., Nature, 2000).

Typical Wnt signaling leads to stabilization of the beta -catenin protein through modulation of protein kinases, thereby transferring into the cell nucleus and transcriptional activity. This transcriptional activity is reported to be caused by transcription factors of the Lef1 / Tcf family (Moon RT et al., Science , 2002; Reya T and Clevers H, Nature , 2005; Wodarz A and Nusse R, Annu Rev Cell Dev Biol ., 1998).

In addition, when the Wnt protein binds to the frizzled receptor and the LRP5 co-receptor, the proteolytic complex loses its activity, causing the transfer of β-catenin into the cell nucleus, which leads to target gene transcription through binding to the Lef1 / Tcf protein. It is reported that activity is promoted (Reya T and Clevers H, Nature, 2005; Tamai K et al., Mol Cell, 2004; Westendorf JJ et al., Gene, 2004).

On the other hand, if there is a gain of function mutation of LRP5, a receptor for the Wnt signal, osteopetrosis, which causes bone density to increase above normal, results in a loss of function mutation. If congenital osteoporosis develops. In addition, it has been reported that overexpression of sFRP-1, which inhibits Wnt signaling, results in a decrease in bone mineral density and promotes apoptosis of osteoblasts.

Based on the importance of the Wnt signaling pathway presented above, in developing drugs that can treat related diseases such as breast cancer, colorectal cancer, bone metabolism and obesity, Wnt signaling molecules are used for drug screening. It is recognized as a good target for this, and many attempts have been made to discover active agents or inhibitors that can control them and develop them as new drugs.

: Moon RT et al., Science, 2002 He XC et al., Nat Genet, 2004 Reya T and Clevers H, Nature, 2005

Accordingly, the present inventors are continuing research to establish an in vitro system for drug screening of Wnt signaling molecules, and the activity of drugs or molecular compounds capable of activating Wnt signaling in vivo. A recombinant vector capable of mass retrieval was prepared, and the transformed cell line into which the expression vector was introduced was confirmed to effectively search for substances involved in the activity of the Wnt protein, thereby completing the present invention.

An object of the present invention is to analyze the quantitative changes and distribution of Wnt protein activity in the cell using fluorescent protein to increase or inhibit the activity of Wnt protein to find a candidate that can be useful as a therapeutic agent related to bone metabolic diseases To provide a way to search.

In order to achieve the above object, the present invention provides a recombinant vector for measuring Wnt activity, comprising a Tcf factor binding site (T cell factor binding site).

More specifically, as a final step of the Wnt signaling system that regulates osteoblast differentiation and promotes bone formation, recombinant 8 × Tcf-Luc can measure activation of the Tcf / Lef promoter through the expression of luciferase. To provide a vector.

The recombinant vector of the present invention comprises a Tcf binding moiety, a TA viral promoter, a luciferase reporter gene, and a neomycin resistance gene in a 5 'to 3' direction, the expression of which is one It is characterized by being controlled by a promoter of.

The recombinant vector of the present invention is characterized by having 8 copies of the Tcf binding moiety.

The present invention provides a transformed cell line wherein the recombinant vector is introduced to stably express the Wnt protein.

In addition, the present invention provides a method for searching for a substance involved in the activity of the Wnt protein using the transformed cell line.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant vector for measuring Wnt activity and a trait stably expressing the compound and gene that can be used as a therapeutic agent related to bone metabolic diseases, which can be usefully used to detect whether stabilization of Wnt protein and its migration to the nucleus. Provide a converting cell line.

More specifically, the recombinant vector of the present invention binds to Tcf after β-catenin present in the cytoplasm reacts with Wnt protein molecules when they bind to intracellular receptors, and then binds to Tcf, and the bound β-catenin / Tcf complex. Focuses on binding to the Tcf response site of the target gene to increase transcription.

Recombinant vector according to the present invention is designed to link the luciferase cDNA to the 3 'position of the nucleotide sequence overlapping the Tcf binding site 8 times and to include a resistance gene for neomycin, so that their expression is controlled by a single promoter See, FIG. 2.

The present invention uses a recombinant vector having the above characteristics to search for a substance involved in the activity of the Wnt protein, a recombinant cell line is inserted into the staining agent of the host cell to stably express the recombinant Wnt protein Offer.

The recombinant vector is a host cell by a conventional transfection method, for example, DEAE-dextran, calcium phosphate method, microinjection method, DNA-containing liposome method, lipofectamine-DNA complex method, etc., preferably by electroporation. , Preferably animal cells, more preferably C2C12 cells, CHO cells or C3H10T1 / 2 cells. Such transfection methods are known in the art ( Molecular Cloning , Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 2001).

The recombinant vector and the transformed cell line stably expressing the same according to the present invention play a crucial role in the differentiation of osteoblasts, screening compounds capable of activating the Wnt signal in bone tissue, and compounds that can be used as therapeutic agents for bone metabolic diseases It can be used very usefully for screening.

Moreover, the recombinant vector of the present invention may be introduced into a transformed cell line, a recombinant adenovirus, a transgenic animal, or the like, and may be usefully used as a biosensor. The cell line into which the recombinant vector is introduced is useful not only for screening osteoporosis treatments, osteoporosis treatment supplements, and bone metabolic diseases treatments, but also for transgenic animals produced using the recombinant vectors, which can observe and evaluate Wnt mobility and stabilization. It may be used as a disease model animal.

Therefore, the screening system of the present invention using the recombinant vector of the present invention and a transformed cell line stably expressing the same is a biosensor for searching for a substance involved in the activity of the Wnt protein, an osteoporosis therapeutic agent and an osteoporosis treatment aid associated with the protein. And it will be a useful tool in the development of therapeutic agents for bone metabolic diseases.

In addition, the present invention provides a method for searching for a substance involved in the activity of the Wnt protein using a transformed cell line stably expressing the recombinant vector.

The search method

(1) culturing the transformed cell line in which the recombinant 8xTcf-Luc vector is stably expressed in the well plate in a well plate;

(2) adding a sample to be searched to each well of the plate and culturing; And

(3) measuring luciferase activity expressed therefrom in the form of a fusion protein.

In the present invention, the sample to increase the amount of Wnt protein can be searched by processing the sample to be searched in the culture cell of the transformed cell line in step (2), and in more detail, the following compound 1 Or compound 2 could be selected.

The selected compound 1 or compound 2 can be used as a therapeutic agent for osteoporosis, and the screening method according to the present invention can easily analyze the stability and mobility of the Wnt protein to the nucleus using recombinant fluorescent protein, and thus, It can be useful for research in related fields such as screening and high speed evaluation system.

The recombinant vector of the present invention and the transformed cell line comprising the same may be effectively used for developing osteoporosis therapeutics, osteoporosis therapeutic supplements, and therapeutic agents for bone metabolic diseases by efficiently searching for substances related to the activity of the Wnt protein.

1 is a schematic diagram showing the schematic diagram of the Wnt signaling system and the action point of the new drug candidate to be discovered in the present invention,
Figure 2 shows a cleavage map of the recombinant vector 8 × Tcf-Luc for measuring the Wnt activity of the present invention,
Figure 3 is a nucleotide sequence after treatment the TopFlash vector comprising the nucleotide sequence of the consensus Tcf binding site 8 copy of the nucleotide sequence (A) and the consensus Tcf binding site 8 copy in accordance with the invention with a restriction enzyme Kpn I and BamH I ( B),
4 is a result confirming that the cell line transformed with the recombinant vector 8 × Tcf-Luc for measuring the Wnt activity according to the present invention is stably expressed.
Figure 5 is the result of measuring the Wnt protein activity of each concentration of the selected material by the method of searching for substances involved in the Wnt protein activity according to the present invention.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[Example] Establishment of high throughput system (HTS) using cell line capable of measuring Wnt activity

(1) Construction of a vector to measure Wnt activity

Wnt activity occurs by binding Wnt to the frizzled receptor (receptor) and then activating β-catenin in the cytoplasm, moving into the nucleus and binding with Tcf / Lef to activate the promoter of the target gene. Therefore, in the present embodiment, a consensus Tcf binding site 8 copy (SEQ ID NO: 1) is inserted into a pTA-Luc vector having a minimal TA viral promoter and a neomycin resistance gene is inserted to insert the recombinant vector (8 × Tcf) of FIG. 2. -Luc vector) was prepared.

Specifically, given the 8 × consensus Tcf then comprises a binding site, and for 1 hour at 37 ℃ the TopFlash vector with Kpn I restriction enzyme 1 ㎕ in the reaction for 30 minutes in Klenow 1 ㎕ 37 ℃ the terminal portion of DNA The blunt-end was treated with 1 μl of BamH I restriction enzyme at 37 ° C. for 1 hour. The DNA treated with the restriction enzyme was prepared using agarose gel extraction kit (Agarose gel extraction Kit, Qiagen) to prepare a 2178 bp insertion DNA fragment (SEQ ID NO: 2).

In addition, pTA-Luc vector, a backbone vector, was treated with 2 μg of pCI-neo vector (Promega E1841) with 1 μl of Bgl II and Sma I restriction enzymes at 37 ° C. for 1 hour, followed by agarose gel separation kit (Agarose gel). A backbone of 4350 bp recombinant vector was prepared using an extraction kit (Qiagen).

2 μl of the prepared 4350 bp backbone vector and 3 μl of the 2178 bp inserted DNA fragment were placed in an EP-tube, 1 μl of T ₄ ligase was added, and the reaction was performed at 4 ° C. over night. The reaction solution was placed in DH5α competent cells, reacted at 42 ° C. for 1 minute, and then spread on an agarose medium plate containing ampicillin to overnight at 37 ° C. . After confirming the E. coli colonies of the cells containing the recombinant vector DNA, 8 × Tcf-Luc recombinant vector was prepared by growing a large amount of E. coli in agarose liquid medium and treating the restriction plasmid DNA with various restriction enzymes. Confirmed.

(2) Construction of Transgenic Cell Line

After transfection of the 8 × Tcf-Luc vector prepared in (1) to Chinese Hamster Ovary (CHO) cells, 1,000 ng / mL of the G418 antibiotic was selected and transformed by selecting resistant cells. Prepare a cell line (stable cell line).

(3) Investigation of the characteristics of the transformed cell line

Factors activating Wnt proteins, including constitutively-active β-catenin adenovirus, sFRP (soluble frizzled-related proteins) to verify the characteristics of the transformed cell line selected in (2) above , Wnt (Wnt inhibitory factors), Dkk-1 (Dickkopf-1) including Wnt and Frizzled receptors to inhibit the binding of the Wnt protein activity was measured and compared.

In detail, the transgenic cell line selected in (2) was passaged once every 3 days in a 100 mm dish plate, and 500 ng / ml G418 antibiotic was added to the culture medium during passage. The cultured transformed cell line was seeded at 2 × 10 ⁴ cells / well in a 24 well plate, and the following various factors were treated by concentration. After 2 days luciferase activity was measured.

The assay was performed by removing the medium of the culture medium to be measured and washing with PBS, lysing the cells with a cell lysis buffer, and adding luciferase to which 10 ul of lysate and luciferine were added. After mixing 50 ul of luciferase reagent, the degree of luciferase activity was measured by a luminometer.

The measured concentrations were 1, 10, 100 ng / ml for Wnt-1, Wnt-3A, 4, 40, 100 mM for LiCl, and 1, 5, 10 mol of constitutively-active β-catenin adenovirus.

As a result, as can be seen in Figure 4, Wnt-1, Wnt-3A, LiCl, increased the activity of luciferase by 4 to 14 times the treatment of the factor activating the Wnt protein, including constitutively-active β-catenin adenovirus, In addition, it was confirmed that this increase in activity is suppressed by Dkk-1 treatment.

From the above results, it was confirmed that the transformed cell line prepared with the 8 × Tcf-Luc vector of this example can be usefully used for measuring Wnt activity, and furthermore, it was confirmed that the cell-based high throughput system (HTS) was useful. Do.

[ Test Example  One] HTS Using Wnt  Activation Low molecular weight  Exploration of matter

In order to search for osteoporosis therapeutic substances using the cell-based HTS of the above example, a small-molecule library of representative compounds was screened by receiving 6,400 representative compounds from the Korea Research Institute of Chemical Technology.

The transformed cell line of the example was treated with the total 6,400 representative compounds and the activity of luciferase was measured. As a result, the following two compounds were selected that significantly increased Wnt activity.

For the two selected compounds, treatment with various concentrations (100 ng / ml for Wnt-3A, compounds 1 and 2: 1, 5, 10, 50, 100 nM) to confirm more accurate Wnt activity. As a result, as can be seen in Figure 5 it was confirmed that the reporter activity, that is, Wnt activity is increased in a concentration-dependent manner.

From the above results, the recombinant 8 × Tcf-Luc vector prepared according to the present invention and the transformed cell line including the same may be very useful for searching for compounds, genes and proteins related to the activity of Wnt protein or studying their mechanism of action. It was confirmed that there is.

Attach an electronic file to a sequence list

Claims (9)

(1) culturing the transformed cell line in a well plate with a recombinant vector for measuring Wnt activity, comprising a Tcf factor binding site;
(2) adding and incubating a sample to be searched to each well of the plate; And
(3) measuring luciferase activity expressed therefrom in the form of a fusion protein;
Searching for a substance involved in Wnt protein activity, including. The method of claim 1,
The vector is involved in the Wnt protein activity, comprising a Tcf binding portion, a TA viral promoter, a luciferase reporter gene, and a neomycin resistance gene in the 5 'to 3' direction. How to search for substances. 3. The method according to claim 1 or 2,
The vector is a method for searching for substances involved in Wnt protein activity, characterized in that the Tcf binding portion has eight copy numbers. delete The method of claim 1,
And said cell is a Chinese Hamster Ovary (CHO) cell. delete The method of claim 1,
Searching for a material involved in Wnt protein activity, characterized in that for searching the sample to increase the amount of Wnt protein by processing the sample to be searched in the culture medium of the transformed cell line in step (2). 8. The method of claim 7,
The substance is a method of searching for a substance involved in Wnt protein activity, characterized in that Compound 1 or Compound 2.

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