G-quadruplex RNA fluorescent ligand, preparation method and application thereof
By developing G-quadruplex RNA fluorescent ligands and a high-throughput fluorescent screening system, the problem of low screening efficiency in existing technologies has been solved, achieving highly sensitive and efficient ligand screening, rapidly discovering high-affinity compounds, and promoting the research and development of anti-tumor drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHONGSHAN POLYTECHNIC UNIVERSITY INSTITUTE OF TECHNOLOGY INNOVATION
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-23
AI Technical Summary
There are few specific small molecule ligands developed for G4-RNA in existing technologies, resulting in low screening efficiency and difficulty in quickly discovering high-affinity ligands.
A fluorescent ligand for G-quadruplex RNA is provided, which uses a highly sensitive fluorescence signal to screen out small organic molecule ligands with high affinity for G-quadruplex RNA, and utilizes a high-throughput fluorescence screening system to evaluate the binding ability of a large number of compounds in a short time.
It improves the accuracy and efficiency of screening, enables the detection of ligand binding to G-quadruplex RNA at low concentrations, rapidly determines the affinity and bioactivity of candidate compounds, simplifies the operation process, and is suitable for large-scale production.
Smart Images
Figure CN122255127A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of a G-quadruplex RNA fluorescent ligand in the screening of antitumor drugs, belonging to the field of biomedicine. Background Technology
[0002] G-quadruplex RNA is a secondary structure formed from guanine-rich RNA sequences, characterized by high stability and structural complexity. In recent years, research on G4-RNA has deepened, particularly in the field of cancer biology. The regulation of many cancer-related genes is closely related to the formation of G4-RNA.
[0003] For example, G4 structures are commonly found in the promoter regions of oncogenes, such as c-MYC, KRAS, and HRAS. These G4 structures can regulate gene expression by inhibiting transcription factor binding or affecting RNA polymerase activity, thus playing an important role in cancer development. Therefore, G4-RNA is considered a potential target for cancer therapy, and scientists are working to understand its specific mechanisms of action in tumorigenesis in order to develop new therapeutic strategies.
[0004] The development of ligands targeting G4 RNA is of great significance in cancer therapy. Ligands are molecules that can specifically bind to the structure of G4 RNA. These molecules can regulate gene expression and cellular function by inducing or stabilizing G4 RNA formation. In recent years, significant progress has been made in the development of several G4 ligands, some of which have entered clinical trials. For example, the ligand CX5461 is currently in a phase II clinical trial in BRCA1 / 2-deficient breast and ovarian cancer patients. CX5461 interferes with ribosomal RNA synthesis by stabilizing the G-quadruplex structure, inhibiting topoisomerase II (Top2), and inducing synthetic lethal effects, leading to DNA damage and death in cancer cells.
[0005] Since there are currently few specific small-molecule ligands developed for G4-RNA, improving the specificity of small molecules for G-quadruplex RNA and discovering more new small molecules are important directions for future research. Therefore, constructing a ligand-G-quadruplex RNA fluorescence system to screen ligands with high affinity for G-quadruplex RNA has become a new method and approach. Using high-throughput fluorescence screening systems, researchers can evaluate the binding affinity of a large number of compounds in a short time, significantly improving screening efficiency. At the same time, high-throughput fluorescence screening also supports diverse compound libraries, enabling a broader range of exploration for new ligands, thereby accelerating the discovery and development of specific small-molecule ligands and promoting the progress of G-quadruplex RNA research. Summary of the Invention
[0006] Therefore, the first objective of this invention is to provide a G-quadruplex RNA fluorescent ligand that can specifically bind to G-quadruplex RNA and generate a highly sensitive fluorescent signal.
[0007] A second objective of this invention is to provide a method for preparing G-quadruplex RNA fluorescent ligands.
[0008] The third objective of this invention is to provide applications for G-quadruplex RNA fluorescent ligands, utilizing a fluorescence competition mechanism to screen for small organic molecule ligands with high affinity for G-quadruplex RNA. This method has the advantages of being simple to operate, highly sensitive, and fast in screening.
[0009] Therefore, the first technical solution provided by this invention is as follows:
[0010] A G-quadruplex RNA fluorescent ligand, the structural formula of which is shown below:
[0011] .
[0012] The second technical solution provided by this invention is a method for preparing the above-mentioned G-quadruplex RNA fluorescent ligand, which includes the following steps in sequence:
[0013] (1) Weigh 2-methylbenzothiazole and iodomethane in a mass ratio of 1.5-2:2, then add them to a solvent and reflux at 80-100℃ for 3-5 hours. After the reaction is completed, cool to room temperature, filter and wash to obtain compound a.
[0014] (2) 1,1,2-trimethyl-1H-benzo[e]indole and 2-iodoacetic acid were mixed in a molar ratio of 1:4 and refluxed at 70-80℃ for 20-28 hours. After cooling to room temperature, the mixture was filtered and washed to obtain compound b.
[0015] (3) Mix the compounds a and b in equal molar amounts, reflux them in a solvent at 60-80°C for 10-14 hours, cool to room temperature, filter and wash to obtain the target compound G-quadruplex RNA fluorescent ligand.
[0016] The synthetic route for the G-quadruplex RNA fluorescent ligand is as follows:
[0017]
[0018] Furthermore, in the above-mentioned method for preparing G-quadruplex RNA fluorescent ligands, the solvent used in steps (1) and (3) is acetonitrile.
[0019] Furthermore, in the above-mentioned method for preparing G-quadruplex RNA fluorescent ligands, the washing in steps (1) and (2) involves washing twice with ethyl acetate.
[0020] Furthermore, in the above-mentioned method for preparing G-quadruplex RNA fluorescent ligands, the washing step (3) involves washing twice with ethyl acetate and then twice with ethanol.
[0021] The third technical solution provided by this invention is the application of the above-mentioned G-quadruplex RNA fluorescent ligand as a probe for screening antitumor drugs.
[0022] The fourth technical solution provided by this invention is the application of the above-mentioned G-quadruplex RNA fluorescent ligand for recognizing G-quadruplex RNA.
[0023] The fifth technical solution provided by this invention is the above-mentioned method for screening antitumor drugs using G-quadruplex RNA fluorescent ligands. The G-quadruplex RNA fluorescent ligand compound described in the first technical solution is diluted and added to a 96-well plate. Then, G-quadruplex RNA is added, and fluorescence signals are collected. Next, different antitumor drugs to be detected are added to each well, and fluorescence signals are collected again. The two collected fluorescence signals are compared, and organic compounds with strong binding ability to G-quadruplex RNA are screened based on the changes in fluorescence signals.
[0024] Furthermore, in the above-mentioned method for screening antitumor drugs using G-quadruplex RNA fluorescent ligands, the G-quadruplex RNA sequence is 5'-GGCGGCGGCAGUGGCGGCGG-3'.
[0025] Furthermore, in the above-mentioned method for screening antitumor drugs using G-quadruplex RNA fluorescent ligands, the collection of fluorescence signals is achieved by using an enzyme-linked immunosorbent assay (ELISA) reader to collect fluorescence signals with an emission wavelength of 540 nm at an excitation wavelength of 460 nm.
[0026] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:
[0027] 1. The G-quadruplex RNA fluorescent ligand provided by this invention can specifically bind to G-quadruplex RNA and generate a highly sensitive fluorescence signal, improving the accuracy and reliability of screening. Simultaneously, the highly sensitive fluorescence signal allows researchers to detect the binding of the ligand to G-quadruplex RNA at low concentrations, further enhancing the sensitivity and efficiency of screening.
[0028] 2. The fluorescent ligands provided by this invention can be used as probes for screening antitumor drugs. By evaluating changes in fluorescence signals, the affinity and bioactivity of candidate compounds can be quickly determined, thereby accelerating the research and development process of antitumor drugs.
[0029] 3. The method for preparing G-quadruplex RNA fluorescent ligands provided by this invention is simple and easy to implement, the raw materials are readily available, the reaction conditions are mild, and it is conducive to large-scale production.
[0030] 4. The screening system constructed in this invention has the advantages of simple operation and fast screening efficiency. It can screen a large number of compounds in a short time and quickly identify small organic molecule ligands with high affinity for G-quadruplex RNA, providing a new and powerful tool for the development of anti-tumor drugs. Attached Figure Description
[0031] Figure 1 This is the 1H NMR spectrum of compound BYBC-1.
[0032] Figure 2 A bar chart showing the fluorescence data of various nucleic acids titrated with compound BYBC-1.
[0033] Figure 3 HCT116 live-cell imaging of compounds BYBC-1, QUMA-1, and Hoechst33342.
[0034] Figure 4 This is a bar graph showing the fluorescence signal changes of compound (BYBC-1) after it binds to G-quadruplex RNA (rKRAS) in a solution system, followed by the addition of organic compounds from an FDA-approved drug library.
[0035] Figure 5 Cellular imaging of compound (BYBC-1) after it binds to G-quadruplex RNA (rKRAS) in a cellular system and is subsequently added to an organic compound in a drug library approved by the U.S. Food and Drug Administration (FDA).
[0036] Figure 6 This is an immunoblot map showing the expression of G-quadruplex RNA-associated protein (KRAS protein) in colon cancer cells (HCT116) by 11 organic compounds from an FDA-approved drug library selected through high-throughput screening using BYBC-1 as a fluorescent probe. Detailed Implementation
[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0038] It should be noted that:
[0039] The library of 3,158 organic small molecule drugs approved by the U.S. Food and Drug Administration (FDA) described in this application is from Targetmol, catalog number L1010;
[0040] Website: https: / / www.targetmol.com / compound-library / fda_approved-pharmacopeia_drug_library.
[0041] Example 1
[0042] This embodiment provides a method for synthesizing G-quadruplex RNA fluorescent ligand (BYBC-1), which includes the following steps in sequence:
[0043] (1) 1.8 g of 2-methylbenzothiazole and 2 g of iodomethane were mixed in 25 mL of acetonitrile and refluxed at 90 °C for 4 hours. After cooling to room temperature, a white solid precipitated out. After filtration through 800 mesh, the solid was washed twice with ethyl acetate to obtain compound a.
[0044] (2) 1 g of 1,1,2-trimethyl-1H-benzo[e]indole and 4 g of 2-iodoacetic acid were refluxed at 75 °C for 24 hours. After cooling to room temperature, a white solid precipitated out. After filtration through 800 mesh, the solid was washed twice with ethyl acetate to obtain compound b.
[0045] (3) Mix compound a prepared in step (1) and compound b prepared in step (2) in equal molar amounts, and reflux in acetonitrile at 70 °C for 12 hours. After cooling to room temperature, a yellow solid precipitates out. After filtration, wash twice with ethyl acetate and twice with ethanol to obtain compound G-quadruplex RNA fluorescent ligand (BYBC-1).
[0046] The compound BYBC-1 obtained in this example was analyzed using nuclear magnetic resonance (NMR). The 1H NMR spectrum of compound BYBC-1 is shown in the attached image. Figure 1 The 1H NMR spectrum results were obtained: 1H NMR (400 MHz, DMSO) δ 8.21 (d, J = 8.2Hz, 2H), 8.05 (d, J = 8.6 Hz, 2H), 8.01 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.8Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 8.2 Hz, 2H), 7.52 (t, J = 7.5Hz, 1H), 6.17 (s, 1H), 5.18 (s, 2H), 4.05 (s, 3H), 2.05 (s, 6H).
[0047] To verify the performance of compound BYBC-1 provided in this application, the following verification experiment of compound BYBC-1 is given.
[0048] 1. Specific fluorescent recognition of G-quadruplex RNA in aqueous solution system.
[0049] The nucleic acid samples were purchased from Invitrogen Biotechnology Ltd.
[0050] The method includes the following steps in sequence:
[0051] 1) Dissolve the nuclei shown in Table 1 in Tris-HCl buffer (pH=7.4, 100 mM Tris, 60 mM KCl), heat at 95℃ for 5 min and then slowly cool and anneal to room temperature to obtain 0.1 mM oligonucleotides as nucleic acid storage solution, and store at 4℃.
[0052] Table 1 Nucleic Acid Sequences
[0053]
[0054] 2) 1 mM of the compound BYBC-1 prepared in Example 1 was diluted to a stock solution of 1 μM in Tris-HCl buffer (pH=7.4, 100 mMtris, 60 mM KCl). Then, different types of nucleic acid stock solutions were added to achieve a final nucleic acid concentration of 10 μM. The fluorescence intensity for different nucleic acids was measured using a fluorescence spectrophotometer (slit width=10, scan speed=200 nm, Ex=460 nm). A bar chart of the fluorescence data from the probe (BYBC-1) titration of various nucleic acids is provided. Figure 2 .
[0055] from Figure 2It can be seen that after compound BYBC-1 binds to G-quadruplex RNA (rTERRA, rTRF2, rVEGF, rBcl2, rNRAS, and rKRAS), the fluorescence intensity is 15 to 60 times higher than that of double-stranded DNA (dDs26), ribosomal RNA (rRNA), ribosomal double-stranded RNA fragment (rRNA hairpin), double-stranded RNA (rHP18), and single-stranded RNA (rSS26). This proves that compound BYBC-1 can specifically fluorescently recognize G-quadruplex RNA in an aqueous system.
[0056] II. Cell Imaging Experiments
[0057] Cell culture: After seeding colon cancer cells (HCT116) into culture flasks, they were cultured in DMEM medium containing 10% fetal bovine blood for 48 hours at 37 ℃ and 5% CO2.
[0058] Cell seeding: Seed the cultured cells in a confocal culture dish to a density of approximately 1 × 10⁻⁶ cells / mL. 5 The cells were collected at a concentration of 1 / mL and then incubated at 37 °C in a 5% CO2 environment for 24 hours.
[0059] Staining: Cells were stained with the commercial nuclear probe Hoechst33342 for 30 minutes in the dark, washed 3 times with 1×PBS, and then incubated with 5 μM of the commercial G-quadruplex RNA probe QUMA-1 in the dark for 3 hours. Finally, the cells were incubated with 1 μM of the compound BYBC-1 in the dark for 30 minutes.
[0060] Laser confocal microscopy: During the detection process, the commercial nuclear probe Hoechst33342 was detected under blue light, the commercial G-quadruplex RNA probe QUMA-1 under red light, and compound BYBC-1 under green light. HCT116 live-cell imaging images of compounds BYBC-1, QUMA-1, and Hoechst33342 obtained in Example 1 are shown below. Figure 3 .
[0061] from Figure 3 (Scale bar is 20 micrometers) It can be seen that BYBC-1 and the commercial G-quadruplex RNA probe QUMA-1 have the same fluorescence imaging target, and the colocalization coefficient between the two is 0.92, which proves that BYBC-1 has the ability to detect G-quadruplex RNA in live cells. Compared with the commercial G-quadruplex RNA probe QUMA-1, the time required for BYBC-1 to enter the cell is only 30 minutes, which is much less than the staining time required by QUMA-1 (3 hours). In the imaging study of G-quadruplex RNA in live cells, BYBC-1 requires a lower concentration and enters the cell faster.
[0062] III. High-throughput screening experiments using solution systems
[0063] The compound BYBC-1 prepared in Example 1 was diluted to a concentration of 1 μM with Tris-HCl buffer (pH=7.4, 100 mM Tris, 60 mM KCl). 100 μL of this solution was added to each well of a 96-well plate, followed by 1 μM of G-quadruplex RNA (rKRAS: sequence 5'-GGCGGCGGCAGUGGCGGCGG-3', SEQ ID NO: 10). Fluorescence was collected using a microplate reader at an excitation wavelength of Ex=460 nm and an emission wavelength of Em=540 nm. Subsequently, 3158 organic compounds from the FDA-approved drug library (final concentration in the 96 wells was 20 μM) were added sequentially to each well of the 96-well plate. Fluorescence was collected again using a microplate reader at an excitation wavelength of Ex=460 nm and an emission wavelength of Em=540 nm. The fluorescence signals before and after the addition of the organic compounds from the drug library were compared, and a bar graph of fluorescence signal change was plotted based on the degree of fluorescence signal attenuation. (See reference). Figure 4 The 11 selected compounds are shown in Table 2.
[0064] Table 2. Compounds selected by high-throughput screening
[0065]
[0066] pass Figure 4 It can be seen that the 11 selected compounds significantly reduced or enhanced the fluorescence signal after BYBC-1 bound to G-quadruplex RNA (rKRAS), indicating that the selected compounds may have a strong affinity for G-quadruplex RNA (rKRAS) and compete with BYBC-1, leading to significant changes in fluorescence signal. This result supports the ability of BYBC-1 to screen for G-quadruplex RNA binding ligands in solution systems.
[0067] IV. High-throughput screening experiments in cell systems (cell systems)
[0068] Cell culture: After seeding colon cancer cells (HCT116) into culture flasks, they were cultured in 10% fetal bovine blood DMEM medium at 37 ℃ and 5% CO2 for 48 hours.
[0069] Cell seeding: Seed the cells in 96-well plates to a density of approximately 8 × 10⁶ cells / well. 4 The cells were collected at a concentration of 1 / mL and then incubated at 37°C with 5% CO2 for 24 hours.
[0070] Staining: Cells were incubated with 1 μM of the compound BYBC-1 prepared in Example 1 in a dark environment for 30 minutes, and then incubated with an organic compound from the drug library for 1 hour.
[0071] Laser confocal microscopy detection: During the detection process, the excitation wavelength is 488 nm, and the collected emission wavelength range is 520~560 nm.
[0072] Figure 5 Fluorescence imaging images of compound BYBC-1 obtained in Example 1 and 11 organic compounds screened from the drug library in HCT116 live cells are shown, with a scale bar of 20 micrometers. Figure 5 As can be seen, the 11 selected compounds significantly altered the fluorescence signal intensity of BYBC-1 in HCT116 cells. Some compounds caused a significant decrease in fluorescence signal, while others enhanced it. This suggests that the selected compounds may have a strong affinity for G-quadruplex RNA (rKRAS) and may influence the binding ability of BYBC-1 to G-quadruplex RNA through competitive binding. In summary, these results support the effectiveness of the selected compounds as potential G-quadruplex RNA binding ligands in HCT116 cells.
[0073] V. Immunoblotting experiment
[0074] First, the effects of the 11 compounds screened in Example 3 on colon cancer cells (HCT116) were measured, and then the effects on the KRAS protein, which is downstream of G-quadruplex RNA, were further measured.
[0075] Colon cancer cells (HCT116) were cultured in 96-well plates at a rate of 8 × 10⁻⁶. 4 Each well was incubated overnight, followed by incubation with 11 compounds screened in Example 3 at different concentrations for 48 hours. The half-maximal inhibitory concentration (IC50) was determined using the MTT assay (tetrazole salt reduction method). 50 ), to obtain the IC50 of these 11 compounds. 50 As shown in Table 3.
[0076] Table 3. Half-inhibitory concentrations of 11 compounds selected by high-throughput screening after 48 hours of treatment on colon cancer cells (HCT116).
[0077]
[0078] KRAS protein immunoblotting assay
[0079] Colon cancer cells (HCT116) were cultured in 6-well plates to the logarithmic growth phase. Cells were then cultured either untreated or treated with one of the 11 compounds screened in Example 5 using a 1x IC50 assay. 50(As shown in Table 3) Incubate for 48 hours. Collect cells by centrifugation and wash three times with PBS. After thoroughly mixing the cells with RIPA lysis buffer, incubate on ice for 20 minutes, then centrifuge at 10,000 rpm for 20 minutes at 4°C. Total protein concentration is quantified using a BCA kit. Load the same amount of protein onto an SDS-PAGE gel and transfer the separated protein to a PVDF membrane. After blocking with 5% skim milk powder, the PVDF membrane is incubated with KRAS (Abcam, catalog number ab275876, dilution 1:1000) and endogenous control primary antibody β-actin (Affinity, AB_2839420, dilution 1:1000) at 37°C for 1 hour. Wash the membrane three times with TBST buffer for 30 minutes each time, then incubate with goat anti-rabbit IgG (H+L) HRP secondary antibody (Affinity, AB_2839429, dilution 1:5000) for 2 hours. The membrane was washed three times with TBST buffer, each time for 30 minutes. Finally, the PVDF membrane was scanned and the data was processed using the ECL system.
[0080] Figure 6 The image shows the immunoblot of 11 organic compounds from the FDA-approved drug library selected through high-throughput screening in Example 6, and their effects on the expression of G-quadruplex RNA-associated protein (KRAS protein) in colon cancer cells (HCT116). The results show that 8 of the 11 compounds significantly inhibited KRAS protein, achieving an accuracy of 73% in the initial screening. This confirms that BYBC-1 has the ability to screen compounds with strong affinity for G-quadruplex RNA.
[0081] In summary, the G-quadruplex RNA fluorescent ligands provided by this invention possess a significant ability to screen small organic molecules with high affinity for G-quadruplex RNA. In a library of 3158 FDA-approved small organic molecule drugs, changes in fluorescence signals effectively identified drugs with strong G-quadruplex RNA binding affinity. Further immunoblotting experiments showed that 8 out of 11 organic compounds screened in the high-throughput screening significantly downregulated the expression of key downstream proteins related to G-quadruplex RNA, achieving a screening efficiency of 73%. Furthermore, the method developed in this invention for rapidly screening G-quadruplex RNA-binding ligands in solution and cell systems provides a new tool for G-quadruplex RNA-related biological research, accelerating the screening and development of anti-tumor drugs.
Claims
1. A G-quadruplex RNA fluorescent ligand, characterized in that, The structural formula of the G-quadruplex RNA fluorescent ligand is shown below: 。 2. The method for preparing the G-quadruplex RNA fluorescent ligand according to claim 1, characterized in that, The steps are as follows: (1) Weigh 2-methylbenzothiazole and iodomethane in a mass ratio of 1.5-2:2, then add them to a solvent and reflux at 80-100℃ for 3-5 hours. After the reaction is completed, cool to room temperature, filter and wash to obtain compound a. (2) 1,1,2-trimethyl-1H-benzo[e]indole and 2-iodoacetic acid were mixed in a molar ratio of 1:4 and refluxed at 70-80℃ for 20-28 hours. After cooling to room temperature, the mixture was filtered and washed to obtain compound b. (3) Mix the compounds a and b in equal molar amounts, reflux them in a solvent at 60-80°C for 10-14 hours, cool to room temperature, filter and wash to obtain the target compound G-quadruplex RNA fluorescent ligand.
3. The method for preparing G-quadruplex RNA fluorescent ligands according to claim 2, characterized in that, The solvent used in steps (1) and (3) is acetonitrile.
4. The method for preparing G-quadruplex RNA fluorescent ligands according to claim 2, characterized in that, The washing described in steps (1) and (2) involves washing twice with ethyl acetate.
5. The method for preparing G-quadruplex RNA fluorescent ligands according to claim 2, characterized in that, The washing process described in step (3) involves washing twice with ethyl acetate and then twice with ethanol.
6. The application of the G-quadruplex RNA fluorescent ligand as described in claim 1 as a probe for screening antitumor drugs.
7. The application of the G-quadruplex RNA fluorescent ligand according to claim 1 for recognizing G-quadruplex RNA.
8. The method for screening antitumor drugs using the G-quadruplex RNA fluorescent ligand as described in claim 1, characterized in that, The G-quadruplex RNA fluorescent ligand of the compound described in claim 1 was diluted and added to a 96-well plate, followed by the addition of G-quadruplex RNA. Fluorescence signals were collected, and then different antitumor drugs to be detected were added to each well, and fluorescence signals were collected again. The two collected fluorescence signals were compared, and organic compounds with strong binding ability to G-quadruplex RNA were screened based on the changes in fluorescence signals.
9. The method for screening antitumor drugs using G-quadruplex RNA fluorescent ligands according to claim 1, characterized in that, The G-quadruplex RNA sequence is 5'-GGCGGCGGCAGUGGCGGCGG-3'.
10. The method for screening antitumor drugs using G-quadruplex RNA fluorescent ligands according to claim 1, characterized in that, The fluorescence signal was collected using an enzyme-linked immunosorbent assay (ELISA) reader at an excitation wavelength of 460 nm to collect the fluorescence signal emitted at an emission wavelength of 540 nm.