A histidine methyltransferase METTL9 inhibitor and screening method, reaction system and application thereof
By constructing a METTL9 reaction system and a fluorescence signal detection method, the challenges of METTL9 enzyme activity detection and inhibitor screening have been solved, enabling efficient and safe high-throughput screening and inhibitor discovery, and promoting the development of METTL9 target drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CENT SOUTH UNIV
- Filing Date
- 2026-03-05
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies lack efficient, sensitive methods for detecting METTL9 enzyme activity suitable for high-throughput screening, and also lack highly specific small molecule inhibitors, resulting in slow progress in the development of METTL9-targeted drugs.
A non-radioactive homogeneous method was used to construct a reaction system for histidine methyltransferase METTL9 and screen METTL9 inhibitors by combining fluorescence signal detection. This process included incubation, enzyme catalysis, fluorescence signal changes, and comparisons, achieving high-throughput screening.
This study provides a highly sensitive, easy-to-operate, safe, and low-cost method for detecting METTL9 activity, enabling the screening of inhibitors with both activity and selectivity, thus providing a tool for the development of METTL9-targeted drugs.
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Figure CN122146844A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a histidine methyltransferase METTL9 inhibitor, its screening method, reaction system, and application. Background Technology
[0002] In recent years, the role of epigenetic modifications in disease development has become increasingly prominent. As one of the important regulatory mechanisms, protein methylation, catalyzed by specific methyltransferases, participates in the precise regulation of various cellular processes. METTL9 is a novel methyltransferase identified in recent years that specifically mediates methylation modification at the histidine Nπ site. Studies have shown that this modification plays a crucial role in fundamental physiological processes such as cell proliferation, zinc ion homeostasis, mitochondrial respiration, and immune responses.
[0003] Functional studies have revealed that METTL9 is significantly upregulated in tissues and cells of various malignant tumors, such as colorectal cancer (CRC) and hepatocellular carcinoma (HCC). In prostate and colon cancer models, knockdown or elimination of METTL9 effectively inhibited tumor cell growth, suggesting its important role in maintaining the malignant phenotype of tumors. These findings collectively establish METTL9 as a potential target for disease intervention, providing a new direction for the development of cancer treatment strategies.
[0004] A thorough understanding of METTL9 function and the development of targeted drugs heavily rely on efficient and reliable activity detection technologies. However, current research and drug development targeting METTL9 face the following major bottlenecks: 1) Lack of activity detection and screening tools: Currently, there is a lack of efficient, sensitive, and suitable methods for high-throughput screening (HTS) of METTL9 enzyme activity. Traditional radiolabeling or mass spectrometry methods are cumbersome, have low throughput, are costly, or pose safety risks, making them unsuitable for rapid screening of large-scale compound libraries; 2) Severe lack of specific inhibitors: To date, there are no publicly reported small molecule compounds that can effectively and selectively inhibit METTL9 activity. Existing broad-spectrum methyltransferase inhibitors may suffer from weak activity, poor selectivity (easily affecting other methyltransferases), or poor cell permeability, failing to meet the needs for in-depth biological research and drug development as lead compounds or tool molecules. 3) Weak foundation in drug development: Due to the lack of efficient screening methods and high-quality lead compounds, the development of innovative drugs targeting METTL9 is progressing slowly.
[0005] Therefore, there is an urgent need to develop a highly specific, sensitive, and easy-to-operate method for detecting METTL9 activity, and to screen inhibitors based on this method. This is of vital importance for promoting research on the biological function of METTL9 and developing drugs that target it. Summary of the Invention
[0006] This invention provides a histidine methyltransferase METTL9 inhibitor, its screening method, reaction system, and application. The purpose is to provide a non-radioactive, homogeneous, easy-to-operate, and high-throughput screening method for detecting METTL9 activity. Through the screening system, a series of novel METTL9 small molecule inhibitors with certain activity and selectivity are discovered and provided.
[0007] To achieve the above objectives, the present invention provides a method for screening histidine methyltransferase METTL9 inhibitors, comprising the following steps: S1. Construct a reaction system for histidine methyltransferase METTL9; the reaction system is incubated with a methyl donor, a polypeptide substrate and histidine methyltransferase METTL9, and the reaction is catalyzed by enzyme to transfer the methyl group of the methyl donor to the polypeptide substrate to generate the transfer product. S2. Divide the reaction system into a test system and a control system, and add S-adenosylhomocysteine hydrolase and a fluorescent dye that can undergo Michael addition reaction with homocysteine and cause changes in fluorescence signal, respectively. S3. Add the candidate compound to the test system, and do not add the candidate compound to the control system, and detect the fluorescence signals of the test system and the control system; S4. Compare the fluorescence signals of the test system and the control system, and determine whether the candidate compound is a METTL9 inhibitor based on the comparison results.
[0008] The method in this application is based on the principle that the net increase in fluorescence signal reflects enzyme activity, and its main contents are as follows: 1) In the reaction system, METTL9 catalyzes the transfer of methyl groups from the donor substrate S-adenosylmethionine (SAM) to the peptide acceptor substrate, generating the byproduct S-adenosylhomocysteine (SAH). 2) SAH is specifically hydrolyzed by SAHH enzyme to generate homocysteine; 3) Homocysteine undergoes a Michael addition reaction with the fluorescent dye in the reaction system, leading to a quantitative change in the fluorescence signal; 4) By detecting the fluorescence intensity of the reaction system (e.g., measuring the fluorescence growth value using the endpoint method) and comparing it with the control, the enzyme activity of METTL9 can be quantitatively evaluated, thereby rapidly and efficiently screening out candidate compounds that can inhibit the enzyme activity.
[0009] Compared with existing technologies, the present invention has significant advantages in terms of method innovation, high efficiency and convenience. The screening method provided by the present invention couples the enzyme-catalyzed reaction with fluorescence signal transduction, which has the advantages of high sensitivity, simple operation, good safety (no need for radioactive labeling), low cost and is particularly suitable for automated high-throughput screening.
[0010] Preferably, the methyl donor in step S1 includes at least one of S-adenosylmethionine, an S-adenosylmethionine analogue, or other chemical substances suitable for the METTL9 catalytic reaction, with a concentration range of 0.1 ~ 10 K. m The polypeptide substrate mentioned in step S1 is a polypeptide containing the xHxH motif, where x represents any small side chain amino acid, H represents histidine, and the concentration range is 0.1 ~ 10 K. m Peptide substrates can be chemically modified, fluorescently labeled, or structurally optimized to improve detection sensitivity or specificity.
[0011] Preferably, the histidine methyltransferase METTL9 reaction system in step S1 further includes any buffer suitable for protein catalytic reactions, with a pH range of 5.5 to 10.5; More preferably, the buffer solution is at least one of 10 mM Tris-HCl, 20 mM Tris-HCl, 50 mM Tris HCl or 150 mM NaCl, with a pH range of 5.5 to 10.5; The histidine methyltransferase METTL9 reaction system also contains an anti-interference substance, which includes one or more of thiol compounds, chelating agents, or protein stabilizers.
[0012] Preferably, the histidine methyltransferase METTL9 in step S1 includes: Histidine methyltransferase METTL9 from any species, including but not limited to: METTL9 derived from one or more of mammals, birds, amphibians, reptiles, plants, fungi, bacteria or prokaryotes; Or any wild-type METTL9 with catalytic activity or a mutant METTL9 with catalytic activity, including genetically engineered METTL9 variants; Among them, mutant METTL9 may have altered amino acid sequences, enhanced or weakened catalytic activity, or different substrate specificity. The concentration range of the histidine methyltransferase METTL9 is 0.1 ~ 500 μM.
[0013] Preferably, the incubation temperature range in step S1 is 15 ~ 50℃, and the incubation time is 1 ~ 120 min; the enzyme catalytic reaction temperature range is 15 ~ 50℃, and the reaction time is 1 ~ 120 min.
[0014] Preferably, the S-adenosine homocysteine hydrolase in step S2 comprises: S-adenosylhomocysteine hydrolase SAHH from any species, including but not limited to: SAHH derived from one or more of mammals, birds, amphibians, reptiles, plants, fungi, bacteria or prokaryotes; Or any wild-type SAHH with catalytic activity or a mutant SAHH with catalytic activity, including genetically engineered SAHH variants; Among them, mutant SAHH may have altered amino acid sequences, enhanced or weakened catalytic activity, or different substrate specificity. The concentration range of the histidine methyltransferase SAHH is 0.1 ~ 500 μM.
[0015] Preferably, the general structural formula of the fluorescent dye in step S2 is: ; R1 is at least one of the following: hydroxyl group, straight-chain or branched alkyl group with 1-10 carbon atoms, aryl group, halogenated alkyl group, or heterocyclic group containing oxygen, nitrogen, or sulfur. R5 is at least one of ester, amide, cyano, sulfonamide, sulfonate, or a derivative thereof; R2, R3 and R5 include at least one of hydrogen, alkyl, aryl or substituent, said substituent including but not limited to halogen, hydroxyl, methoxy, nitro or amino; The fluorescent dye may contain hydrophilic or hydrophobic groups.
[0016] Preferably, the fluorescence signal in step S3 includes one or more of fluorescence intensity, fluorescence lifetime, or fluorescence polarization; Step S4, comparing the fluorescence signals of the test system and the control system, specifically includes: calculating the fluorescence intensity change rate or the final fluorescence signal value based on a signal processing algorithm, using the following formula:
[0017] The test well, negative control well, and positive control well refer to the fluorescence intensity at each control well. Enzyme activity is determined based on the calculation results. When the inhibition rate is greater than 80%, the substance to be tested is preliminarily determined to be an inhibitor.
[0018] Under the same technical concept, the present invention also provides a reaction system for screening histidine methyltransferase METTL9 inhibitors, wherein the reaction system is combined with an automated high-throughput screening platform, and the reaction conditions and fluorescence signal detection are controlled by microplates, multichannel pipettes or microfluidic devices.
[0019] Under the same technical concept, the present invention also provides a histidine methyltransferase METTL9 inhibitor, wherein the histidine methyltransferase METTL9 inhibitor comprises at least one of the compounds with the chemical structures shown below: S9042, S1116, S4845, S4425, S3666, S7781, and S4008. .
[0020] Under the same technical concept, the present invention also provides the use of a histidine methyltransferase METTL9 inhibitor in the preparation of a pharmaceutical product for the prevention or treatment of diseases related to abnormal METTL9 activity, comprising the histidine methyltransferase METTL9 inhibitor or a pharmaceutically acceptable derivative thereof.
[0021] Under the same technical concept, the present invention also provides the application of a histidine methyltransferase METTL9 inhibitor in the study of the biological function of METTL9 or its related signaling pathways, comprising the histidine methyltransferase METTL9 inhibitor or a pharmaceutically acceptable derivative thereof.
[0022] The above-described solution of the present invention has the following beneficial effects: (1) The screening method provided by the present invention couples the enzyme-catalyzed reaction with fluorescence signal transduction, which has the advantages of high sensitivity, simple operation, good safety (no need for radioactive labeling), low cost and is particularly suitable for automated high-throughput screening. (2) The inhibitors screened by this invention have both activity and selectivity, providing new tools and candidate compounds for the research and development of drugs targeting METTL9-related diseases, and can be used for the development of medical drugs targeting METTL9. Attached Figure Description
[0023] Figure 1 The SDS-PAGE detection results of the prepared METTL9 protein in the embodiments of the present invention are shown in the figure; the lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during sample loading), W (waste liquid flushed down from Buffer A), Elu (target protein), Elu' (target protein after tag removal), and Marker; Figure 2The SDS-PAGE detection results of SAHH prepared in the embodiments of the present invention are shown in the figure; the lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during sample loading), W (waste liquid flushed down from Buffer A), Elu (target protein), Elu' (target protein after tag removal), and Marker; Figure 3 This invention aims to evaluate the effect of buffer solution pH on enzyme activity in this embodiment. Figure 4 To test the linear range of the reaction between -SH and ThioGlo4 in this embodiment of the invention, and to determine the effective range of experimental fluorescence signal values; Figure 5 The Km value of the substrate SAM in this embodiment of the invention; Figure 6 The value is the Km value of the polypeptide substrate in the embodiments of the present invention. Detailed Implementation
[0024] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0025] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.
[0026] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.
[0027] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0028] One of the standard operating conditions for the method and system described in this invention is: 1) The concentration of METTL9 was 4 μM; 2) The SAHH concentration was 15 μM; 3) The fluorescent dye is ThioGlo4, with a concentration of 200 μM; 4) The peptide substrate was LAPPGHQGHSHGHQGG, at a concentration of 80 μM; 5) The SAM concentration was 360 μM; 6) The buffer solution consists of 50 mM Tris-HCl, 50 mM NaCl, 1 mM MgCl2, and pH 7.5; 7) The incubation and testing temperature is 37℃, the incubation time is 60 min, and the testing time is 60 min; 8) The excitation and emission wavelengths are 400 nm and 465 nm, respectively. One of the standard operating procedures for the method and system described in this invention is as follows: 1) Buffer solution preparation: Prepare buffer solutions to ensure the concentrations of each component are: 4 μM METTL9, 15 μM SAHH, 360 μM SAM, and 80 μM peptide substrate.
[0029] The buffer solution was formulated as follows: 50 mM Tris-HCl, 50 mM NaCl, 1 mM MgCl2, pH 7.5.
[0030] The prepared buffer solution must be thoroughly mixed before use, and the concentration of each component must be accurate.
[0031] 2) Incubation steps: Take 98 μL of the prepared buffer solution and distribute it into each well of a 384-well plate.
[0032] Place the 384-well plate in a 37°C incubator and incubate in the dark for 60 minutes.
[0033] During incubation, ensure the well plate is placed horizontally to guarantee thorough mixing of the solution in each well.
[0034] 3) Addition of fluorescent dyes: Take 2 μL of 10 mM ThioGlo4 and carefully add it to each well using a pipette.
[0035] Use a pipette to mix the solution from top to bottom to ensure that ThioGlo4 is evenly distributed in each well. The mixing operation needs to be performed 10 times to ensure that the solution is completely mixed.
[0036] 4) Fluorescence monitoring: Place the 384-well plate in the fluorescence module of the multi-functional microplate reader, and set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Using the fluorescence intensity mode, start the kinetic monitoring program and record the fluorescence intensity change of each well every minute; The monitoring lasted for 60 minutes, during which fluorescence data was collected every 3 minutes to ensure that the fluorescence changes during the reaction were reflected in real time.
[0037] 5) Data Analysis: This invention uses the endpoint fluorescence signal method for data processing; The net increase in fluorescence intensity was calculated and correlated with METTL9 enzyme activity to achieve activity quantification. By analyzing this quantitative data, the effects of different conditions on enzyme activity can be assessed.
[0038] Example 1: Prokaryotic expression, isolation, and purification of METTL9 1.1) Transformation of METTL9 plasmid: The METTL9 plasmid was transformed into competent cells of protein expression strain BL21(DE3), plated on a plate containing kanamycin sulfate, and incubated in a 37°C incubator. 1.2) Small culture: Pick a single colony and transfer it to 100 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. Incubate at 37°C and 180 rpm for 16 hours. 1.3) Scale-up culture: Take two Erlenmeyer flasks containing 1 L LB liquid medium, add 1 mL of 1000× kanamycin sulfate to each flask to a final concentration of 50 μg / mL, and mix well. Then add 50 mL of bacterial culture after 16 h of small-scale culture to each flask, and place them in a shaker at 37℃ and 180 rpm for incubation.
[0039] 1.4) Induction of expression: When the expanded culture of *E. coli* reached the logarithmic growth phase, i.e., the OD value of the bacterial culture was 0.6-0.8, the bacterial culture was cooled in a chromatography cabinet at 4°C, and a small amount of the bacterial culture was taken as a sample (uninitiated). Isopropyl thiogalactoside (IPTG) was added to the cooled bacterial culture to a final concentration of 0.2 mM, and the culture was placed in a shaker at 16°C and 180 rpm for 16 h to induce full protein expression.
[0040] 1.5) Centrifugation to collect bacterial cells: Centrifuge the induced bacterial culture at 4°C and 4000 rpm for 10 minutes, and remove the supernatant. Resuspend the bacterial cells in 30 mL of Buffer A, and add PMSF to a final concentration of 1 mM and mix well. Disrupt the resuspended bacterial culture using a high-pressure homogenizer cooled at 4°C until the culture becomes clear and transparent.
[0041] 1.6) Centrifugation to remove cell debris: Centrifuge the disrupted bacterial culture at 12,000 rpm for 30 minutes to remove cell debris. Repeat this centrifugation step three times to ensure complete removal of any remaining cells.
[0042] 1.7) Ni-NTA Affinity Chromatography Purification: METTL9 protein was isolated and purified using an AKTA protein purification system via a Ni-NTA affinity chromatography column. Equilibration was performed using Buffer A. The lysate was loaded, and impurities were eluted with Buffer A until the UV absorbance at A280 nm reached zero, ensuring the absence of contaminants. Buffer B was then used for elution of the target protein. Buffer A: (50 mM Tris-HCl, 400 mM NaCl, 25 mM imidazole, pH 7.5); Buffer B: (50 mM Tris-HCl, 400 mM NaCl, 400 mM imidazole, pH 7.5) 1.8) Dialysis to remove imidazole: The eluted target protein was transferred to a dialysis bag and placed in 100 times the volume of dialysis buffer (50 mM Tris-HCl, 400 mM NaCl, pH 7.5) and dialyzed at 4°C for 4 hours to remove imidazole and other small molecule impurities.
[0043] 1.9) Removal of the SUMO tag: ULP1 enzyme was added to the dialyzed METTL9 protein at a ratio of METTL9:ULP1 = 50:1. The protein solution was placed in 100 volumes of dialysate (50 mM Tris-HCl, 400 mM NaCl, pH 7.5) and subjected to enzymatic digestion at 4°C for 16 hours. The SUMO tag of the METTL9 protein was removed by ULP1, yielding the final purified protein.
[0044] 1.10) Purity Verification: Protein purity was verified using 12% SDS-PAGE and Coomassie Brilliant Blue staining. After concentration determination, the protein was stored at -80℃ for later use. The test results are as follows: Figure 1As shown; the lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during sample loading), W (waste liquid flushed down from Buffer A), Elu (target protein), Elu' (target protein after tag removal), and Marker; Example 2: Prokaryotic expression, isolation, and purification of SAHH 2.1) Transformation of SAHH plasmid: The SAHH plasmid was transformed into competent cells of protein expression strain BL21(DE3), plated on a plate containing ampicillin, and incubated in a constant temperature incubator at 37°C.
[0045] 2.2) Small culture: Pick a single colony and place it in 100 mL LB liquid medium containing 50 μg / mL ampicillin. Incubate in a shaker at 37℃ and 180 rpm for 16 h.
[0046] 2.3) Scale-up culture: Take two Erlenmeyer flasks containing 1 L LB liquid medium, add 1 mL of 1000× ampicillin to each flask to a final concentration of 50 μg / mL, and mix well. Then add 50 mL of bacterial culture after 16 h of small-scale culture to each flask, and place them in a shaker at 37℃ and 180 rpm for incubation.
[0047] 2.4) Induction of expression: When the expanded culture of *E. coli* reached the logarithmic growth phase, i.e., the OD value of the bacterial culture was 0.6-0.8, the bacterial culture was cooled in a chromatography cabinet at 4°C, and a small sample of the bacterial culture was retained (uninitiated). Isopropyl thiogalactoside (IPTG) was added to the cooled bacterial culture to a final concentration of 0.4 mM, and the culture was placed in a shaker at 30°C and 180 rpm for 16 h to induce full protein expression.
[0048] 2.5) Centrifugation to collect bacterial cells: Centrifuge the bacterial solution at 4℃ and 4000 rpm for 10 min. After centrifugation, discard the supernatant, add 30 mL of Buffer A to resuspend the bacterial cells, and then add PMSF to a final concentration of 1 mM and mix well. The resuspended bacterial solution is then homogenized using a high-pressure homogenizer cooled at 4℃. The homogenized bacterial solution is clearer and more transparent than the unhomogenized solution.
[0049] 2.6) Centrifugation to remove cell debris: Centrifuge the disrupted bacterial culture at 12,000 rpm for 30 minutes to remove cell debris. Repeat this centrifugation step three times to ensure complete removal of any remaining cells.
[0050] 2.7) Ni-NTA Affinity Chromatography Purification: SAHH protein was purified using an AKTA protein purification system via a Ni-NTA affinity chromatography column. The Ni-NTA was pre-equilibrated, and the column was rinsed with Buffer A for equilibration. Sample loading was performed using a sample pump, repeated twice. Subsequently, the column was rinsed with Buffer A until no contaminating protein was observed in the eluent (monitored by UV absorption at A280 nm). The target protein was then rinsed with Buffer B. Buffer A: (25 mM HEPES, 500 mM NaCl, 20 mM imidazole, pH 8.0); Buffer B: (25 mM HEPES, 500 mM NaCl, 400 mM Mimidazole, pH 8.0) 2.8) Dialysis to remove imidazole: The target protein was placed in a dialysis bag and immersed in 100 times its volume of dialysis buffer (the final concentration of NaH2PO4 / Na2HPO4 at pH 7.0 was 50 mM, and the final concentration of NaCl was 500 mM). Dialysis was performed in a chromatography cabinet at 4°C for 4 h to remove imidazole from the protein.
[0051] 2.9) Removal of the His tag: TEV enzyme was added to the dialyzed SAHH protein at a ratio of SAHH:TEV = 50:1, and placed in 100 times the volume of dialysate (20 mM KH2PO4 / K2HPO4 pH 7.2, 100 mM NaCl). The protein was digested at 4°C for 2 h, and then the same dialysate was used for digestion at 4°C for 16 h.
[0052] 2.10) Purity Verification: Protein purity was verified using 12% SDS-PAGE and Coomassie Brilliant Blue staining. After concentration determination, the protein was stored at -80℃ for later use. The test results are as follows: Figure 2 As shown; the lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during sample loading), W (waste liquid flushed down from Buffer A), Elu (target protein), Elu' (target protein after tag removal), and Marker; Example 3: Determining the optimal conditions, such as the type of buffer solution, ion concentration, and pH, to be used. 3.1) Prepare different types of buffer solutions Prepare a buffer solution with the following components: 4 µM METTL9; 15 µM SAHH; 360 µM SAM; 80 µM peptide substrate. Buffer solution formulation: A: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 B: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 3.2) Incubation of samples Add 98 μL of the prepared buffer solution to a 384-well plate. Incubate at 37°C in the dark for 60 minutes.
[0053] 3.3) Add 200 µM ThioGlo4 Prepare a 10 mM ThioGlo4 solution. Add 2 μL of ThioGlo4 solution to each well of a 384-well plate. Mix the solution thoroughly using a pipette, moving it up and down 10 times to ensure homogeneity.
[0054] 3.4) Fluorescence intensity measurement On the multi-functional microplate reader, select the Fluorescence Intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Perform kinetic cycling, acquiring data every 3 minutes, and continuously monitor for 60 minutes.
[0055] 3.5) Data Analysis The effect of the two buffer solutions on enzyme activity was evaluated based on the fluorescence intensity increase value of each buffer solution. The buffer solution with the highest fluorescence intensity increase value was selected as the optimal condition, and the evaluation results are as follows: Figure 3 As shown. Figure 4 To test the linear range of the reaction between -SH and ThioGlo4 in this embodiment of the invention, and to determine the effective range of experimental fluorescence signal values; Example 4: Determination of the K-value of the substrate methyl donor SAM m value A fixed peptide substrate was used at a saturation concentration (100 µM), and the concentration of SAM was varied in the range of 0–800 µM to determine the K+ of SAM. m .
[0056] 4.1) Prepare buffer solution Prepare a buffer solution with the following components: 4 µM METTL9; 15 µM SAHH; 80 µM peptide substrate. Buffer solution formulation: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5; 4.2) Dilute the SAM solution SAM was diluted 2-fold starting at a concentration of 800 µM to obtain a series of concentrations (0 ~ 800 µM).
[0057] 4.3) Add different concentrations of SAM and incubate the samples. Add 88 μL of the prepared buffer solution to a 384-well plate; add 10 μL of SAM solution of different concentrations to each well of the 384-well plate. Mix the solution up and down 10 times with a pipette to ensure homogeneity, and incubate at 37°C in the dark for 60 minutes.
[0058] 4.4) Add 200 µM ThioGlo4 Prepare a 10 mM ThioGlo4 solution. Add 2 μL of ThioGlo4 solution to each well of a 384-well plate. Mix the solution thoroughly using a pipette, moving it up and down 10 times to ensure homogeneity.
[0059] 4.5) Fluorescence intensity measurement On a multi-functional microplate reader, select the Fluorescence Intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Perform kinetic cycling, acquiring fluorescence intensity every 3 minutes for 60 minutes.
[0060] 4.6) Data Analysis and K m Value Calculation The enzyme activity of METTL9 was calculated based on the fluorescence intensity increase value of each well. The Kc of SAM was determined based on the enzyme kinetic curve fitting data. m The value, the calculation result is as follows Figure 5 As shown.
[0061] Example 5: Determining the K of peptide substrates m value With a fixed substrate SAM at a saturation concentration (360 µM), the concentration of the peptide substrate was varied in the range of 0 to 200 µM to determine the Km value of the peptide substrate.
[0062] 5.1) Prepare buffer solution Prepare a buffer solution with the following components: 4 µM METTL9; 15 µM SAHH; 3600 µM SAM Buffer solution formulation: 50 mM Tris-HCl, pH 7.5; 5.2) Dilute the peptide substrate solution The peptide solution was diluted 2-fold starting from a concentration of 200 µM to obtain a series of concentrations (0 ~ 200 µM).
[0063] 5.3) Add different concentrations of peptide substrate and incubate the samples. Add 96 µL of the prepared buffer solution to a 384-well plate; add 2 μL of peptide solutions of different concentrations to each well of the 384-well plate. Mix the solutions up and down 10 times with a pipette to ensure homogeneity, and incubate at 37°C in the dark for 60 minutes.
[0064] 5.4) Add 200 µM ThioGlo4 Prepare a 10 mM ThioGlo4 solution. Add 2 μL of ThioGlo4 solution to each well of a 384-well plate. Mix the solution thoroughly using a pipette, moving it up and down 10 times to ensure homogeneity.
[0065] 5.5) Fluorescence intensity measurement On a multi-functional microplate reader, select the Fluorescence Intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Perform kinetic cycling, acquiring fluorescence intensity every 3 minutes for 60 minutes.
[0066] 5.6) Data Analysis and K m Value Calculation The enzyme activity of METTL9 was calculated based on the fluorescence intensity increase value of each well. The Kc of SAM was determined based on the enzyme kinetic curve fitting data. m The value, the calculation result is as follows Figure 6 As shown.
[0067] Example 6: Screening, taking the screening of 80 small molecule compounds from compound library L1300 as an example. 6.1) Take out the purified proteins METTL9 and SAHH, methyl donor SAM, polypeptide substrate, fluorescent dye, prepared reaction buffer, and enzyme-free water obtained in Examples 1 and 2. The table below shows the amount of each reactant required in the reaction well of a single compound in the in vitro methylation reaction. Calculate and prepare the total reaction volume including 80 test compound wells and 8 positive control wells according to Table 1 below, and premix.
[0068] Table 1
[0069] 6.2) Using the same operating steps as in 6.1), calculate and prepare the total mixture required for the reaction of 8 negative control wells according to the amount of each component in a single reaction well as listed in Table 2 below.
[0070] Table 2
[0071] 6.3) Take out the compound library purchased from TargetMol, thaw, vortex, centrifuge, and then use a multi-channel pipette to take 1 μL of each compound in sequence to make the final concentration of 100 μM in the final detection system; and add the same volume of DMSO as the compound to the last two wells of each row of the 384-well plate of the detection system as positive control and negative control wells.
[0072] 6.4) Mixing 6.1) Using a pipette, add 97 μL of the enzyme reaction complex to each well of a 384-well plate containing 1 μL of different small molecule compounds and 1 μL of DMSO positive control in 6.3). Swish the pipette up and down about 10 times to ensure thorough mixing.
[0073] 6.5) Mix the prepared manifold from 6.2) Using a single-channel pipette, add 97 μL of the enzyme reaction complex to each well of the 384-well plate containing 1 μL LDMSO negative control in the last column of 6.3), and pipette up and down about 10 times to ensure thorough mixing.
[0074] 6.6) Place the 384 reaction plate in a 37°C incubator and incubate in the dark for 60 min. During incubation, ensure that the plate is placed horizontally to ensure that the solution in each well is thoroughly mixed.
[0075] 6.7) After incubation, take 2 μL of 10 mM fluorescent dye and add it to each well using a pipette. Swish the pipette up and down about 10 times to ensure thorough mixing.
[0076] 6.8) Place the 384-well plate in the fluorescence module of the multi-functional microplate reader, set the excitation wavelength to 400 nm and the emission wavelength to 465 nm; use the fluorescence intensity mode, start the kinetic monitoring program, and continuously monitor for 60 min. During this process, collect fluorescence data every 3 min to ensure real-time fluorescence changes and the final fluorescence signal growth value.
[0077] 6.9) Based on signal processing algorithms, calculate the rate of change of fluorescence intensity or the final fluorescence signal value. The calculation formula is as follows: (1.1) The test well, negative control well, and positive control well refer to the fluorescence intensity at each control well. The inhibition rate of the test well is calculated according to Formula 1.1. By analyzing this data, the effect of different compounds on the activity of METTL9 enzyme is evaluated, and compounds with an inhibition rate greater than 80% are screened out and preliminarily identified as primary screening inhibitors, which will then be further validated.
[0078] Example 7: Secondary validation of compounds with a single-point concentration inhibition rate >90% at 100 μM from the compound library screening results. 7.1) Take the compounds to be validated, numbered S9042, S1116, S4845, S3666, S7781, S4008, and S4425, dissolve them in a suitable solvent, and prepare stock solutions of the required concentrations. Use a 3-fold dilution factor to perform serial dilutions of the compounds in a 96-well plate, setting a total of 10 concentration points. For each concentration, use a pipette to transfer 1 μL of the compound to a 384-well plate; simultaneously, add an equal volume of DMSO to two adjacent wells as a solvent control.
[0079] 7.2) Take out the purified proteins METTL9 and SAHH, methyl donor SAM, polypeptide substrate, fluorescent dye, prepared reaction buffer, and enzyme-free water obtained in Examples 1 and 2. The table below shows the amount of each reactant required in the reaction well of a single compound in the in vitro methylation reaction. Calculate and prepare the total reaction volume including 90 test compound wells and 9 positive control wells according to Table 3 below, and premix.
[0080] Table 3
[0081] 7.3) Following the same procedure as 7.1), calculate and prepare the total mixture required for the reaction of 9 negative control wells according to the amount of each component in a single reaction well listed in Table 4 below.
[0082] Table 4
[0083] 7.4) Mix the prepared manifold from 7.1). Using a pipette, add 97 μL of the enzyme reaction complex to each well of a 384-well plate containing 1 μL of different concentrations of small molecule compounds and 1 μL of DMSO positive control from 7.1). Swish the pipette up and down about 10 times to ensure thorough mixing.
[0084] 7.5) Mix the prepared manifold from 7.2) Using a single-channel pipette, add 97 μL of the enzyme reaction complex to the last column of the 384-well plate containing 1 μL LDMSO negative control from 7.1), and pipette up and down about 10 times to ensure thorough mixing.
[0085] 7.6) Place the 384 reaction plate in a 37°C incubator and incubate in the dark for 60 min. During incubation, ensure that the plate is placed horizontally to ensure that the solution in each well is thoroughly mixed.
[0086] 7.7) After incubation, take 2 μL of 10 mM fluorescent dye and add it to each well using a pipette. Swish the pipette up and down about 10 times to ensure thorough mixing.
[0087] 7.8) Place the 384-well plate in the fluorescence module of the multi-functional microplate reader, set the excitation wavelength to 400 nm and the emission wavelength to 465 nm; use the fluorescence intensity mode, start the kinetic monitoring program, and continuously monitor for 60 min. During this process, collect fluorescence data every 3 min to ensure real-time fluorescence changes and the final fluorescence signal growth value.
[0088] 7.9) The inhibition rate of the test well was calculated using Formula 1.1, and the IC of the compound was obtained by fitting a curve. 50 The values were used to systematically evaluate the inhibitory efficacy of each compound on enzyme activity.
[0089] 7.10) Analysis of the data shows that the verified compounds exhibit dose-concentration inhibitory activity against METTL9. The following compounds were screened using this method: S9042 (IC50 = 19.05 µM), S1116 (IC50 = 20.10 µM), S4845 (IC50 = 28.79 µM), S4425 (IC50 = 23.93 µM), S3666 (IC50 = 21.23 µM), S7781 (IC50 = 23.75 µM), and S4008 (IC50 = 153 µM). These compounds effectively inhibiting METTL9 were identified.
Claims
1. A method for screening inhibitors of histidine methyltransferase METTL9, characterized in that, Includes the following steps: S1. Construct a reaction system for histidine methyltransferase METTL9; the reaction system is incubated with a methyl donor, a polypeptide substrate and histidine methyltransferase METTL9, and the reaction is catalyzed by enzyme to transfer the methyl group of the methyl donor to the polypeptide substrate to generate the transfer product. S2. Divide the reaction system into a test system and a control system, and add S-adenosylhomocysteine hydrolase and a fluorescent dye that can undergo Michael addition reaction with homocysteine and cause changes in fluorescence signal, respectively. S3. Add the candidate compound to the test system, and do not add the candidate compound to the control system, and detect the fluorescence signals of the test system and the control system; S4. Compare the fluorescence signals of the test system and the control system, and determine whether the candidate compound is a METTL9 inhibitor based on the comparison results.
2. The screening method as described in claim 1, characterized in that, The methyl donor mentioned in step S1 includes at least one of S-adenosylmethionine, an S-adenosylmethionine analogue, or other chemical substances suitable for the METTL9 catalytic reaction, with a concentration range of 0.1 ~ 10 K. m The polypeptide substrate mentioned in step S1 is a polypeptide containing the xHxH motif, where x represents any small side chain amino acid, H represents histidine, and the concentration range is 0.1 ~ 10 K. m .
3. The screening method as described in claim 1, characterized in that, The histidine methyltransferase METTL9 reaction system in step S1 further includes any buffer suitable for protein catalytic reactions, with a pH range of 5.5 to 10.5; the histidine methyltransferase METTL9 reaction system further includes an anti-interference substance, which includes one or more of thiol compounds, chelating agents, or protein stabilizers.
4. The screening method as described in claim 1, characterized in that, The histidine methyltransferase METTL9 mentioned in step S1 includes: Histidine methyltransferase METTL9 from any species, including but not limited to: METTL9 derived from one or more of mammals, birds, amphibians, reptiles, plants, fungi, bacteria or prokaryotes; Or any wild-type METTL9 with catalytic activity or a mutant METTL9 with catalytic activity, including genetically engineered METTL9 variants; Among them, mutant METTL9 may have altered amino acid sequences, enhanced or weakened catalytic activity, or different substrate specificity. The concentration range of the histidine methyltransferase METTL9 is 0.1 ~ 500 μM.
5. The screening method as described in claim 1, characterized in that, The incubation temperature range in step S1 is 15 ~ 50℃, and the incubation time is 1 ~ 120 min; the enzyme catalytic reaction temperature range is 15 ~ 50℃, and the reaction time is 1 ~ 120 min.
6. The screening method as described in claim 1, characterized in that, The S-adenosine homocysteine hydrolase mentioned in step S2 includes: S-adenosylhomocysteine hydrolase SAHH from any species, including but not limited to: SAHH derived from one or more of mammals, birds, amphibians, reptiles, plants, fungi, bacteria or prokaryotes; Or any wild-type SAHH with catalytic activity or a mutant SAHH with catalytic activity, including genetically engineered SAHH variants; Among them, mutant SAHH may have altered amino acid sequences, enhanced or weakened catalytic activity, or different substrate specificity. The concentration range of the histidine methyltransferase SAHH is 0.1 ~ 500 μM.
7. The screening method as described in claim 1, characterized in that, The general structural formula of the fluorescent dye mentioned in step S2 is: ； R1 is at least one of the following: hydroxyl group, straight-chain or branched alkyl group with 1-10 carbon atoms, aryl group, halogenated alkyl group, or heterocyclic group containing oxygen, nitrogen, or sulfur. R5 is at least one of ester, amide, cyano, sulfonamide, sulfonate, or a derivative thereof; R2, R3, and R5 include at least one of hydrogen, alkyl, aryl, or substituents, including but not limited to halogens, hydroxyl groups, methoxy groups, nitro groups, or amino groups.
8. The screening method as described in claim 1, characterized in that, The fluorescence signal mentioned in step S3 includes one or more of fluorescence intensity, fluorescence lifetime, or fluorescence polarization; Step S4, comparing the fluorescence signals of the test system and the control system, specifically includes: calculating the fluorescence intensity change rate or the final fluorescence signal value based on a signal processing algorithm, using the following formula: ； The test well, negative control well, and positive control well refer to the fluorescence intensity at each control well. Enzyme activity is determined based on the calculation results. When the inhibition rate is greater than 80%, the substance to be tested is preliminarily determined to be an inhibitor.
9. A reaction system for screening histidine methyltransferase METTL9 inhibitors as described in any one of claims 1-8, characterized in that, The reaction system is combined with an automated high-throughput screening platform, which controls reaction conditions and detects fluorescence signals through microplates, multichannel pipettes or microfluidic devices.
10. A histidine methyltransferase METTL9 inhibitor, characterized in that, The histidine methyltransferase METTL9 inhibitor comprises at least one compound with the chemical structures shown in S9042, S1116, S4845, S4425, S3666, S7781, and S4008: 。 11. The use of a histidine methyltransferase METTL9 inhibitor in the preparation of pharmaceutical products for the prevention or treatment of diseases related to abnormal METTL9 activity, characterized in that, The histidine methyltransferase METTL9 inhibitor comprises the histidine methyltransferase METTL9 inhibitor as described in claim 10, or the histidine methyltransferase METTL9 inhibitor screened by the screening method described in any one of claims 2-8, or a pharmaceutically acceptable derivative thereof.
12. The application of a histidine methyltransferase METTL9 inhibitor in studying the biological function of METTL9 or its related signaling pathways, characterized in that, The histidine methyltransferase METTL9 inhibitor comprises the histidine methyltransferase METTL9 inhibitor as described in claim 10, or the histidine methyltransferase METTL9 inhibitor screened by the screening method described in any one of claims 2-8, or a pharmaceutically acceptable derivative thereof.