A method for screening drugs targeting histone deacetylase SIRT6 regulators

Abstract

The present invention belongs to the field of biomedical technology and discloses a method for screening drugs targeting histone deacetylase SIRT6 regulators. Based on testing the affinity between SIRT6 protein and small molecules with SYPRO Orange Protein Gel Staining Solution, this method constructs a drug screening model to screen for SIRT6 regulator-targeting drugs. The drug screening model constructed by the present invention is applicable to screening monomeric compound drugs with strong affinity effects (including agonists and inhibitors) on histone deacetylase SIRT6, and the action sites of the screened compounds are clear; the screening of monomeric compound drugs using this drug screening model has the advantages of high throughput, small protein consumption, low concentration, low cost, strong specificity, and convenient operation, providing a new and effective option for the screening of histone deacetylase SIRT6 regulators in this field, having good application prospects, and successfully screening out monomeric compound drugs with strong targeting effects on SIRT6 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a method for screening drugs targeting histone deacetylase SIRT6 regulators Background Art

[0002] Sirtuins are NAD+-dependent histone lysine deacetylases that are involved in regulating many physiological processes in prokaryotes and eukaryotes, such as the cell cycle, metabolism, stress response, and aging processes. There are seven sirtuins in mammals (i.e., SIRT1 - SIRT7), which differ in their subcellular localization and the substrate proteins they deacetylate. Among these sirtuin family members, SIRT6 is particularly important. The basic functions of SIRT6 include hydrolyzing long-chain acylated substrates, catalyzing the deacetylation of acetyl-lysine at positions 9, 18, and 56 of histone H3 (H3K9ac, H3K18ac, and H3K56ac, respectively), and are related to many physiological and pathological phenotypes. Activation of SIRT6 function may play an important role in extending lifespan and intervening in related diseases, while inhibition of SIRT6 function may also play an important role in the treatment of leukemia. A recent study showed that SIRT6 is responsible for more efficient DNA double-strand break repair in long-lived species, and an increase in SIRT6 activity corresponds to a longer lifespan. Many studies have also shown that SIRT6 is involved in various diseases, such as neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. Notably, SIRT6 has been shown to be a tumor suppressor gene in some cancers, which means that activation of SIRT6 may have potential value in the treatment of these cancers [1, Sebastián, C.; Zwaans, B.M.; Silberman, D.M.; Gymrek, M.; Goren, A.; Zhong, L.; Ram, O.; Truelove, J.; Guimaraes, A.R.; Toiber, D. The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism. Cell 2012, 151, 1185 - 1199; 2, Bhardwaj, A.; Das, S. SIRT6 deacetylates PKM2 to suppress its nuclear localization and oncogenic functions. Proc. Natl. Acad. Sci. 2016, 113, E538 - E547; 3, Ghosh, S.; Liu, B.; Wang, Y.; Hao, Q.; Zhou, Z. Lamin A is an endogenous SIRT6 activator and promotes SIRT6-mediated DNA repair. Cell Rep. 2015, 13, 1396 - 1406.].For example, SIRT6 has been found to be crucial for suppressing pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies [Kugel, S.; Sebastián, C.; Fitamant, J.; Ross, K. N.; Saha, S. K.; Jain, E.; Gladden, A.; Arora, K. S.; Kato, Y.; Rivera, M. N. SIRT6 suppresses pancreatic cancer through control of Lin28b. Cell 2016, 165, 1401-1415.]. Inactivation of SIRT6 promotes PDAC progression and metastasis by upregulating Lin28b, a negative regulator of let-7 microRNA. Knockout of the SIRT6 gene results in histone hyperacetylation and c-Myc recruitment at the Lin28b promoter and significantly induces the expression of Lin28b and downstream let-7 target genes, including IGF2BP1 and IGF2BP3. In contrast, SIRT6 activation may inhibit PDAC progression and reduce metastasis formation by downregulating Lin28b. Therefore, SIRT6 activation is considered a promising target for the treatment of PDAC. The development of SIRT6 modulators has become a current research hotspot.

[0003] You et al. reported the first synthetic SIRT6 activator, pyrrolo[1,2-a]quinoxaline derivative (UBCS039), which binds to the fatty acyl substrate site and the distal region of the "C-site" of SIRT6. The EC 50 of this compound for SIRT6 is 38 μM [You, W.; Rotili, D.; Li, T. M.; Kambach, C.; Meleshin, M.; Schutkowski, M.; Chua, K. F.; Mai, A.; Steegborn, C. Structural basis of sirtuin 6 activation by synthetic small molecules. Angew. Chem. Int. Ed. 2017, 56, 1007-1011]. Recently, Huang et al. disclosed a new cell-active SIRT6 activator, methyl 2-(N-(5-bromo-4-fluoro-2-methylphenyl)sulfonyl)-5-(3,5-dichlorobenzenesulfonamide)benzoate (MDL-800), whose EC 50The value is 10.3 μM [Huang, Z.; Zhao, J.; Deng, W.; Chen, Y.; Shang, J.; Song, K.; Zhang, L.; Wang, C.; Lu, S.; Yang, X. Identification of a cellularly active SIRT6 allosteric activator. Nat. Chem. Biol. 2018, 14, 1118 - 1126.]. MDL-800 has significant activity in reducing the levels of H3K9ac and H3K56ac in human hepatocellular carcinoma (HCC) cells. Apparently, from the perspective of a good drug candidate, the potency of these compounds is not sufficient. Therefore, more effective and selective SIRT6 activators with new scaffolds are needed.

[0004] However, the current methods for screening SIRT6 modulators are not yet widespread. Moreover, the current method for screening lead compounds is the fluorescence-labeled lysine modification (FLUOR DE LYS, FDL) test method of histone acylation substrates labeled with 7-amino-4-methylcoumarin (AMC) and the fluorescence resonance energy transfer (FRET) of a pair of fluorescent groups 4-(dimethylaminoazo)benzene-4-carboxylic acid / 5-((2-Aminoethyl)aminonaphth-alene-1-sulfonicacid) [4-(dimethylaminoazo)benzene-4-carboxylic acid / 5-((2-Aminoethyl)aminonaphth-alene-1-sulfonicacid), DABCYL / EDANS] [1, Kokkonen P, Rahnasto-Rilla M, Mellini P, Jarho E, Lahtela-Kakkonen M, Kokkola T. Studying SIRT6 regulation using H3K56 based substrate and small molecules. Eur J Pharm Sci. 2014 Oct 15;63:71-6; 2. Li Y, You L, Huang W, Liu J, Zhu H, He B. A FRET-based assay for screening SIRT6 modulators. Eur J Med Chem. 2015;96:245-9..]. Since fluorescent groups are required in the FDL and FRET methods, the synthesis method of this substrate is difficult, the cost is expensive, and problems such as the dropping of fluorescent groups during improper storage interfering with the test results may occur. Therefore, there is an urgent need to develop a test method with the advantages of high throughput, low protein consumption, low concentration, low cost, strong specificity, convenient operation, and low price for screening modulators of histone deacetylase SIRT6. Summary of the Invention

[0005] To solve the above technical problems, the first object of the present invention is to construct a drug screening model targeting SIRT6 based on labeling histone deacetylase SIRT6 protein with SYPRO Orange Protein Gel Staining Solution.

[0006] The second object of the present invention is to screen out monomeric compound drugs with high affinity for SIRT6 from the compound library.

[0007] To achieve the object of the present invention, a drug screening model is constructed based on testing the affinity between SYPRO Orange protein gel staining solution and SIRT6 protein and small molecules, and drugs targeting SIRT6 regulators are screened.

[0008] The drug screening model targeting SIRT6 is constructed according to the following method:

[0009] (1) Extract and purify SIRT6 protein by using the conventional method for preparing membrane proteins

[0010] Escherichia coli E. coli M15 [pREP4] carrying the pQE80L.1 (the internal pQE80 derivative has a TEV protease cleavage site) or pET151-D-TOPO recombinant plasmid is cultured in a shaker, induced to express, incubated on an ice shaker, and then broken to collect the cell membrane precipitate. The crude extract of SIRT6 protein is separated, and the pure product of SIRT6 protein is obtained by metal ion chelating chromatography and size exclusion chromatography, and the solution components required for storage and testing are optimized.

[0011] (2) Determine the types of dyes required in the differential scanning fluorimetry (DSF) in the present invention

[0012] and the concentration of SYPRO Orange protein gel staining solution

[0013] According to the types of dyes, 5 candidate dyes are selected for comparison, as shown in Table 1.

[0014] Table 1

[0015]

[0016] (3) Select the crystal structure of SIRT6 protein, determine the active screening pocket, and perform docking screening on the prepared virtual compound library.

[0017] (4) According to the solubility of the solid compound and the DMSO tolerance ability of SIRT6 protein, determine the DMSO concentration in the compound to meet the screening conditions.

[0018] (5) Construct a drug screening model targeting histone deacetylase SIRT6 and evaluate the drug screening model

[0019] According to the property that SYPRO Orange protein gel staining solution can bind to SIRT6 protein, as the temperature increases, the fluorescence increases accordingly. After the protein is completely denatured and unable to bind to the fluorescent dye, the fluorescence intensity decreases as the temperature rises. If the monomeric drug compound added to the above system can bind to SIRT6, its stability will be affected, and the critical temperature of protein denaturation will increase, thus causing the fluorescence intensity of the system to change at different critical points with temperature variation. Based on this principle, a drug screening model can be constructed for screening SIRT6 protein regulators. Investigate the effects of conditions such as solvents and buffers on the drug screening model.

[0020] (6) Screening of monomeric drug compounds with strong binding ability to SIRT6

[0021] The main principle of this method is: in a suitable buffer solution for the protein, the protein and its surrounding environment can form a relatively stable thermodynamic system. If there is a change in the thermodynamic energy (i.e., Gibbs free energy difference u G) that alters the equilibrium state of this system, then the stability of the protein will decrease. When the temperature rises, u G will decrease, and the protein will undergo partial unfolding, causing the stability of most proteins to decrease with increasing temperature. When u G = 0, an equal amount of reversible equilibrium state will form between the normally folded part and the unfolded part of the protein, and the corresponding temperature at this time is the melting temperature (melting temperature, T m ). Normally, the protein is in a folded state in the corresponding buffer solution, with hydrophilic amino acids exposed to the environment and hydrophobic amino acids hidden inside. As the environmental temperature increases, the structure of the protein is damaged, and after unfolding occurs, the hydrophobic amino acids of the protein will be exposed. When the buffer solution contains a fluorescent dye with hydrophobic affinity, the two will gradually bind, causing the fluorescence signal intensity of the reaction system to increase. At a certain temperature, the unfolded proteins will gradually aggregate, preventing the fluorescent dye from binding, and the excess fluorescent dye will be quenched, causing the fluorescence signal intensity of the system to decrease. By detecting the change in fluorescence intensity, the T m value of the protein can be calculated. The change in the T m value (T m ) can reflect the binding strength between the protein and the ligand (such as small molecule compounds), that is, the binding of small molecule ligands usually stabilizes the protein structure and increases the T m value of the protein.

[0022] Add the monomeric drug compound to be screened to the above drug screening model, and calculate the melting temperature (melting temperature, T m ) of the monomeric drug compound to SIRT6 based on the change in the fluorescence intensity of the system. According to Tm Based on the difference in size, monomeric compounds with strong binding to SIRT6 protein were screened out. Compounds MDL-800 of equal volume were used to replace the candidate monomeric compounds as the positive control group, and DMSO of equal volume was used to replace the monomeric compounds as the negative control group, and the melting temperature T m was measured. According to the Boltzmann sigmoid equation, the T m value of the sample protein was calculated. Based on the T m values of the solvent control group and the compound test group, the stabilizing ability of each compound to SIRT6 protein was calculated.

[0023]

[0024] ΔT m = V 50 (compound) - V 50 (DMSO) (2)

[0025] This equation describes the change of fluorescence intensity with temperature, which describes the fluorescence intensity (Y) as a function of temperature (X). The fluorescence intensity from "bottom" to "top" is different. V50 is the temperature at which the fluorescence intensity is between "bottom" and "top", that is, T m . ΔT m represents the difference in melting temperature between the melting temperature of SIRT6 protein stability after the monomeric compound binds to SIRT6 and the melting temperature of the negative control group with DMSO of equal volume replacing the monomeric compound.

[0026] The following compounds were screened out in the present invention: Cpd-1: 7-(4-chloro-2-fluorobenzyl)-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)-4-(morpholinomethyl)pyrrolo[1,2-b]pyridazin-2-amine [7-(4-chloro-2-fluorobenzyl)-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)-4-(morpholinomethyl)pyrrolo[1,2-b]pyridazin-2-amine];

[0027] Cpd-2: N-(1,3-dimethyl-2-oxo-6-(piperidin-1-yl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-methoxybenzamide [N-(1,3-dimethyl-2-oxo-6-(piperidin-1-yl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-methoxybenzamide];

[0028] Cpd-3: methyl 6-hydroxy-2,2-dimethyl-4a,10b-dihydro-2H-benzo[h]chromene-5-carboxylate, which has good utility in stabilizing SIRT6 protein.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The drug screening model constructed in the present invention is applicable to screening monomeric drug compounds with strong affinity for histone deacetylase SIRT6, and the action sites of the screened compounds are clear.

[0031] (2) In a 96-well plate, screening monomeric drug compounds using this drug screening model has the advantages of high throughput and convenient operation.

[0032] (3) In the present invention, the process of labeling proteins with SYPRO Orange Protein Gel Staining Solution is not limited to SIRT6 protein only. Other proteins with histone deacetylase activity (HDAC1 - 11 and SIRT1 - 7) can also be labeled by this method, thereby constructing drug screening models targeting different histone deacetylase subfamily receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the SDS - polyacrylamide gel electrophoresis analysis chart of SIRT6 protein;

[0034] Figure 2 It is the influence of DMSO on the activity of SIRT6 protein in the drug screening model;

[0035] Figure 3 It is the influence of buffer components on the activity of SIRT6 protein in the drug screening model;

[0036] Figure 4 It is the influence of 10 monomeric compounds on the stability of SIRT6 protein. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the content of the embodiments in any form. The test methods described in the embodiments are all conventional methods unless otherwise specified; the reagents and biological materials can be obtained from commercial channels unless otherwise specified.

[0038] Embodiment

[0039] 1. Extract and purify SIRT6 protein using the conventional method for preparing membrane proteins

[0040] The Escherichia coli strain BL21(DE3) was purchased from Novagen, and the plasmid was obtained by referring to the literature [You, W.; Rotili, D.; Li, T.-M.; Kambach, C.; Meleshin, M.; Schutkowski, M.; Chua, K.F.; Mai, A.; Steegborn, C. Structural basis of sirtuin 6 activation by synthetic small molecules. Angew. Chem., Int. Ed. 2017, 56, 1007 - 1011]. The plasmid was added to the competent Escherichia coli BL21(DE3), mixed well and placed on ice for 30 min. Then, the competent cells containing the plasmid were heat-shocked at 42 °C in a common water bath for 90 s and quickly placed on ice for 3 min. An LB medium composed of 10 g NaCl, 10 g bactotrypton, and 5 g yeast extract per liter was shaken to an appropriate concentration and then evenly spread on a pre-prepared LB solid medium culture plate containing chloramphenicol and ampicillin. It was transferred to an incubator at 37 °C and cultured overnight.

[0041] Single colonies were selected in a laminar flow hood and added to 20 mL of LB medium containing chloramphenicol and ampicillin resistance. They were placed in a shaker and cultured at 220 rpm and 37 °C for about 4 h. When OD600 = 0.6 - 0.8, they were transferred to a 1 L culture medium shake flask at a standard of 10 mL / 1 L and placed in a shaker and cultured at 220 rpm and 37 °C for about 4 h. When OD600 = 0.6 - 0.8, the LB medium containing Escherichia coli was cooled to 20 °C, and an isopropyl β-D-thiogalactoside (IPTG) solution with a final concentration of 0.5 mM was added. It was cultured overnight at 220 rpm and 20 °C.

[0042] The next day, transfer the bacterial solution to a 1L centrifuge tube, centrifuge at 4000 rpm for 15 min to collect Escherichia coli. After pouring off the upper layer of the culture medium, resuspend the bottom layer of Escherichia coli with resuspension buffer (50 mM Tris-HCl, pH = 7.0 and 200 mM NaCl) at a ratio of 15 mL / 1L. After collection, place it on ice and add a phenylmethanesulfonyl fluoride (PMSF) solution with a final concentration of 1 mM. Then use a high-pressure cell disruptor to disrupt for about 4 min. Transfer the disrupted bacterial solution to an ultracentrifuge tube and centrifuge at 18000 rpm for 30 min at 4 °C. Collect the supernatant, incubate it with a Ni column, and elute the impurity proteins with elution buffer (Eluent 1: 50 mM Tris-HCl, pH = 7.0 and 200 mM NaCl; Eluent 2: 50 mM Tris-HCl, pH = 7.0, 200 mM NaCl and 10 mM imidazole; Eluent 3: 50 mM Tris-HCl, pH = 7.0, 200 mM NaCl and 20 mM imidazole), and finally elute the target protein with Eluent 4 (50 mM Tris-HCl, pH = 7.0, 10 mM NaCl and 250 mM imidazole). Transfer the eluted protein to a dialysis bag, add an appropriate amount of TEV enzyme according to a molar ratio of 1:50, and stir overnight. The next day, hang the protein on the Ni column in reverse and collect the protein that flows through.

[0043] After concentrating the protein solution, load the sample using a sample loop and perform ion exchange chromatography to purify the protein using a cation exchange column (HiTrapSP HP, 5 ml) from GE. Buffer A (50 mM Tris-HCl, pH = 7.0, 10 mM NaCl) and Buffer B (50 mM Tris-HCl, pH = 7.0, 1000 mM NaCl) were used in this experiment, and the target protein was eluted according to different salt concentrations. Collect the protein sample at the peak tip, concentrate it and load it onto a Superdex 200 molecular sieve column from GE for further size exclusion chromatography purification. The running buffer was 20 mM Na-HEPES, pH 7.5, 100 mM NaCl, 2 mM DTT. After collecting the peak tip sample, perform SDS-PAGE electrophoresis to observe the purity of the SIRT6 protein, and use a micro ultraviolet spectrophotometer (Nanodrop 1000) to detect the protein concentration, and aliquot and store it at -80 °C for subsequent affinity tests.

[0044] 2. Preparation of SYPRO Orange Protein Gel Staining Solution

[0045] Comparing five dyes, it was found that SYPRO Orange Protein Gel Stain is the most suitable fluorescent dye for this experimental protocol due to its high signal-to-noise ratio. The excitation wavelength of SYPRO Orange Protein Gel Stain is relatively high, close to 500 nm, reducing the possibility of any small molecules interfering with the optical properties of the dye and resulting in quenching of the fluorescence intensity. In comparison, the spectral properties of the commonly used 1-anilino-8-naphthalenesulfonate (1,8-ANS) are interfered with by many compounds, and its excitation maximum is approximately 350 nm. In addition, taking 1,8-ANS as an example, the signal intensity is relatively small (~80%). Therefore, we purchased from Sigma-Aldrich Trading Co., Ltd. the product with the product number S5692 and the name Orange Protein Gel Stain(5000X Concentrate in DMSO), for testing, diluted from the DMSO stock solution with a mother liquor of 5000X to a final concentration of 50X.

[0046] 3. Docking Screening Based on Crystal Structure

[0047] (1) Treatment of Compound Library

[0048] The compound library for the experiment includes a commercial compound library for virtual screening, and the types of compounds mainly include fragment compound libraries, diverse scaffold compounds, and natural product compound libraries. All the compounds used in this experiment were processed on a virtual screening platform. In the Prepare Ligands menu, the parameter settings mainly include: whether to change the charge of polar atoms in the compound (Change Ionization), whether to generate tautomers (Generate Tautomers), whether to generate isomers (Generate Isomers), and all these parameters were set to not generate. Other parameter settings were set to default parameters. All the compounds used were further processed for drug-likeness, such as (1) Lipinski Rules of Five and Veber Rule; (2) -2.0 < AlogP < 5.0; (3) false positive filtering rules.

[0049] (2) Structure-Based Screening

[0050] Using typical screening methods in computer-aided drug design, a structure-based virtual screening strategy is employed to select small molecule compounds that may target specific disease targets from numerous unknown compound libraries. Five crystal structures of human SIRT6 and agonist complexes have been reported, with PDB IDs being 5Y2F (MDL-800), 5MGN (UBCS038), 5MF6 (UBCS039), 5MFZ (UBCS040), and 5MPF (UBCS058). Among these five structures, the pocket where the compound MDL-800 binds is an allosteric pocket, and this compound has relatively high activity. Therefore, we selected the crystal structure of SIRT6 (PDB: 5Y2F), the crystal structure of the complex containing the cofactor ADP-ribose (ADPR) and the agonist MDL-800 for molecular docking screening. The epigenetics library with known activity, SPECS, and ChemDiv commercial databases used for docking were prepared in the virtual screening platform. Since this site was first reported in the literature and the binding pocket at this site is an allosteric pocket, it is conducive to screening for highly active and highly selective small molecule inhibitors or agonists.

[0051] For the GOLD software program, the pre-defined generic algorithm (GA) was set to "automatic", the matching function was set to "Goldscore", and other parameters were set to default values. According to the scoring ranking and novelty filter of GoldScore, 10 compounds were selected and purchased for testing this screening method, namely Cpd-1: 7-(4-chloro-2-fluorobenzyl)-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)-4-(morpholinomethyl)pyrrolo[1,2-b]pyridazin-2-amine;

[0052] Cpd-2: N-(1,3-dimethyl-2-oxo-6-(piperidin-1-yl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-methoxybenzamide;

[0053] Cpd-3: 6-hydroxy-2,2-dimethyl-4a,10b-dihydro-2H-benzo[h]chromene-5-carboxylate; Cpd-4: (E)-2-cyano-3-(5-(2,5-dichlorophenyl)furan-2-yl)-N-(quinolin-5-yl)acrylamide; Cpd-5: methyl 2-((2-methoxy-2-oxoethyl)amino)-2-oxoacetate;

[0054] Cpd-6: N-(5-(4-((6-cyanopyridin-3-yl)methoxy)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide;

[0055] Cpd-7: 4,5,13-trimethyl-2-((1-(piperidin-4-yl)-1H-pyrazol-3-yl)amino)-4,4a,5,13-tetrahydro-6H-naphtho[2,3-e]pyrimido[5,4-b][1,4]diazepin-6-one;

[0056] Cpd-8: N-(4-(((4-(4-methoxyphenyl)tetrahydro-2H-pyran-4-yl)methyl)carbamoyl)phenyl)furan-2-carboxamide;

[0057] Cpd-9: 1-((1R,4S)-4-((S)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)ethyl)cyclohexyl)-N-methylmethanesulfonamide;

[0058] Cpd-10: (E)-44-(2-(pyrrolidin-1-yl)ethoxy)-6,11-dioxa-3-aza-2(4,2)-pyrimidina-1,4(1,3)-dibenzenacyclododecaphan-8-ene.

[0059]

[0060] 4. Construction and Evaluation of Drug Screening Model

[0061] (1) Influence of Dimethyl Sulfoxide (DMSO)

[0062] Since most drug monomer compounds are dissolved in DMSO, the present invention examines the influence of DMSO on the use of the drug screening model. Reaction conditions: The concentration of SIRT6 protein is 2 μM, the test buffer is 20 mM Hepes-Na, pH = 7.0, 100 mM NaCl. Different gradient concentrations of DMSO are added and mixed to 20 μL. After sealing with plastic wrap, it is placed on a shaker at 35 rpm and incubated at room temperature for 15 min. The dye probe SYPRO orange dye is diluted from the DMSO stock solution with a mother liquor of 5000X to a final concentration of 50X, and stirred and added to the above protein mixture. Then, the 96-well PCR plate is placed in a q-PCR instrument for detection. The excitation and emission wavelengths are adjusted to 587 nm and 607 nm respectively. The heating rate is usually set at 1 °C / min, and the temperature change range is set at 25 - 95 °C. The fluorescence intensity is measured every 1 °C. Finally, using the analysis software GraphPad Prism 5.0, a curve of fluorescence intensity vs. temperature is plotted, and the T m value of the sample protein is calculated using the Boltzmann sigmoid equation (Formula 1). As Figure 2It is shown that DMSO has a certain effect on the SIRT6 protein in the drug screening model. Therefore, according to the test results, we selected DMSO with a mass percentage of 2% as the screening condition.

[0063] (2) Influence of buffer components

[0064] To test whether the buffer components would interfere with the results of this method, the present invention selected the commonly used reagents Hepes-Na and Tris-Na for comparison. Their components were 20 mM Hepes-Na, pH = 7.0, 100 mM NaCl and 20 mM Tris-Na, pH = 7.0, 100 mM NaCl respectively. The remaining conditions were as described above, and the results were viewed by a q-PCR instrument. As Figure 3 shown, Hepes-Na and Tris-Na in the buffer had no obvious influence.

[0065] 4. Screening of drug monomer compounds

[0066] (1) Pretreatment of drug monomers

[0067] The screened traditional Chinese medicine monomer compounds with different functional characteristics were pretreated. DMSO had no obvious influence on the kinase activity assay experiment. Therefore, the present invention uniformly used DMSO as the solvent to dissolve the samples. After weighing the drugs, they were added to DMSO and vortexed to completely dissolve them. The mother liquor concentration was configured to be 10 mM, and then further diluted with the buffer, and added to the reaction system according to the volume required for the final concentration to be tested.

[0068] (2) Test of the stability of monomer compounds on SIRT6 protein

[0069] Differential fluorescence scanning method based on SYPRO orange dye is performed using a q-PCR instrument at room temperature. First, the aliquoted mother solution of SIRT6 protein is taken out from -80 °C and diluted with the test buffer (20 mM Hepes-Na, pH = 7.0, 100 mM NaCl). The final concentration of the protein for testing is 2 μM. Then, different concentrations and different structures of compounds to be tested and the solvent (DMSO) as a blank control are added into a 96-well plate dedicated for PCR. After sealing with plastic wrap, it is placed on a shaker at 35 rpm and incubated at room temperature for 15 min. The dye probe SYPRO orange dye is diluted from the DMSO mother solution with a concentration of 5000X to a final concentration of 50X, and is stirred and added into the mixture of the above-mentioned protein and compounds. The solvent DMSO of the compound is used as the blank control group, and 3 replicates are set. Each test group is also set with 3 replicates. Then, the 96-well PCR plate is put into the q-PCR instrument for detection. The excitation and emission wavelengths are respectively adjusted to 587 nm and 607 nm. The heating rate is usually set at 1 °C / min, and the temperature change range is set at 25 - 95 °C. The fluorescence intensity is measured every 1 °C. Finally, through the analysis software GraphPad Prism 5.0, the curve of the fluorescence intensity changing with temperature is plotted, and the T m value of the sample protein is calculated using the Boltzmann sigmoid equation (Formula 1). According to the T m values of the solvent control group and the compound test group (Formula 2), the stabilizing ability of each compound to the SIRT6 protein is calculated.

[0070] The results of 10 monomeric compounds obtained by screening in the present invention are as Figure 4 shown: Among them, the positive control compound MDL-800 causes the largest change in the T m value of the SIRT6 protein, which is 0.83 °C; Cpd-1 and compound Cpd-2 cause relatively large changes in the T m values of the SIRT6 protein, which are 0.395 and 0.41 °C respectively; compound Cpd-3 causes a general change in the T m value of the SIRT6 protein, which is 0.29 °C; the changes in the T m values of compounds Cpd4 - 10 proteins are relatively small, less than 0.2 °C.

[0071] This result proves that the drug screening model provided by the present invention has good screening effects, and at the same time has advantages such as high throughput, strong specificity, and convenient operation.

Claims

1. Use of a compound with the following structural formula in the preparation of a modulator targeting histone deacetylase SIRT6, characterized in that, It is used to stabilize the SIRT6 protein, 。

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