JP2016202063A - Screening method of molecular targeted drug for treating neuroblastoma, and kit for screening molecular targeted drug for treating neuroblastoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a screening method and a screening kit of a molecular targeted drug for treating neuroblastoma.SOLUTION: There are provided a screening method of a molecular targeted drug for treating neuroblastoma includes a process of selecting a candidate substance by using suppression of VRK1 function as an indicator, and a kit for screening a molecular targeted drug for treating neuroblastoma in which the kit includes a cell expressing VRK1 and at least one selected from the group consisting of a primer for quantitative PCR for measuring suppression of VRK1 function, an anti-VRK1 antibody, and an antibody that specifically recognizes natural or synthetic peptide being a substrate of VRK1 and a phosphorylation site on the natural or synthetic peptide.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a screening method for a molecular target drug for treating neuroblastoma and a kit for screening a molecular target drug for treating neuroblastoma. In particular, (1) a cell expressing VRK1, preferably a cell that strongly expresses VRK1, is used as an indicator for suppressing the function of VRK1, or (2) the presence or absence of binding to isolated VRK1, The present invention relates to a method for screening a molecular target drug for treating neuroblastoma, and a kit for use in the screening method.

  Neuroblastoma is a type of childhood cancer and is a typical cancer that forms in the abdomen of children. Amplification (50-100 copies) of the MYCN gene belonging to the MYC family is observed in about 20% of cases of neuroblastoma, and this MYCN gene amplification is known to strongly correlate with poor prognosis of patients. .

  If the expression of the MYCN gene is directly suppressed by knockdown or the like, it may act not only on cancer cells but also on normal cells, and a drug that acts directly on the MYCN gene may cause side effects. It is done. Therefore, we focus on the “synthetic lethality”, which is a life phenomenon that results in death only when there is an abnormality in both of the combinations of the two genes, and we know that we will search for new genes and molecular targets that are targets for treatment. (See Patent Document 1).

JP2013-230130A

  As a therapeutic agent for neuroblastoma, cisplatin developed as an anticancer agent for adult cancer is currently used. By the way, neuroblastoma is required to be safer than therapeutic drugs for other diseases due to the nature of childhood cancer. Therefore, it is desired to search for various genes / proteins that can be targets for drug discovery, and to develop molecular target drugs with low toxicity and high therapeutic effect using the genes / proteins.

  In addition, there are many cases where therapeutic drugs on the market target a limited class of molecular species such as enzymes, receptors (GPCRs), ion channels, etc., but Smc2 described in Patent Document 1 above. And CAP-D2 etc. are not proteins having enzyme activity. Therefore, when searching for various genes / proteins that can be targets for therapeutic drugs for neuroblastoma, search for genes / proteins related to enzymes, receptors (GPCRs), ion channels, etc. that are preferred as drug targets. It is desirable.

  The present invention has been made in order to solve the above-described conventional problems, and as a result of extensive research, gene knockdown screening was performed in parallel on two types of neuroblastoma cells of MYCN amplification type and single copy type. By carrying out the above, VRK1 (Vaccinia-related kinase 1) was newly found as an index capable of specifically suppressing MYCN-amplified cell growth by synthetic lethality. And, it is possible to screen a molecular target drug for treating neuroblastoma by using inhibition of VRK1 function as an index or presence or absence of binding to isolated VRK1, and to provide a kit for screening The present invention has been completed.

  That is, an object of the present invention is to provide a screening method for a molecular target drug for treating neuroblastoma and a kit for screening a molecular target drug for treating neuroblastoma.

  The present invention relates to a screening method for a molecular target drug for treating neuroblastoma and a kit for screening a molecular target drug for treating neuroblastoma, as described below.

(1) A screening method for a molecular target drug for treating neuroblastoma,
The screening method comprises:
A step of selecting a candidate substance using suppression of the function of VRK1 as an index,
A screening method comprising:
(2) The step of selecting candidate substances using the inhibition of the function of VRK1 as an index,
The screening method according to (1) above, wherein a candidate substance is administered to a MYCN-amplified neuroblastoma cell, and the inhibition of VRK1 function of the MYCN-amplified neuroblastoma cell is measured.
(3) The screening method according to (1) above, wherein the step of selecting the candidate substance replaces suppression of the function of VRK1 and uses the presence or absence of binding between the isolated VRK1 and the candidate substance as an index.
(4) A kit for screening a molecular targeted drug for treating neuroblastoma,
The kit is
Cells expressing VRK1, and
Primers for quantitative PCR for measuring suppression of VRK1 function, anti-VRK1 antibody, and natural or synthetic peptide serving as a substrate for VRK1 and an antibody that specifically recognizes a phosphorylation site on the natural or synthetic peptide At least one selected from
Including kit.
(5) The kit according to (4), wherein the cell expressing VRK1 is a MYCN-amplified neuroblastoma cell.
(6) The kit is
The kit according to (4) above, comprising a VRK1 protein having an enzymatic activity, a natural or synthetic peptide that serves as a substrate for VRK1, and an antibody that specifically recognizes a phosphorylation site on the natural or synthetic peptide.
(7) A kit for screening a molecular targeted drug for treating neuroblastoma,
The kit is
A kit comprising at least labeled VRK1.
(8) In place of the labeled VRK1,
The kit according to (7) above, comprising at least the isolated VRK1 protein and a labeling reagent for labeling the candidate substance.

  According to the present invention, a molecular target capable of specifically suppressing (killing) MYCN-amplified cell proliferation by using inhibition of VRK1 function as an indicator or presence or absence of binding to isolated VRK1 as an indicator. Drugs can be screened, and kits used for the screening can be provided. Therefore, molecular targeted drugs for treating neuroblastoma can be developed.

  Moreover, since VRK1 is a preferable enzyme (kinase) as a drug discovery target, a molecular target drug can be screened using suppression of the function of VRK1 as an index.

FIG. 1A shows SH-SY5Y, a MYCN single copy type cell, IMR32, an MYCN amplification type cell, and VRK1 gene of NB39 cell, after knocking down the VRK1 gene for 3 days. FIG. 1B is a graph showing the survival rate of SH-SY5Y and IMR32 when left for 5 days. 2 is a graph showing the expression levels of VRK1 genes of SH-SY5Y and IMR32 in Example 2. FIG. FIG. 3 shows the results of Western blotting in Example 3.

  Hereinafter, a screening method for a molecular target drug for treating neuroblastoma of the present invention and a kit for screening a molecular target drug for treating neuroblastoma will be described in detail.

  The method for screening a molecular target drug for treating neuroblastoma of the present invention specifically suppresses (kills) MYCN-amplified cell proliferation, but does not significantly affect MYCN single-copy cells. Is focused on. VRK1 is a kind of serine / threonine kinase, and it has been clarified from previous basic studies that cell cycle progression is regulated by phosphorylating transcription factors such as histone H3 and ATF2. In addition, papers (Kim YS et al., PLoS One, 9 (10), e109655, 2014) that screened VRK1 inhibitors from natural product compound libraries have been reported. However, the present inventors have found for the first time that the suppression of VRK1 expression is involved in the synthetic lethality of MYCN-amplified cells.

  In the present invention, “inhibition of function” means to suppress the function of VRK1 by suppressing the expression of VRK1 itself, and to suppress the function of VRK1 that is expressed but VRK1 is expressed. Moreover, "suppression" means not only complete suppression (that is, inhibition) but also partial suppression. Note that “expression” of VRK1 means gene and / or protein expression unless otherwise specified.

  The screening method of the present invention includes a step of selecting a candidate substance using inhibition of VRK1 function as an index. Candidate substances include, for example, natural compounds, organic compounds, inorganic compounds, single compounds such as proteins, antibodies, peptides, etc., as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermentations Examples include microbial products, marine organism extracts, plant extracts, and the like.

  The gene information, cDNA sequence and amino acid sequence of VRK1 are known, for example, NCBI Reference Sequence Gene ID: 7443 gene information, NCBI Reference Sequence: NM_003384.2 mRNA, and NCBI Reference Sequence_N The amino acid sequence information of can be obtained.

  The screening method of the present invention is particularly effective if the candidate substance is brought into contact with a system capable of directly or indirectly detecting whether or not the function of VRK1 can be suppressed, and changes in the function of VRK1 before and after the contact can be detected. There is no limit. For example, a candidate substance may be brought into contact with a cell expressing VRK1, and then inhibition of the function of VRK1 may be measured.

  Examples of cells in which VRK1 is strongly expressed include MYCN-amplified cells such as IMR32, NB39, TNB1, and LA-N-1. Examples of cells expressing VRK1 include MYCN single copy cells such as SH-SY5Y, SH-EP, SK-N-SH, and SK-N-AS; cells transformed with the VRK1 gene, and the like. Can be mentioned. Alternatively, instead of cells expressing VRK1, a candidate substance may be contacted with isolated VRK1 to detect the presence or absence of binding. From the viewpoint of screening for candidate substances, any of the above-mentioned MYCN-amplified cells, MYCN single-copy cells, cells transformed with VRK1 gene, and isolated VRK1 is not a problem, but MYCN-amplified cells When cells are used, it is preferable because cell death can be finally confirmed in addition to suppression of the function of VRK1.

  Examples of the detection method (suppression of expression) for suppressing the function of VRK1 include quantification of mRNA and protein. mRNA can be quantified by a quantitative PCR method. As the quantitative PCR method, a known method such as SYBR Green method, TaqMan probe method, RT-PCR method or the like can be used. In addition, protein quantification is performed using known methods such as ELISA (enzyme immunoassay), Western blot (WB), immunoprecipitation-western blot (IP-WB), LC-MS, etc. using anti-VRK1 antibody. Can be used. A known method can also be used as a method for detecting the presence or absence of binding between the isolated VRK1 and the candidate substance, for example, ELISA, VRK1 or a method for detecting the presence or absence of binding by labeling the candidate substance. It is done.

  As a method for detecting suppression of the function of VRK1 (inhibition of the function of expressed VRK1), purified VRK1 enzyme may be used, or cells expressing VRK1 may be used. When using a purified VRK1 enzyme, first, a VRK1 protein having enzyme activity and a protein that has been shown to be a substrate of VRK1, for example, a synthetic peptide consisting of an amino acid sequence near the phosphorylation site of histone H3, are mixed. . Next, the phosphorylation reaction by VRK1 is detected using an ELISA method or a time-resolved FRET method using an antibody that specifically recognizes a phosphorylation site on the peptide. This system can be used to detect inhibition of VRK1 function as a candidate substance. A commercially available VRK1 protein may be used. Further, as described above, it is efficient to use a synthetic peptide as a substrate for VRK1, but a natural peptide may be used.

  When cells expressing VRK1 are used, first, a candidate substance is treated in cells expressing VRK1, and a protein that has been clarified to become a substrate of VRK1 by WB using a cell extract, For example, the phosphorylation reaction by VRK1 is detected using an antibody that specifically recognizes the phosphorylation site of histone H3. Alternatively, a cell population with a high rate of phosphorylation by a technique such as flow cytometry is identified and quantified using a phosphorylated antibody against a protein that has been fixed and becomes a substrate of VRK1. . These systems can be used to detect inhibition of VRK1 function as a candidate substance. As described above, a natural peptide may be used as the substrate for VRK1.

  Since the MYCN-amplified cells could be effectively killed by suppressing the function of VRK1, as described in the examples described later, PCR primers capable of detecting the VRK1 gene, antibodies capable of detecting the VRK1 protein, specific to VRK1 A kit for screening a molecular target drug for suppressing the function of VRK1 containing a typical substrate or the like can be provided.

  In the case of a kit for detecting the VRK1 gene, in addition to the PCR primer set that can quantify the expression level of the VRK1 gene and the expression level of the VRK1 gene, the kit can be configured to include enzymes, reagents, etc. that are optimal for quantification . As the primer, any sequence may be used as long as VRK1 can be quantitatively measured. In order to normalize the expression level, a control primer such as a primer for amplifying GAPDH may be included.

  In the case of a kit for detecting the VRK1 protein, it is possible to include a cell that expresses the VRK1, an antibody for detecting the VRK1 protein, a molecule that binds to the VRK1 protein such as an aptamer, and a reagent necessary for detection. Good.

  In the case of a kit that detects inhibition of the function of VRK1 expressed using purified VRK1 enzyme, a VRK1 protein having an enzyme activity, a natural or synthetic peptide that serves as a substrate for VRK1, and a phosphorylation site on the natural or synthetic peptide are specified. In addition to the antibody to be recognized, a configuration including a reagent necessary for detection may be used.

  In the case of a kit for detecting inhibition of the function of VRK1 expressed using cells expressing VRK1, cells expressing VRK1, natural or synthetic peptides serving as substrates for VRK1, and phosphorus on natural or synthetic peptides What is necessary is just to set it as the structure containing the reagent required for a detection other than the antibody which recognizes an oxidation site | part specifically.

In addition, when detecting the presence or absence of binding between the isolated VRK1 protein and the candidate substance, it is sufficient to include at least a reagent for detecting the isolated VRK1 and the binding between VRK1 and the candidate substance. For example,
(1) a reagent for detecting a labeled VRK1 protein labeled with a fluorescent substance, an enzyme, etc., and a labeled VRK1 as necessary, or
(2) Fluorescence for labeling candidate substances, labeling reagents such as enzymes, isolated VRK1 protein, and reagents for detecting the label as necessary,
May be included.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

[Search for genes that specifically suppress cell growth of MYCN amplification by synthetic lethality]
IMR32, a MYCN-amplified cell, was obtained from the JCRB cell bank of the Pharmaceutical Research Institute. SH-SY5Y, a MYCN single-copy cell, was obtained from FIRC Institute of Molecular Oncology, Italy. IMR32 and SH-SY5Y were comprehensively knocked down using a genome-wide shRNA library (manufactured by SIGMA: SHPH01). Next, using next-generation sequencing technology (NGS analysis), IMR32, which is a MYCN-amplified cell, dies or cell growth is suppressed, but the survival of SH-SY5Y, which is a MYCN single-copy cell, is suppressed. As a result of searching for genes that do not have a significant effect, VRK1 was selected as a candidate gene.

[Measurement of VRK1 gene knockdown and cell viability in human neuroblastoma cells]
<Example 1>
It was confirmed by the following procedure whether MYCN-amplified cells could be synthetically killed by suppressing the expression of the selected VRK1.

(1) Cell lines and reagents NB39: Obtained from the Medical Cell Resource Center of Tohoku University Institute of Aging Medicine.
-SiRNA against VRK1: Stealth RNAi siRNA (Life Technologies, HSS111308)
SiRNA introduction reagent: Lipofectamine RNAiMAX Transfection Reagent (Life Technologies, 13778150)
SiRNA dilution medium: Opti-MEM (Life Technologies, 31985-070)
-Medium for IMR32: MEM (Sigma-Aldrich, M4655)
-Medium for SH-SY5Y: MEM + Ham's F12 (1: 1) * Ham's F12 (Life Technologies, 11765-054)
-Medium for NB39: RPMI-1640 (Sigma-Aldrich, R8758)
・ FBS: Biosera, 515-99055
-Tissue culture plate: 6 well (IWAKI, 3810-006)
Fluorescent reagent: Resazurin sodium salt (Sigma-Aldrich, R7017)

(2) Experimental method (a) Dilution of siRNA and complex formation with siRNA introduction reagent First, 500 μl of Opti-MEM was added to each well of a 6-well tissue culture plate. Next, 2 μl of 5 μM siRNA was added to each well, and further 4 μl of siRNA introduction reagent (RNAiMAX) was added to each well. After the reagent addition, the plate was left at room temperature for 15 minutes.
(B) Cell preparation, knockdown of VRK1 gene Human neuroblastoma cell lines SH-SY5Y (MYCN single copy type), IMR32 (MYCN amplification type), and NB39 (MYCN amplification type) were added in their respective compatible media (10 % FBS) and 0.08 × 10 ⁶ cells / ml.
Next, 2.5 ml of the cell suspension prepared to 0.08 × 10 ⁶ cells / ml was added to Opti-MEM containing siRNA and siRNA introduction reagent for VRK1 prepared in (a) above. The tissue culture plate was left in a CO ₂ incubator for 72 hours (3 days). Moreover, about SH-SY5Y and IMR32, what was left to stand for 120 hours (5 days) was also implemented.
(C) Measurement of cell viability Resazurin sodium salt was dissolved in phosphate buffered saline to prepare a 1200 μM solution. Next, 600 μl of Resazurin solution was added to each well of a 6-well tissue culture plate, and then the tissue culture plate was left in a CO ₂ incubator for 1 hour. Next, 120 μl each of the resazurin solution and medium mixture was taken, and the fluorescence intensity was measured using POWERSCAN4 (DS PHARMA BIOMEDICAL) under conditions of an excitation wavelength of 560 nm and a detection wavelength of 590 nm. Since resazurin is converted into a fluorescent substance in viable cells, the fluorescence intensity is proportional to the number of viable cells. Cell viability was determined from the measured fluorescence intensity.

  FIG. 1A shows SH-SY5Y, a MYCN single copy type cell, IMR32, an MYCN amplification type cell, and VRK1 gene of NB39 cell, after knocking down the VRK1 gene for 3 days. FIG. 1B is a graph showing the survival rate of SH-SY5Y and IMR32 when left for 5 days. As shown in FIG. 1A and FIG. 1B, the survival rate of MYCN-amplified cells could be reduced by knocking down VRK1. Moreover, from the survival rate of IMR32 in FIG. 1A and FIG. 1B, it became clear that even when VRK1 is knocked down, cells do not die immediately but gradually die over time.

  In addition, even in MYCN single copy type SH-SY5Y, cell death could be confirmed by knocking down VRK1. Since VRK1 is expressed in actively proliferating cells and is thought to play some role in the progression of the cell cycle, it is considered that the inhibition of VRK1 has somewhat affected MYCN single-copy SH-SY5Y. In addition, the reason why the survival rate of MYCN-amplified cells did not reach 0% was that some cells were not killed even when VRK1 was knocked down when the cell culture time was 72 to 120 hours, and siVRK1 This is probably because some cells were not transfected. From the above results, it has been clarified that VRK1 can be an index for specifically suppressing the growth of MYCN-amplified cells by synthetic lethality.

[VRK1 gene knockdown and measurement of gene expression level in human neuroblastoma cells]
<Example 2>
Next, the gene expression level when the VRK1 gene was knocked down in human neuroblastoma cells was measured using a real-time PCR method.

(1) Reagents, etc./RNA purification reagent: ISOGEN (NIPPON GENE, 311-02501)
CDNA synthesis reagent: ReverseTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, FSQ-301)
-Reagent for rPCR: THUNDERBIRD SYBR qPCR Mix (TOYOBO, QPS-201)
The cell lines and reagents used in the experimental method of Example 2 are the same as in Example 1 except for the above.

(2) Experimental method Knockdown of the VRK1 gene of the cell was carried out in the same procedure as in (2) Experimental method (a) and (b) of Example 1 except that only SH-SY5Y and IMR32 were used as cell lines. went.
Next, 500 μl of ISOGEN per well was added to lyse the cells, and total RNA was purified according to the manual. CDNA synthesis was performed from 0.5 μg of total RNA using RiverTra Ace qPCR RT Master Mix with gDNA Remover. Using this cDNA sample as a template, real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix, and the gene amount of the siVRK1 sample relative to the control sample was calculated. The measuring instrument used was Mx3005P manufactured by Agilent Technologies. Primer sequences for gene detection are as follows.
VRK1_Fw: 5′-GCCTCGTGTAAAAGCAGCTC (SEQ ID NO: 1)
VRK1_Rv: 5′-GTGCATCACTGCCAACTGAC (SEQ ID NO: 2)
GAPDH_Fw: 5′-ATCATCCCTGCCCTCACTGG (SEQ ID NO: 3)
GAPDH_Rv: 5′-CCCTCCGACGCCCTCTTCAC (SEQ ID NO: 4)

  2 is a graph showing the expression levels of VRK1 genes of SH-SY5Y and IMR32 in Example 2. FIG. As shown in FIG. 2, it was revealed that the expression of the VRK1 gene was suppressed in both SH-SY5Y and IMR32 transfected with siVRK1, as compared with the control (siNC).

[VRK1 gene knockdown and measurement of protein content in human neuroblastoma cells]
<Example 3>
Next, the amount of VRK1 protein when the VRK1 gene was knocked down in human neuroblastoma cells was measured using Western blotting.

(1) Reagents, etc. NET-N buffer: 20 mM Tris-HCl pH = 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40
Protein concentration measurement kit: Pierce BCA Protein Assay Kit (Thermo Scientific, 23225)
The cell lines and reagents used in the experimental method of Example 3 are the same as in Examples 1 and 2 except for the above.

(2) Experimental method In the same procedure as in Example 2, knockdown of the VRK1 gene of the cells was performed. Next, the collected cells were dissolved in NET-N buffer, and the protein concentration was measured using Pierce BCA Protein Assay Kit. Electrophoresis was performed such that the total protein amount per well of a 10% polyacrylamide gel was 10 μg. As an antibody,
Anti-VRK1 antibody: SIGMA, HPA000660
Monoclonal Anti --- Actin antibody: SIGMA, A5441
Western blotting was performed by a conventional method. Further, β-actin was used as a control.
Next, using an image analyzer LAS-4010 (GE Healthcare), the band intensity of the obtained image was quantified by the attached ImageQuant TL.

  FIG. 3 shows the results of Western blotting in Example 3. As is clear from FIG. 3, compared to the control (siNC), the SH-SY5Y and IMR32 transfected with siVRK1 both had about half the amount of VRK1 protein.

  From the results of Examples 1 to 3, it was revealed that only MYCN-amplified cells can be specifically killed by suppressing the expression of VRK1. Therefore, by using inhibition of VRK1 function as an index, it is possible to screen for a molecular target drug for treating neuroblastoma.

  By using the method for screening a molecular target drug for treating neuroblastoma of the present invention and a kit for screening a molecular target drug for treating neuroblastoma, neuroblastoma can be specifically suppressed. Molecular targeted drugs can be screened. Therefore, it is useful for the drug discovery industry.

Claims (8)

A screening method for a molecular targeted drug for treating neuroblastoma, comprising:
The screening method comprises:
A step of selecting a candidate substance using suppression of the function of VRK1 as an index,
A screening method comprising: The step of selecting candidate substances using the inhibition of the function of VRK1 as an index,
The screening method according to claim 1, wherein a candidate substance is administered to MYCN-amplified neuroblastoma cells, and inhibition of VRK1 function of the MYCN-amplified neuroblastoma cells is measured.   The screening method according to claim 1, wherein the step of selecting the candidate substance uses, as an index, the presence or absence of binding between the isolated VRK1 and the candidate substance instead of suppressing the function of VRK1. A kit for screening a molecular targeted drug for treating neuroblastoma,
The kit is
Cells expressing VRK1, and
Primers for quantitative PCR for measuring suppression of VRK1 function, anti-VRK1 antibody, and natural or synthetic peptide serving as a substrate for VRK1 and an antibody that specifically recognizes a phosphorylation site on the natural or synthetic peptide At least one selected from
Including kit.   The kit according to claim 4, wherein the cell expressing VRK1 is a MYCN-amplified neuroblastoma cell. The kit is
The kit according to claim 4, comprising a VRK1 protein having enzyme activity, a natural or synthetic peptide that serves as a substrate for VRK1, and an antibody that specifically recognizes a phosphorylation site on the natural or synthetic peptide. A kit for screening a molecular targeted drug for treating neuroblastoma,
The kit is
A kit comprising at least labeled VRK1. Instead of the labeled VRK1,
The kit according to claim 7, comprising at least the isolated VRK1 protein and a labeling reagent for labeling the candidate substance.