KR100847149B1 - Screening method of fast biophysical active substance using reporter adenovirus with SeaForce gene - Google Patents

Abstract

The present invention relates to a recombinant reporter adenovirus that can be used for evaluating the bioefficiency of a ligand of a G protein-coupled receptor (GPCR), and is applicable to a wider variety of cells than the conventional technology. It may be applicable to ligand discovery of unknown orphan G protein-linked receptors (oGPCR), faster and more accurate than conventional methods, and save time and money. In order to achieve the above object, the present invention is to provide a technology capable of measuring receptor-specific activity with high efficiency by making a reporter adenovirus that can measure the various activities of GPCRs.

GPCR, cfos, reporter gene, β-galactosidase, lactamase, GFP, luciferase, adenovirus, cell based assay

Description

Reporter adenovirus having c-fos promoter for high throughout screening system for G protein-coupled receptor (GPCR) modulators}

1 is a diagram illustrating a process of recombining adenovirus into which a promoter and a reporter gene are inserted.

Figure 2 is a diagram showing the nucleotide sequence of each gene promoter recombined into an adenovirus vector.

FIG. 3 is a diagram showing luciferase activity after infection of reporter adenovirus produced by recombination with an adenovirus vector with increasing MOI in U-2OS cell lines, and after treatment with each marker-specific agent. FIG.

4 is a diagram showing luciferase activity after infection of reporter adenoviruses with different MOIs in CV-1 cell lines and treatment with marker gene specific agents. Figure 4b is a GnRH treatment after infecting the CV-1 cell line with a second type of gonadotropin-releasing hormone (GnRH) receptor of bullfrog, which has been well known for signaling between adenovirus and a receptor. It is a figure which shows luciferase activity after doing so.

FIG. 5 is a diagram illustrating the actual ligand of oGPCR called GPR92 by actual screening of oGPCR ligand using reporter adenovirus. FIG. 5A shows CRE-luciferase activity significantly increased by E-50% when GPR92-infected cells were treated with ligand candidate groups extracted from two-month-old fetuses, and FIG. 5B shows this activity. In order to confirm correctly, the change of CRE-luciferase activity was observed by varying the concentration of the E-50% extract. In FIGS. 5C and 5D, after extracting and extracting the E-50% extract showing specific activity against GPR92 with finer materials, the sample called E50-6 is treated with GPR92-infected cells. This figure confirms that the CRE-luciferase activity by GPR92 also increases significantly with the change of concentration.

6 is a genetic map of cfos luciferase adenovirus.

The large family of G protein-coupled receptors (GPCRs) penetrates the cell membrane seven times and occupies the largest range of cell membrane receptors. Its reproductive function, growth, metabolism, homeostasis, appetite, It regulates most human activities, including sleep and psychology. GPCRs exhibit various activities of the receptor by ligands such as various signals outside the cell, such as ions, amino acids, nucleic acids, neurotransmitters and neuropeptides, hormones. In general, ligand-bound GPCRs cause structural changes to induce the activity of different types of G proteins (G _s , G _{q / 11} , G _{i / o} , G _12/13 ) in cells and activate G Proteins regulate several signaling substances in cells.

GPCR large families are largely classified into Rhodopsin (Class A), Secretin Receptor (Class B), and Metabotropic Receptor (Family C) by the chemical properties of ligands and structural similarities of receptors. (Bockaert et al ., EMBO J vol. 18 pp 1723-1729, 1999). From the completion of the Human Genome Project, it has been found that over 300 genes encode GPCRs, with the exception of the GPCRs involved in the sense of smell and taste. About half of these ligands or bioactive functions on GPCRs have not yet been identified. These receptors are called orphan GPCRs (oGPCRs) and these oGPCRs have structural characteristics similar to those of GPCRs that have already been identified, but show less than 45% identity in amino acid sequences.

GPCR is one of the most important targets in the drug development market, with the top 20% of pharmaceutical sales in the drug development sector and about 50% of all pharmaceuticals. Thus, oGPCRs whose ligands or functions have not yet been identified are targeted for drug development with high probability of success (Neubig et al ., Nat Rev Drug Discov vol. 1 pp 187-97, 2002). There are currently about 85 different target oGPCRs available for drug discovery, and discovering their function by identifying new ligands for each oGPCR can be a tremendous economic benefit in the medical / pharmaceutical industry.

The discovery of oGPCR ligand has been in the spotlight in Korea and abroad since the Human Genome Project, and it is actively being conducted in about 30 universities and private enterprise research institutes around the world. In particular, there are only a handful of companies with relevant technologies, and large multinational pharmaceutical companies such as GlaxoSmithKline, Pfizer and Merck are intensively investing in preemptively related technologies through cooperation with small and medium sized bio venture companies. About 50 ligand-oGPCR pairs have been identified by traditional and reverse pharmacology methods. The first step in making oGPCR a target for drug development is to discover the function of oGPCR through de-orphanization of ligand discovery. De-orphanization of the receptor is a very difficult task that requires a lot of effort. De- orphanization involves predicting the receptor's function or properties based on tissue or cellular distribution of oGPCRs and developing and using effective drug screening devices with protein extracts or hundreds or thousands of chemical combinations from tissues.

In order to discover the ligands of oGPCRs and analyze their function successfully, three important factors must be reviewed first: 1) whether oGPCR is normally expressed in the cell membrane and 2) purified ligand library or good quality. Tissue extract, 3) is a high-speed / mass screening method that can measure receptor activity using ligand candidate groups (Howard et al ., Trends Pharmacol Sci vol. 22 pp 132-140, 2001). Of these three factors, it is well known by the present inventors and other researchers to secure cell membrane expression of oGPCR and various ligand candidates, and a high efficiency method is used. Depending on whether you choose a large difference in efficiency. This is because, in the case of GPCR, the type of G protein to be activated for each receptor is different, and according to the type of the G protein, a different activity evaluation system is required, and in particular, in the case of oGPCR, information on which G protein expresses the activity of the receptor is shown. There is no systemic approach to various proteins because there is no.

Currently, a method for measuring the activity of GPCRs by screening ligands at high speed and in bulk in a cell-based system for ligand discovery of oGPCR

1) cytosensor microphysiometer for measuring pH changes in cell culture

2) measuring K ⁺ current using Xenopus oocyte voltage clamp,

3) Method to confirm the change of pigmentation reaction of colored cells according to GPCR activity using Xenopus melanophore, 4) Ca ²⁺ sensing system using FLIPR or Aquarin, 5) b-arrestin with fluorescent substance attached to GPCR There is a method of analyzing an image moving to the cell membrane according to the activity. However, it is possible to measure the pH change in cell culture in real time, but it is not suitable for high throughput screening (HTS), and it is possible to measure K ⁺ current using Xenopus oocyte voltage clamp. The measuring method can measure only the activity of a receptor that binds to G _{i / o} among several GPCRs and is not suitable for HTS. The assay of oGPCR activity through the staining of cells using Xenopus melanophore staining cells can be used to test the activity of receptors that bind to different G proteins (G _s , G _{q / 11} , G _{i / o} ). Although there is a number, there is a disadvantage that the sensitivity is low and there is a limit to the introduction of the gene into the pigmented cells. The Ca ²⁺ sensing system, which measures the sensitivity of Ca ²⁺ by oGPCR using FLIPR or Aquarin, which is sensitive, is suitable for HTS but not for oGPCR that does not show Ca ²⁺ response. The method of analyzing the image of the b-arrestin with fluorescent substance transported to the cell membrane according to the GPCR activity requires a special image analysis device, and is not suitable for the GPCR to which the b-arrestin does not work and also for the inhibitory inhibitory ligand. There is a weakness that does not react. Various screening methods for such oGPCR ligand screening have been developed and used by various companies and are already patented. However, since there are problems and disadvantages in measuring oGPCR activity, there is a need for the development of a new system that can measure various activities of GPCR in cell-based systems. will be. It is estimated that the method using adenovirus can effectively overcome these difficulties.

Gene transfer vectors derived from adenoviruses (hereinafter referred to as adenovirus vectors) have many features that are useful for introducing a desired gene into a host specific cell or tissue. For example, the biology of adenoviruses has been characterized in detail, and adenovirus vectors lack replication of the virus itself by deletion in the initial region 1 (E1) of the viral genome, thus providing no pathological factor in humans. . The virus can be produced in large quantities (10 ¹¹ pfu / ml) at high titers (10 ¹¹ pfu / ml), with its DNA inserted into the host cell very efficiently, with a wide range of hosts, and relatively easily at the laboratory level. The El region of the adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcription units, the E1A and E1B genes. The main action of the E1A gene product is to 1) resume the synthesis of DNA in the cell by allowing the stationary cell to enter the cell cycle, and 2) The other initial regions (E2, E3, E4) are activated by the transcription. In most cases, the expression of E1A causes the death of programmed cells and sometimes causes non-limiting proliferation (Jochemsen et al ., EMBO J vol. 6 pp 3399-3405, 1987). Therefore, the recombinant adenoviruses currently used for transduction have been prepared by a method of replacing the E1 region essential for the DNA replication process of the adenovirus with the genetic material desired by the researcher, which supplies a protein in which the recombinant adenovirus is expressed from the E1 region. It is intended to be replicated as a virus only in the packaging cell line, and to prevent replication and virus granulation in other cells. Currently used packaging cell lines include HEK293, 911 cells, C6 cells and the like (Hitt MM et al ., Advances in Pharmacology vol. 40 pp 137-206, 1997). Therefore, adenovirus can be said to be preferable for the preparation of a vector for heterologous cloning and expression.

 In the present invention, the c-fos promoter and the reporter gene were inserted into the E1 region of the adenovirus vector so as to measure the activity of GPCR. Was intended.

In order to detect ligands of oGPCRs and analyze their function, the present inventors can measure the activity of receptors by various ligand candidate groups at high speed and in large quantities, and recombine label genes capable of measuring the activity of GPCRs into adenovirus vectors. Luciferase activity was measured after infecting specific cells with GPCR recombined with adenovirus vectors. As a result, the adenovirus-labeled genes acted specifically for different signaling processes, and thus, the activity of the oGPCR-ligand interaction could be analyzed in a large amount within a short time, thereby completing the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides for the development of signaling processes specific for signaling pathways recombined with adenovirus vectors to discover ligands of oGPCRs. 1 shows a process for recombining a marker gene into an adenovirus vector. Site-specific recombination of a master clone containing the firefly luciferase marker gene into a promoter containing a responsive element that can be finally activated through a signaling process in which the receptor is activated by a ligand. It produced by the method. The prepared master clone is confirmed the correct sequence through sequencing. Cyclic viral genomic DNA is digested with restriction enzymes to produce a linear viral genomic DNA flanked by site-specific recombination sites, followed by in vitro masterclones and site- flanked by site-specific recombination sites. When specific recombination is performed, the target gene, the receptor activity measuring marker gene, is inserted at the E1 region of the adenovirus. When the adenovirus vector recombined with the marker gene is introduced into the HEK293 cell line, which is a packaging cell for supplying the protein expressed from the E1 site, a large amount of virus expressing the marker gene can be obtained while forming a virus plaque. Figures 2a to 2d shows a promoter sequence containing a gene response element (responsive element) that can be finally activated through the signaling process of the marker gene sequence for measuring receptor activity. 2a shows that when GPCR activates G _{q / 11} protein by ligand, phospholipase C (PLC) is reactivated and phospholipase C (protein kinase C, PKC), which is an intracellular secondary transporter, is activated. The c-fos promoter sequence, which is regulated by the signal transducing agents of Gq _{/ 11} and shows the activity of GPCR linked to G _{q / 11} , is indicated by the arrow region of the human c-fos promoter sequence, followed by the luciferase gene. You can see that it is connected. Figure 2b is a promoter sequence with an NF-kB binding site that reacts when various cytokine-like substances are activated by GPCR, and there are five NF-kB binding sites ( GGGGACTTTCC ) underlined in the sequence. . FIG. 2C is a promoter including one SRE sequence ( CC (A / T) ₆ GG ) to which a gene such as SRF (serum response factor) regulated when G _{q / 11} protein is activated by a receptor. to be. Figure 2d is a promoter containing four CRE sequences ( TGACGTCA ) that can bind to genes such as CREB, ATF-2, which are regulated when the G _s protein is activated by the receptor, and luciferase is linked to it. Able to know. The use of the marker gene system introduced into the adenovirus allows the short-term measurement and analysis of various activities of the receptor.

When infected with each signaling process-specific marker gene using U-2OS cell line, which is human osteosarcoma cells, the MOI (multiplicity of expression) can be expressed at high levels without toxicity to the cells. infection, multiples of infection; a unit that represents the number of viruses infected by a single cell, high MOI means that a single cell is infected with multiple viruses, and low MOI means when there are multiple infected cells per virus In order to determine the cell state and expression level of the gene of interest, the MOI used in the experiment was increased.Increase the MOI in the U-2OS cell line in the units indicated at the bottom of the graph X-axis. After infection, each marker-specific agent is treated to give the marker genes specific signaling pathways. It was intended to determine whether the difference in activity level of the marker gene according to whether it works or increases the MOI (Fig. 3). In the case of c-fos-luciferase, NF-kB-luciferase, and SRE-luciferase, the expression level increased according to infection with cell line by increasing MOI, and the signaling process connected with G _{q / 11} protein by receptor It can be seen that the TPA, which is an agent activating the enzyme, was treated with 200 nM (black bar graph) and significantly higher in the activity of each marker gene than the group without the agent (white bar graph) (FIGS. 3A and 3B, 3c). CRE- the same manner even in the case of luciferase with increasing MOI in the cell lines was the expression levels increased with the Sikkim infection of agents that activate signaling pathways associated with protein G _s by forskolin receptor (forskolin, FKN) 10 mM treatment It can be seen that the activity of the marker gene is significantly higher than the group not treated with the agent (Fig. 3d). Therefore, these experimental results suggest that a signaling system specific for each signaling process recombined with the adenoviruses prepared by the present inventors works well at the cellular level, and that a screening system for discovering ligands of oGPCRs using the same may be useful.

Infected marker gene prepared by the inventor with increasing MOI to CV-1 cell line, a kidney cell of African green monkey, a cell line to be used for ligand discovery of oGPCR. Delivery process specificity and MOI levels appropriate for infection with the CV-1 cell line were identified. In the case of CRE-luciferase, the activity of phospholine, which is an agonist, was significantly increased at 50 MOI, while SRE-luciferase was 25 MOI and NF-kB-luciferase. In the case of and c-fos-luciferase it can be seen that it shows a significant activity by the TPA agonist at 50 MOI (Fig. 4a). In order to use the label gene system developed by the present inventors in discovering oGPCR ligands, the inventors recombined dozens of oGPCRs already possessed to adenoviruses in the same manner as the method for producing label genes. When the recombinant oGPCR and the marker gene are infected with the CV-1 cell line together, the inventors have already found that the marker gene is not toxic and that the marker gene works well by receptor-ligand interaction as confirmed by the agent. Cloned by adenovirus and well-known for its function, the second type of receptor, the gonadotropin-releasing hormone (GnRH) of bullfrog, was infected with CV-1 cells along with marker genes. . The second type of GnRH receptor in bullfrog is known to activate both G _{q / 11} and G _s proteins by the ligand GnRH (Oh et al ., Mol Cell Endocrinol vol. 205 pp 89-98, 2003). It can be seen that the activities of CRE-luciferase and SRE-luciferase were both significantly increased compared to those not treated with GnRH (FIG. 4B). Thus, it can be confirmed once again that the marker gene recombined with the adenovirus developed by the present inventors can selectively measure not only the agent in the signaling process but also the activity exhibited by the interaction of the GPCR and its ligand.

In order to discover the ligand of oGPCR and to measure its activity, high-speed / bulk ligand screening was performed using a newly developed adenovirus-labeled gene and oGPCRs recombined with the adenovirus. . Among the 35 adenoviruses currently recombined, oGPCRs belong to the class Rhodopsin subfamily and show approximately 37% identity with the purine P2Y receptor (Lee et al ., Gene vol. 275 pp). 83-91, 2001) When cells were infected with oGPCR, designated GPR92, we found a significant increase in CRE-luciferase activity against E-50% of ligand candidates extracted from 2-month-old fetuses (FIG. 5A). ). In order to confirm this activity more accurately, the change of CRE-luciferase activity was observed by increasing the concentration of the E-50% extract, and it was confirmed that the CRE-luciferase activity by GPR92 increased as the concentration was increased. . On the other hand, for cells not infected with GPR92, there was no effect on the CRE-luciferase response to E-50%. 5B), the E-50% extract was separated and extracted again with finer materials. The six samples thus extracted were treated with GPR92-infected cells, and the sample E50-6 was treated with CRE- by GPR92. It was confirmed that significantly increased luciferase activity (FIG. 5C). Treatment of cells infected with GPR92 with increasing concentrations of E50-6 showed that CRE-luciferase activity was significantly increased only in cells infected with GPR92 as the concentration was increased (FIG. 5D). Therefore, the screening system for discovering ligands of oGPCR using the adenovirus-labeled gene system developed by the present inventor not only works well, but is simpler than other methods developed by other researchers, and various activities of the receptor are not required. It is useful for studying.

The present invention will be explained in more detail with reference to the following examples.

Example

Example 1 Construction of Recombinant Reporter Adenovirus Vectors

BP from Invitrogen (USA) using the CRE: Luc, SRE: Luc, c-fos: Luc, and NFkB: Luc vectors as templates for the production of marker genes recombined with adenoviruses. The first site-specific recombination was carried out using a recombination enzyme called clonase, and the resulting recombinant master clone was subjected to sequencing and then described by Neurogenex (PCT / KR / 03/00453). The recombinant adenovirus was generated. In detail, the linear adenovirus genomic DNA, which binds the terminal protein (TP DNA) at both ends, is treated with restriction enzymes, digested into two fragments, and the master clone is added to the site-specific recombinant enzyme (LR clonase from Invitrogen). When site-specific recombination is performed in vitro with master clones, the target genes CRE: Luc, SRE: Luc, c-fos: Luc, and NFkB: Luc are each inserted into the E1 region of the adenovirus. do. The recombinant adenovirus DNA thus produced is introduced into HEK293 cell line, which is a packaging cell for supplying the protein expressed from the E1 site, and when the adenovirus is propagated, the plaque is purified and then amplified. Luc, Ad-c-fos: Luc, Ad-NFkB: Luc Adenovirus will be obtained in large quantities.

Example 2 Signaling Process Specific Activity Analysis of Recombinant Reporter Adenovirus

When GPCR activates Gq / 11 protein by ligand, phospholipase C (PLC) is reactivated and signal transduction material when phospholipase C (protein kinase C, PKC) is activated. Are regulated by the C-fos promoter, a promoter with an NF-kB binding site, and a SRE site that can bind genes such as a serum response factor (SRF) that is regulated when the G _{q / 11} protein is activated. Activation of the promoter increases expression of the luciferase gene linked to it, and activation of the G _s protein activates a promoter that contains a CRE sequence to which genes such as regulated CREB and ATF-2 can bind. The expression of the luciferase gene is increased. The label developed by the present inventors was measured by measuring the expression level of intracellular luciferase gene in the treatment of a specific signal transduction agent, a known GPCR ligand, and several ligand candidates for screening ligands for oGPCR. Not only does the gene system work well, but also the activity of the receptor can be investigated, which can be useful for screening oGPCR ligands.

2-1: Activity analysis of marker genes after agent treatment for specific signaling processes

Human osteosarcoma cells purchased from the American Cell Line Bank (ATCC) to determine MOIs that can exhibit high levels of marker gene expression without toxicity to cells when infected with each signaling process specific marker gene. U-2OS cell line (ATCC no. HTB-96) and CV-1 cell line, an kidney cell of African green monkey, purchased from Korea Cell Line Bank, were cultured in 96-well plates by 5x103 cell number. The recombinant marker gene was infected with 1-100 MOI / well of the serum-free medium for 3 hours, then changed to a new culture medium containing serum, and then cultured for 24 hours to 48 hours, followed by Ad-SRE: Luc, Ad. -C-fos: Luc, Ad-NFkB: Luc Adenovirus virus infected cells received 200 nM of TPA from Sigma (USA), a signaling pathway agonist linked to Gq / 11 protein, and Ad-CRE: Luc After the no-virus infected cells is treated with a 10 mM forskolin purchased from Sigma Co. (United States) the signaling pathway agonist associated with a G _s protein was examined for six hours luciferase activity.

2-2: Analysis of luciferase activity by GnRH after infection of CV-1 cell line with adenovirus marker gene and bullfrog second type GnRH receptor

In order to use the label gene system developed by the present inventors to discover ligands of oGPCR, the present inventors recombined 35 kinds of oGPCRs already possessed by adenoviruses in the same manner as the method of producing label genes. When the recombinant oGPCR and the marker gene are infected with the CV-1 cell line together, the inventors have already found that the marker gene is not toxic and that the marker gene works well by receptor-ligand interaction as confirmed by the agent. Cloned by adenovirus and well-known for its function, the second type of GnRH receptor 50 MOI of bullfrog, with the marker genes of 50 MOI CRE-luciferase and 25 MOI SRE-luciferase in 1x10 ⁴ cell number After infecting the CV-1 cells in a 96-well plate for 3 hours, the culture medium was changed and cultured for another 48 hours, and then GnRH-2 purchased from Anygen (Gwangju, Korea Gwangju Institute of Science and Technology) Treated for 6 hours and luciferase activity was measured

2-3: Activity analysis of adenovirus marker genes as screening system for ligand discovery of oGPCR

In order to discover the ligand of oGPCR and to measure its activity, high-speed / mass ligand screening was performed using a label gene recombinantly recombined with the adenovirus developed by the present inventors and oGPCRs recombined with the adenovirus currently possessed. After adenovirus oGPCR 50 MOI and the marker gene virus were infected with CV-1 cells cultured in 96-well, the culture was carried out in the same manner as above, and the ligand candidate groups extracted from the two-month-old fetuses were treated for 6 hours. Luciferase activity was measured. Among the ligand candidates, the activity was selected to measure the concentration-dependent luciferase activity, and the fetal extract was further separated and processed for further analysis of the substance in the candidate group, thereby narrowing the scope for oGPCR ligand discovery.

The present invention relates to the development of a fast / bulk ligand screening system using marker genes recombined with adenovirus to discover ligands of oGPCRs and analyze the function of receptors. The development of this technique can be used directly commercially and also for oGPCR, the ligand of which has not yet been identified, and can be used for new ligand discovery studies.

<110> Neurogenex Co., Ltd <120> cfos promoter reporter adenovirus <130> P <160> 1 <170> KopatentIn 1.7 <210> 1 <211> 811 <212> DNA <213> synthetic construct <400> 1 tgcagcccaa gcttgcatgc ctgcaggtcg agcccgaggg ctggaggtta ggggatgaag 60 gtctgcttcc acgctttgca ctgaattagg gctagaattg gggatggggg taggggcgca 120 ttccttcggg agccgaggct taagtcctcg gggtcctgta ctcgatgccg ttctcctatc 180 tctgagcctc agaactgtct tcagtttccg tacaagggta aaaaggcgct ctctgcccca 240 tcccccccga ctcggggaac aagggtccgc attgaaccag gtgcgaatgt tctctctcat 300 tctgcgccgt tcccgcctcc cctcccccag ccgcggcccc cgcctccccc cgcactgcac 360 cctcggtgtt ggctgcagcc cgcgagcagt tcccgtcaat ccctcccccc ttacacagga 420 tgtccatatt aggacatctg cgtcagcagg tttccacggc ctttccctgt agccctgggg 480 ggagccatcc ccgaaacccc tatcttgggg ggcccacgag acctctgaga caggaactgc 540 gaaatgctca cgagattagg acacgcgcca aggcgggggc agggagctgc gagcgctggg 600 gacgcagccg ggcggccgca gaagcgccca ggcccgcgcg ccacccctct ggcgccaccg 660 tggttgagcc cgtgacgttt acactcattc ataaaacgct tgttataaaa gcagtggctg 720 cggcgcctcg tactccaacc gcatctgcag gagcaactga gaagccaaga ctgagccggc 780 ggcctctaga gtcgaactct agaggatctc c 811

Claims (3)

Overlapping the target cells with the recombinant adenovirus of FIG. 6 and the G-protein coupled receptor (GPCR) gene recombinant adenovirus including the c-fos promoter of SEQ ID NO: 1 and luciferase bound thereto; Treating a search drug on the infected target cell; And Biological activity screening method of GPCR activity modulating drugs comprising the step of measuring the activity of luciferase. delete delete

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