KR102043303B1 - Method for quantitative analysis of SIRT7-deacetylation activity and method for screening SIRT-7 inhibitors using the same - Google Patents
Method for quantitative analysis of SIRT7-deacetylation activity and method for screening SIRT-7 inhibitors using the same Download PDFInfo
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Abstract
The present invention relates to a method for quantitating the deacetylation activity of SIRT7 protein using Fluorescence polarization and to a screening method of an inhibitor that inhibits the activity using the method, and more specifically, histone H3 protein having a fluorescent probe attached thereto. After preparing a peptide containing the acetylated lysine No. 18, the method of quantitative analysis of the formation of the conjugate by inhibiting the binding pattern of the fluorescently labeled peptide and SIRT7 by polarized fluorescence value and inhibited the deacetylation activity of SIRT7 using the same It relates to a method for screening inhibitors.
The method according to the present invention provides a new method for quantitatively analyzing the activity of SIRT7 protein, an important mechanism for cancer cells, and thus can be usefully used for screening various drugs that inhibit binding of anticancer agents or differentiation inducing agents. have.
Description
The present invention relates to a method for quantitatively analyzing the deacetylation activity of SIRT7 protein using Fluorescence polarization and a screening method for inhibitors that inhibit the activity using the method, and more specifically, histone H3 having a fluorescent probe attached thereto. After preparing the protein, the method of quantitatively analyzing the deacetylation activity of the SIRT7 protein by measuring the binding pattern between the fluorescently labeled protein and the SIRT7 by polarization fluorescence value, and the screening method of the inhibitor inhibiting the deacetylation activity of the SIRT7 using the same It is about.
SIRT (sirtuin) has been shown to be related to aging in various species from yeast and has nicotinic acid amide (NAD) -dependent deacetylation activity. To date, a total of seven species have been identified, from Sirt1 to Sirt7. Most of them are involved in aging, metabolic diseases and gene stability. In 1992, the yeast function was found to extend yeast lifespan by extending the DNA end, and the subject was extended to vertebrates and related to metabolic diseases. Sirt1 was found to inhibit appetite when the activity was inhibited, and it has been reported that sirt1 expression is reduced in fat cells of obese model rats. Indeed, model rats overexpressed sitrt1 inhibited the production of adipocytes and increased fat degradation. In fact, sirt1 is expressed twice as much in subcutaneous fat of skinny women than obese women. Sirt2 and Sirt3 are also involved in adipocytes and lipolysis. Sirt4 is highly expressed in beta cells and has been reported to be involved in insulin secretion. sirt6 is involved in glucose degradation and mitochondrial function. In fact, mice genetically removed from sirt6 are born with low blood sugar and die early.
In the case of SIRT7, its function was revealed in 2012. SIRT7 is a class III hisotone deacetylase that is dependent on NAD + to deacetylate histones and target proteins to regulate apoptosis, stress response, DNA repair, cell cycle and metabolism, and is associated with cancer, diabetes and heart disease.
SIRT7 is mainly present in the nucleus, binds to the rRNA gene (rDNA), activates RNA polymerase I to regulate ribosomal RNA (rRNA), and is associated with thyroid, breast and liver cancers. (Houtkooper RH et al., Sirtuins as regulators of metabolism and healthspan.Nat Rev Mol Cell Biol 2012; 13: 225-238 .; Bosch-Presegue L et al., The dual role of sirtuins in cancer.Genes Cancer 2011; 2 : 648-662 .; Ford E et al., Mammalian
Sir2 homolog SIRT7 is an activator of RNA polymerase I ranscription.
Genes Dev 2006; 20: 1075-1080 .; Grob A et al., Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis. J Cell Sci 2009; 122: 489-498 .; De Nigris F et al., Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues. Br J Cancer 2002; 87: 1479 .; Ashraf N et al. Altered sirtuin expression is associated with node-positive breast cancer. Br J Cancer 2006; 95: 1056-1061.)
SIRT7 deacetylates the 18 lysine residue of histone H3 and contributes to tumorigenesis by inhibiting the expression of tumor suppressor genes such as COPS2 and NME1. (Matthew F. Barber et al., SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.Nature 2012; 487: 114-120.)
SIRT7 specifically deacetylates lysine 18 (H3K18) of histone H3 protein. H3K18 is known to be involved in the cancerous process of human cells caused by viruses. For this reason, SIRT7 regulates the expression of NME1 CIPS2 RPS20, a cancer-related gene, and furthermore, it has been found that cancer cell growth is inhibited when SIRT7-specific siRNA is treated to cancer cells. Therefore, by inhibiting the activity of SIRT7 it can inhibit the growth and production of cancer.
Therefore, quantitative analysis of the deacetylation activity of SIRT7 is very important for cancer-related life phenomena research, and these binding or inhibitory assays are essential for the development of disease treatments.
It is an object of the present invention to provide a method for simple and accurate quantitative analysis of deacetylation activity of SIRT7, and a method for screening an inhibitor that inhibits activity using the analytical method.
The present invention to achieve the above object, a) attaching a fluorescent material to the acetylated histone H3 protein to prepare a fluorescent probe; b) reacting the fluorescently labeled acetylated histone H3 protein with SIRT7 protein; c) reacting an antibody recognizing the acetylated histone H3 protein with the reactant of step b); And d) measuring a fluorescence polarization value of the reactant of step c) and comparing the fluorescence polarization value of the fluorescence probe itself to analyze a change in the fluorescence polarization value; It provides a method for quantitatively deacetylating activity of SIRT7 comprising a.
Preferably, the protein of step a) is an acetyl group bound to lysine No. 18 of the histone H3 protein, and the probe of step a) has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The fluorescent material may be attached to any one or more of the N- terminal and C- terminal of the H3 protein. In step b), the fluorescently labeled histone H3 protein and SIRT7 protein are preferably mixed in a molar ratio of 2: 1 to 50: 1.
Phosphors that can be labeled on the probes are fluoresceinamide (FAM), fluoresceincarboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxy Coumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6- It is preferably selected from the group consisting of one.
In addition, the present invention to achieve the above object, a) a step of reacting a mixture of SIRT7 protein and inhibitor candidate; b) reacting by adding a fluorescent probe having a fluorescent substance attached to the histone H3 protein acetylated to the reactant of step a); c) b) reacting the reactant by mixing an antibody recognizing the acetylated histone H3 protein; d) measuring the fluorescence polarization value of the reactant of step c), and determining the deacetylation activity inhibitor when the fluorescence polarization value of step c) is high compared to the fluorescence polarization value of the fluorescent probe itself; It provides a method for screening an inhibitor that inhibits the deacetylation activity of SIRT7.
Preferably, the protein of step a) is an acetyl group bound to lysine No. 18 of the histone H3 protein, and the probe of step b) has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The fluorescent substance may be attached to any one or more of the N- terminal and C- terminal of the H3 protein. In step b), the fluorescently labeled histone H3 protein and SIRT7 protein are preferably mixed in a molar ratio of 2: 1 to 50: 1.
In another aspect, the present invention provides a pharmaceutical composition for treating cancer containing an inhibitor that inhibits the deacetylation activity of SIRT7 as an active ingredient. The inhibitor is to inhibit the binding of the SIRT7 protein and histone H3 protein to inhibit the activity of SIRT7 to leave an acetyl group in the histone H3 protein, and to increase the expression of tumor suppressor genes such as COPS, NME1. It is characterized in that any one of liver cancer, thyroid cancer and breast cancer.
Hereinafter, the present invention will be described in more detail.
The present invention comprises the steps of: a) attaching a fluorescent substance to the acetylated histone H3 protein to prepare a fluorescent probe; b) reacting the fluorescently labeled acetylated histone H3 protein with SIRT7 protein; c) reacting an antibody recognizing the acetylated histone H3 protein with the reactant of step b); And d) measuring a fluorescence polarization value of the reactant of step c) and comparing the fluorescence polarization value of the fluorescence probe itself to analyze a change in the fluorescence polarization value; It provides a method for quantitatively deacetylating activity of SIRT7 comprising a.
SIRT-7 is a protein that binds to the acetyl group of lysine No. 18 of the histone H3 protein and deacetylates it, and the deacetylation test did not affect the acetyl group of other histone proteins. Therefore, the histone H3 protein of step a) is an acetyl group bonded to lysine 18 (H3K18Ac).
In particular, it is preferable that the histone H3 protein to which the acetyl group is bound has the sequence shown in Table 1 below.
The sequence of the histone H3 protein in which the fluorescent substance FAM is attached to the N-terminus and the acetyl group (Ac) is bound is shown in Table 2 below.
The fluorescent probe of the present invention binds specifically to SIRT7 protein with histone H3 protein labeled with a fluorescent dye to measure the fluorescence polarization value. Fluorescent probes have an inverse that can hybridize with histone H3 proteins and can normally be appropriately adjusted in length depending on the target protein and can be shorter or longer. The present invention preferably may be a probe having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and any amino acid sequence may be included in addition to the amino acid sequence as long as it does not affect the binding ability of SIRT7 and histone H3.
In addition, the fluorescent material may be attached to any one or more of the N- terminal and C- terminal of the H3 protein, and preferably is attached to the N- terminal.
Phosphors that can be labeled on the probes are fluoresceinamide (FAM), fluoresceincarboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxy Coumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6- It is preferably selected from the group consisting of one.
In the reaction of step b), the fluorescent probe-attached protein and the SIRT7 protein are preferably reacted by mixing 2: 1 to 50: 1 in an appropriate buffer solution. When the protein attached to the fluorescent probe is mixed less than 2: 1 may not be sufficient binding reaction, if more than 50: 1 mixed reaction occurs irrespective of the amount of the protein, the additional amount may be unnecessary.
The fluorescent probe may be synthesized by selecting a specific sequence including a site that binds to SIRT7 among known sequences of histone H3, and may be prepared by labeling a fluorescent material at the end thereof. Preparation or selection of probes can be facilitated by those skilled in the art and can be modified using known conditions.
In addition, antibodies that recognize the acetylated histone H3 protein of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.
In the present invention, an antibody means a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to an acetylated histone H3 protein, which is encoded by the marker gene by cloning each gene into an expression vector according to conventional methods. Proteins can be obtained and prepared from conventional proteins by conventional methods.
After the binding reaction between the SIRT7 protein and the histone H3 using the fluorescent probe of step a), the fluorescence polarization value of the reactant may be measured to quantitatively analyze the presence or absence of the binding. The measurement of the fluorescence polarization value is not particularly limited, and a fluorescence microscopy, a fluorescence spectrometer or a well plate based well plate reader may be used. When the light of a specific wavelength is exposed, it absorbs the light and emits light of longer wavelength with lower energy, so the wavelength of the tube of the fluorescent microscope can be changed according to the fluorescent dye.
Fluorescent probes of the histone H3 protein to which the fluorescent substance is attached have relatively small fluorescence polarization values, but when combined with an antibody that recognizes the acetylated histone H3 protein, the conjugate is slowed to move relative. It has a large fluorescence polarization value. Since the SIRT7 protein deacetylates the acetylated histone H3 protein, an antibody that recognizes the acetylated histone H3 protein cannot bind and has a small fluorescence polarization value. Thus, the changes in the fluorescence polarization values can be used to analyze the formation of conjugates between histone H3 protein and SIRT7 protein. For example, when there is little change in the fluorescence polarization value, it can be seen that SIRT7 is bound and deacetylated, and when it is significantly increased, it can be analyzed that they are not bound to form a binder. Therefore, by analyzing these changes in the fluorescence polarization value, the formation of the conjugate can be predicted, and as a result, the presence or absence of deacetylation activity of SIRT7 can be quantitatively analyzed.
In addition, the present invention comprises the steps of a) mixing and reacting the SIRT7 protein and the inhibitor candidate; b) reacting by adding a fluorescent probe having a fluorescent substance attached to the histone H3 protein acetylated to the reactant of step a); c) b) reacting the reactant by mixing an antibody recognizing the acetylated histone H3 protein; d) measuring the fluorescence polarization value of the reactant of step c), and determining the deacetylation activity inhibitor when the fluorescence polarization value of step c) is high compared to the fluorescence polarization value of the fluorescent probe itself; It provides a method for screening inhibitors that inhibit the deacetylation activity of SIRT7.
If the inhibitor candidate binds to the SIRT7 protein and inhibits the deacetylation activity of the protein, the antibody does not deacetylate the acetylated histone H3 protein. Will combine. As a result, since the fluorescence polarization value is higher than the fluorescence polarization value of the fluorescence probe itself, since the fluorescence polarization value is high, the deacetylation reaction does not occur, so it may be determined as an inhibitor.
Preferably, the protein of step a) is an acetyl group bound to lysine No. 18 of the histone H3 protein, and the probe of step b) has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The fluorescent substance may be attached to any one or more of the N- terminal and C- terminal of the H3 protein. In step b), the fluorescently labeled histone H3 protein and SIRT7 protein are preferably mixed in a molar ratio of 2: 1 to 50: 1.
Phosphors that can be labeled on the probes are fluoresceinamide (FAM), fluoresceincarboxylic acid (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxy Coumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6- It is preferably selected from the group consisting of one.
Although not particularly limited to the inhibitor candidate of step b), it is preferable to use an antibody, peptide, oligonucleotide, or synthetic compound capable of competitively attaching to the histone H3 protein to which the fluorescent substance is attached to inhibit the binding of SIRT7 protein. Do.
In addition, the present invention provides a pharmaceutical composition for treating cancer, which contains an inhibitor of binding of SIRT7 protein and histone H3 protein as an active ingredient. The inhibitor inhibits the binding of SIRT7 protein and histone H3 protein to inhibit the activity of SIRT7 and the acetyl group in histone H3 protein to promote the expression of tumor suppressor genes such as COPS and NME1. Preferably, the cancer is liver cancer, It is characterized by any one of thyroid cancer and breast cancer.
The inhibitor may be administered as the sole active pharmaceutical agent, but may also be used in combination with one or more inhibitors of the invention or other agents. When administered in combination, the therapeutic agents may be formulated as separate compositions administered simultaneously or sequentially at different times, or provided as a single composition. Administration of such inhibitors may be combined with additional therapies known to those of skill in the art in anticancer agent treatment, such as radiation therapy or administration of cytostatic agents or cytotoxic agents.
The present invention provides a new method for quantitatively analyzing the activity of SIRT7 protein, which is an important mechanism for cancer cells, and thus may be usefully used for screening various drugs that inhibit binding of anticancer agents or differentiation inducing agents.
1 shows a reaction step of an antibody that recognizes a fluorescent probe, a SIRT7 protein, and an acetylated histone H3 protein by attaching a fluorescent substance to the acetylated histone H3 protein.
2 is a graph showing absorbance values of Example 2. FIG.
Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely examples and are not limited to the examples.
Example 1 Preparation of Fluorescent Probe
After synthesis of the histone H3 protein of SEQ ID NO: 1, an acetyl group was bound to lysine 18, and a fluorescent probe was prepared by attaching a fluorescent substance FAM to the N-terminus. SIRT7 protein (SignalChem, Cat. No. S41-30H) was prepared.
The acetylated histone H3 protein labeled FAM (FAM-Histone H3 Lys18-Ac, SEQ ID NO: 1) has the sequence of FAM-RKSTGGKAPRK (-Ac) QLATKAARK.
Example 2 Reactivity Analysis of FAM-labeled Acetylated Histone H3 Protein (FAM labeled Histone H3 Lys 18-acetyl) and Antibody
Acetyl FAM-labeled in 384 well plates (Corning, Cat.No. 3676.) in buffer (50 mM Tris-Hcl pH8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 5 mM DTT, 0.05% BSA) The purified histone H3 protein was immobilized at 10 nM and 1 specific antibody (anti-acetyl Histone H3 (Lys 18), Millipore, Cat. No. 07-354) that recognizes the 18 Lysine residue of acetylated histone H3 was detected. After dilution with / 200, the reaction mixture was shaken at a low speed for 2 hours at room temperature, and the fluorescence value was measured at an excitation wavelength of 493 nm and an emission wavelength of 518 nm.
In addition, 10 nM of acetylated histone H3 protein labeled with FAM and acetylated histone H3 protein labeled with biotin instead of FAM were added to the buffer solution together with 20 nM and 40 nM to recognize the acetylated histone H3 protein No. 18 Lysine residue. Specific antibodies were diluted and added at 1/200 and then reacted at low speed for 2 hours at room temperature. Fluorescence values were measured at an excitation wavelength of 493 nm and an emission wavelength of 518 nm.
In Example 1, the amount of protein of histone H3 labeled with biotin as a competitor with the protein of histone H3 labeled with FAM was performed in the same manner as in Table 2 below. Absorbance values were measured as shown in Table 3 below.
K18-
(competitor)
The table shows that the absorbance values of the FAM-Histone H3 K18 protein, the fluorescent probes, were fixed at 10 nM and the same as the fluorescent probes, but the biotin-binding protein (competitor) was increased by 2 times and 4 times. . As can be seen from the table above, the absorbance value was 5.2434 without the antibody, but the absorbance value was increased to 130.2664 when the antibody was added. In addition, when the biotin-binding protein was added to the competitor, the absorbance value decreased according to the content. Figure 2 shows the absorbance values in Table 3 graphically.
From the above results, it was confirmed that the reaction between the protein and the antibody was specific.
The above description is merely illustrative of the technical idea of the present invention, and those skilled in the art to which the present invention pertains may make various modifications and changes without departing from the essential characteristics of the present invention. Therefore, since the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, the scope of the technical idea of the present invention will not be limited to the present embodiment. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent to the technical configuration described in the claims should be interpreted as belonging to the scope of the present invention.
<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY
<120> Method for quantitative analysis of SIRT7-deacetylation activity
and method for screening SIRT-7 inhibitors using the same
<130> P13020910254
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<170> KopatentIn 2.0
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<223> amino acid sequence of Histone H3 peptide
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Arg Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys Gln Leu Ala Thr Lys
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| Kim JK et al., 'Sirtuin7 oncogenic potential in human hepatocellular carcinoma and its regulation by the tumor suppressors MiR-125a-5p and MiR-125b', Hepatology, (2013.02.11.), Vol. 57, pp* |
| Matthew F. Barber et al., 'SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation', Nature, 2012, Vol. 487, pp 114-120. 1부.* |
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