Small molecule compound for regulating and controlling catalytic activity of tetrahymena ribozyme as well as screening method and application of small molecule compound

Abstract

The invention discloses a small molecule compound for regulating and controlling the catalytic activity of tetrahymena ribozyme and a screening method and application thereof, based on FRET (Fluorescence Resonance Energy Transfer) high-throughput entity screening, virtual screening, a biological membrane interference technology, a freezing electron microscope structure analysis technology and other technologies are combined to form a set of complete and efficient RNA (Ribonucleic Acid) targeted small molecule compound extraction method; the tetrahymena ribozyme is specifically subjected to small molecule compound screening; aiming at research and regulation of tetrahymena ribozyme; meanwhile, the invention aims to develop a high-throughput and systematic small-molecule compound screening method which is used for rapidly and efficiently screening potential small-molecule inhibitors so as to accelerate the discovery and research and development process of new drugs.

Description

Technical Field

[0001] The present application relates to the technical field of small molecule compound screening, and in particular to a small molecule compound that regulates the catalytic activity of a Tetrahymena ribozyme, a screening method, and uses. Background Art

[0002] The Tetrahymena ribozyme, one of the earliest discovered ribozymes, possesses the property of self-splicing, allowing it to cleave its own RNA molecules under specific conditions. This cleavage mechanism has been extensively studied in the RNA field and has shed important light on RNA splicing and modification. The unique properties and readily available accessibility of the Tetrahymena ribozyme have enabled further research on Tetrahymena, providing a crucial tool for understanding ribozyme mechanisms and RNA biology. In recent years, circular RNA has gained increasing attention as a potential vaccine vector. The self-splicing nature of the Tetrahymena ribozyme results in a circular intronic RNA as its final product. Researchers have successfully rearranged the structure of the Tetrahymena ribozyme, enabling its use in the production of circular RNA. This discovery has sparked renewed interest in and research on the Tetrahymena ribozyme, providing new possibilities for the application of circular RNA and enriching the research landscape of ribozymes and RNA.

[0003] The above reasons fully demonstrate that the research on Tetrahymena group I introns is still of broad importance. The addition of small molecule inhibitors / activators can further deepen people's understanding of the splicing mechanism of Tetrahymena group I introns and introduce regulatory factors for the production of circular RNA. Therefore, the study of Tetrahymena ribozyme catalysts is also of considerable importance. Existing Tetrahymena ribozyme inhibitors have disadvantages such as poor activity and poor specificity, which greatly limit their effectiveness and targetability in subsequent applications. At the same time, there is also a lack of explanation of the mechanism of compound inhibition of catalytic activity based on the three-dimensional structure of RNA. On the other hand, there is no high-throughput, systematic compound screening method in existing research, which makes the research on Tetrahymena ribozyme inhibitors still in a relatively preliminary stage, and it is impossible to conduct more in-depth research on the three-dimensional structure of Tetrahymena ribozymes. Summary of the Invention

[0004] The present invention aims to provide a small molecule compound that modulates the catalytic activity of the Tetrahymena ribozyme, as well as a screening method and uses thereof. Based on the aforementioned research status, the present invention aims to develop a highly effective, active, and specific small molecule inhibitor for the study and regulation of the Tetrahymena ribozyme. Furthermore, the present invention aims to develop a high-throughput, systematic screening method for small molecule compounds to rapidly and efficiently screen for potential small molecule inhibitors, thereby accelerating the discovery and development of new drugs.

[0005] In order to solve the above technical problems, the present invention adopts the following solutions:

[0006] A method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes comprises the following steps:

[0007] S1, obtain all the docking binding sites of Tetrahymena ribozyme, perform virtual screening of the entity compounds in the CSTAR compound library through Dock6 molecular docking, and obtain the top-ranked small molecule compounds to be screened;

[0008] S2, designing a fluorescent group substrate Cy3-CCCUCUAAACC-FAM, and detecting the activity of the small molecule compounds to be screened by fluorescence resonance energy transfer. First, a dual-concentration inhibition rate test was performed, and then a concentration-inhibition rate dependence test was performed to analyze the inhibitory effect of the small molecule compounds to be screened, and obtain the secondary screening small molecule compounds;

[0009] S3, using biomembrane interferometry to analyze the affinity between the secondary screening small molecule compounds and the Tetrahymena nuclear membrane loaded on the sensor, and fitting and calculating the affinity constant K D ;

[0010] S4, through cryo-electron microscopy analysis of the complex structure of secondary screened small molecule compounds with both inhibitory activity and affinity activity and Tetrahymena ribozyme, the inhibitory mechanism of the compound was analyzed based on the complex structure, and the small molecule compound that regulates the catalytic activity of Tetrahymena ribozyme was obtained.

[0011] Furthermore, step S1 includes the following steps:

[0012] S10, convert the entity compounds in the CSTAR compound library into Mol2 format, and use Open Babel3.1.1 to remove the non-main part of the entity compounds;

[0013] S11, removing the interfering docking region of the Tetrahymena ribozyme model in the protein database to obtain the modified Tetrahymena ribozyme model;

[0014] S12, adding hydrogen atoms to the modified Tetrahymena ribozyme model and assigning AM1-BCC charges to generate the binding site;

[0015] S13, visual inspection was used to exclude potential nonspecific docking regions and the size was obtained. The grid is used for Dock6 molecular docking. The docking results are ranked according to the grid scores and screened using molecular visualization tools to obtain small molecule compounds to be screened.

[0016] Furthermore, step S2 includes the following steps:

[0017] S20, extracting Tetrahymena ribozyme RNA, denaturing and renaturing it, and then adding GTP and shearing buffer to obtain a reaction mixture;

[0018] S21, dissolving the small molecule compound to be screened, adding it to the reaction mixture, and detecting the fluorescence interference of the compound I int , while detecting the fluorescence background I0 in the wells without compound addition;

[0019] S22, after adding the fluorescent group substrate, the mixture was incubated at 25°C for 15 minutes, and the fluorescence intensity I in this state was detected. cpd ;

[0020] Among them, the wells without Tetrahymena ribozyme RNA were used as positive controls. pos ; Add Tetrahymena ribozyme RNA to the well as a negative control 1 neg ;

[0021] The activity of the small molecule compounds to be screened is expressed by the relative FAM fluorescence intensity value;

[0022] S23, obtaining a secondary screening small molecule compound having a concentration-dependent effect on Tetrahymena ribozyme RNA.

[0023] Furthermore, the calculation formula for the relative FAM fluorescence intensity of the small molecule compound to be screened is:

[0024]

[0025] Furthermore, a dose-response experiment was conducted on the small molecule compounds to be screened whose inhibitory activity against Tetrahymena ribozyme exceeded 60% at a concentration of 1000 μM. Each compound was tested at 6 concentrations ranging from 1000 μM to 31 μM. The inhibition rate at each concentration was calculated as follows:

[0026]

[0027] IC 50 Values were calculated by dose-response inhibition analysis using the following formula:

[0028]

[0029] Among them, Top and Bottom represent the platform values of the Y-axis unit, and HillSlope describes the steepness of the curve family.

[0030] Furthermore, step S3 includes the following steps:

[0031] S30, Tetrahymena ribozyme RNA was labeled with biotin and coupled to the surface of the superstreptavidin biosensor;

[0032] S31, analysis cycle: including obtaining a baseline, binding of the superstreptavidin biosensor to the secondary screening small molecule compound, and dissociation in the binding buffer; after the superstreptavidin biosensor is cleaned, the next analysis cycle is entered;

[0033] S32, affinity constant K was calculated by fitting using Octet Data Analysis HT software D .

[0034] Furthermore, the calculation formula is:

[0035]

[0036] Among them, K D The value is calculated by steady-state kinetic analysis, [Compound] represents the concentration of the compound, R eq is the estimated equilibrium response value, k on is the binding rate constant, k off is the dissociation rate constant, R max Maximum binding of Tetrahymena ribozyme RNA to secondary screening small molecule compounds.

[0037] Furthermore, step S4 includes the following steps:

[0038] S40, the renatured Tetrahymena ribozyme RNA was mixed with the secondary screening small molecule compound and loaded onto a copper grid. The frozen copper grid was loaded into a Titan Krios electron microscope and images were collected.

[0039] S41,

[0040] MotionCorr2 was used to correct the motion of the collected photos, and then Fourier transform analysis was performed using CTFFIND4 to remove photos of poor quality;

[0041] S42, the remaining photos are sequentially input into EMAN2 and Relion4 for neural network particle selection and two-dimensional classification of particles, respectively, to remove invalid particles and obtain valid particles;

[0042] S43, the effective particles were three-dimensionally classified using CryoSPARC to obtain a complex density map;

[0043] S44, the density map of the composite image was optimized by Relion4 and CryoSPARC to obtain the resolution-optimized structural density of the composite;

[0044] S45, performing density-atomic model differential analysis on the structure density of the complex after resolution optimization, to identify small molecule compounds that modulate the catalytic activity of the Tetrahymena ribozyme. A small molecule compound that modulates the catalytic activity of the Tetrahymena ribozyme was screened using the above-mentioned screening method for small molecule compounds that modulate the catalytic activity of the Tetrahymena ribozyme, including ZPT01 and ZPT70. The binding site of ZPT01 is site 1 of the Tetrahymena ribozyme, with an inhibitory activity of 9.8 μM and a binding activity of 25 μM.

[0045] The binding site of ZPT70 is site 2 of Tetrahymena ribozyme, with an inhibitory activity of 230 μM and a binding activity of 190 μM.

[0046] A use of a small molecule compound for regulating the catalytic activity of a Tetrahymena ribozyme in inhibiting the cleavage activity of the Tetrahymena ribozyme.

[0047] The beneficial effects of the present invention are:

[0048] This invention combines high-throughput FRET-based real-world screening with virtual screening, biomembrane interferometry (BLI), and cryo-electron microscopy structure analysis to create a comprehensive and efficient method for extracting RNA-targeted small-molecule compounds. It specifically screens small-molecule compounds targeting Tetrahymena ribozymes. All compounds in the CSTAR compound library are real compounds and can be purchased directly, eliminating the time-consuming molecular synthesis required in a virtual compound library. This significantly shortens the time from screening to experimental verification, providing greater timeliness for drug discovery.

[0049] In the present invention, the complete structure of the target is input into Dock6, and all possible binding sites are identified through virtual docking, which solves the problem of screening targets with unknown binding sites. At the same time, the present invention can comprehensively cover potential binding sites, avoiding data omissions that may result from screening of a single binding site.

[0050] The present invention integrates cryo-electron microscopy structure analysis technology, which can not only clarify the activity of small molecule compounds, but also deeply reveal their mechanism of action, providing a structural basis and theoretical basis for subsequent drug optimization, significantly improving the scientific nature and operability of drug development, and ultimately screening out two small molecule compounds, ZPT01 and ZPT70, to inhibit the cleavage activity of Tetrahymena ribozyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the Tetrahymena ribozyme binding site after molecular docking of the present invention;

[0052] Figure 2Schematic diagram of the present invention's screening of compounds ZPT01 and ZPT70 with inhibitory and binding activities using the HitSTARS process. a is a schematic diagram of the process, b is a compound with a high score, c is ZPT01 and ZPT70 bound to site 1 and site 2 of the Tetrahymena ribozyme, respectively, and d is the structure, inhibitory activity, and binding activity of ZPT01 and ZPT70, respectively.

[0053] Figure 3 This is a schematic diagram of the complex structure analyzed using cryo-electron microscopy in the present invention, a and b are the binding pockets composed of ZPT70 binding to the Tetrahymena ribozyme, c is a schematic diagram of the new conformation formed, and d is a schematic diagram of the inhibitory matrix of ZPT70. DETAILED DESCRIPTION

[0054] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Unless otherwise specifically stated, the relative arrangement of components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention.

[0056] Technologies, methods and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, such technologies, methods and equipment should be considered part of the authorization specification.

[0057] In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0058] Example

[0059] The present application is a method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes, referring to Figure 2 a, including the following steps:

[0060] (1) Virtual screening: All docking binding sites of Tetrahymena ribozyme were obtained, and the entity compounds in the CSTAR compound library were virtually screened through Dock6 molecular docking to obtain the top-ranked small molecule compounds to be screened.

[0061] The entity compounds in the CSTAR compound library (cstar.zsulab.com) were converted to Mol2 format and processed using Open Babel 3.1.1 to remove non-main components such as HCl and BH3 in the entity compounds and retain only the largest continuous fragments.

[0062] The Tetrahymena ribozyme model was obtained from the Protein Data Bank (PDB code 7EZ0) and modified to remove the single-stranded 5' end (nucleotides 22-27), P6 (nucleotides 227-248), and P9.2 (nucleotides 372-399) to generate the grid.

[0063] Hydrogen atoms were added to the modified Tetrahymena ribozyme model and AM1-BCC charges were assigned. Spherical clusters were generated on its surface, representing the space where the compound might bind, i.e., the site to be bound.

[0064] The clusters were visually inspected to exclude potential nonspecific docking regions, and the resulting clusters formed a cluster with a size of The grid was used for molecular docking in DOCK6, with all other parameters set to default values. Docking results were ranked according to grid score, and the top 10,000 small molecule complexes with the Tetrahymena ribozyme target were visually analyzed in Chimera to identify binding pockets and generate small molecule compounds for screening.

[0065] Reference Figure 1 After molecular docking, 6 binding sites were found, among which site 1 and 2 bound more than 99% of the compounds and were the main binding sites. Site 3-6 could accommodate the docking of compounds, but the scores were poor.

[0066] (2) Fluorescence resonance energy transfer (FRET) detection of the activity and inhibitory effect of the small molecule compounds to be screened: a fluorescent group substrate Cy3-CCCUCUAAACC-FAM was designed, and the activity of the small molecule compounds to be screened was detected by fluorescence resonance energy transfer. Then, a concentration-inhibition rate dependence test was performed to analyze the inhibitory effect of the small molecule compounds to be screened, and the secondary screening small molecule compounds were obtained.

[0067] Extraction of Tetrahymena ribozyme RNA: Tetrahymena ribozyme RNA was denatured in 50 mM Na-HEPES (pH 8.0) at 90°C for 3 min, then cooled to room temperature for 10 min, and MgCl2 was added to a final concentration of 10 mM. The sample was incubated at 50°C for 30 min and then cooled to 25°C for 10 min.

[0068] Add Cy3 / FAM-labeled substrate: Transfer a mixture containing buffer and renatured Tetrahymena ribozyme RNA (final concentration: 100 nM) to the assay plate, followed by the addition of 100 μM GTP. Add Cy3 / FAM-labeled substrate (final concentration: 200 nM) to the reaction mixture and initiate the reaction. Seal the plate with film, protect from light, and incubate at 25°C for 15 minutes. After brief centrifugation, perform fluorescence detection.

[0069] Cy3- and FAM-labeled RNA oligonucleotide substrates (5'-Cy3-CCCUCUAAACC-FAM-3') were purchased from Sangon Biotech. FRET screening experiments were performed in 384-well black polypropylene plates (Corning, Cat. No. 3677) in a 10 μl reaction volume containing 50 mM Tris-HCl (pH 7.5), 100 mM (NH₄)₂SO₄, and 5 mM MgCl₂.

[0070] Add the small molecule compound to be screened: Dissolve the small molecule compound to be screened in DMSO and dilute it with DMSO to 20 times the final concentration, then add it to the reaction mixture. The final DMSO concentration is 5%. Before adding the fluorescent group substrate, perform a fluorescence reading to evaluate the fluorescence interference of the compound (I int , indicating interference intensity) and the fluorescence background (I0, indicating background intensity) in the wells without adding the compound. After adding the substrate, the mixture was incubated at 25°C for 15 minutes, and then the fluorescence was detected again according to the same steps (I cpd , indicating the compound strength). In the compound inhibitory activity detection experiment, the wells without Tetrahymena ribozyme were used as positive controls (I pos , indicating 100% inhibition), while the wells to which Tetrahymena ribozyme was added served as negative controls (I neg , indicating 0% inhibition). Both controls contained 5% DMSO.

[0071] Fluorescence detection was performed using a CLARIOstar Plus (BMG LABTECH) microplate reader with an excitation filter of 482 nm (bandwidth 16 nm) and an emission filter of 530 nm (bandwidth 20 nm).

[0072] The relative FAM intensity of each small molecule compound to be screened was calculated using the following formula:

[0073]

[0074] Compounds with L-16 inhibitory activity exceeding 60% at a concentration of 1000 μM were further subjected to dose-response experiments. Each small molecule compound to be screened was tested at 6 concentrations (1000 μM to 31 μM). The inhibition rate at each concentration was calculated using the following formula:

[0075]

[0076] IC 50 Values were calculated by dose-response inhibition analysis using the following formula:

[0077]

[0078] Among them, Top and Bottom represent the platform values of the Y-axis unit, and HillSlope describes the steepness of the curve family.

[0079] Secondary screening of small molecule compounds was performed based on relative FMA intensity and inhibition rate.

[0080] (3) Use biomembrane interferometry (BLI) to analyze the affinity between the secondary screening small molecule compounds and the sensor loaded with the Tetrahymena nuclear membrane, and fit and calculate the affinity constant K d .

[0081] Biotinylation of Tetrahymena ribozyme RNA: 1 nmol of Tetrahymena ribozyme RNA was mixed with 10 μl of 0.5 M NaIO4 and 10 μl of 0.5 M NaOAc (pH 5.2) and diluted to 50 μl with RNase-free water. The reaction mixture was incubated at room temperature in the dark for 1.5 h. After incubation, the reaction was terminated and the 3'-oxidized RNA was recovered by isopropanol precipitation. The precipitate was washed with isopropanol, dried, and dissolved in 40 μl of RNase-free water. Subsequently, 10 μl of 50 mM biotin hydrazide was added, and the mixture was incubated at room temperature in the dark for 12 h. After incubation, phenol extraction was performed, and the biotinylated Tetrahymena ribozyme RNA was recovered by isopropanol precipitation. The precipitate was washed with isopropanol, dried, and dissolved in RNase-free water. The precipitate was quantified using a NanoDrop spectrophotometer (ThermoScientific) and stored at -80°C until use.

[0082] Calculation of biotinylation efficiency: For analysis by gel shift assay, 20 ng of biotinylated Tetrahymena ribozyme RNA was incubated with excess streptavidin (1 μg) in a buffer containing 10 mM Tris (pH 7.4), 2.5 mM MgCl2, and 100 mM NaCl for 15 min. The mixture was then mixed with loading buffer (250 mM Na-HEPES, pH 7.5, 5 mM EDTA, pH 8.0, 50% glycerol, 0.1% xylene cyanol, and 0.1% bromophenol blue). The sample was loaded onto a 6% 29:1 acrylamide:bisacrylamide polyacrylamide gel and electrophoresed at 10 W for 1 h. The gel was stained with SYBR-Gold (Invitrogen) for 5 min and imaged using a Bio-Rad ChemiDoc XRS+ UV-transmitter molecular imager. The biotinylation efficiency was calculated by comparing the band intensity of the biotinylated RNA-streptavidin complex with that of unmodified RNA.

[0083] Analysis cycle: Tetrahymena ribozyme RNA was renatured according to the method in step (2) and coupled to the surface of superstreptavidin biosensor (SSA, Sartorius) with a coupling time of 600 seconds. The optimal response value was maintained between 0.5 and 2 nm, and the response value difference of sensors in the same row should not exceed 0.2 nm. A typical analysis cycle includes: obtaining a baseline in 1× binding buffer (50mM Na-HEPES (pH 8.0), 10mM MgCl2, 0.02% Tween-20) (60s), binding in the wells containing secondary screening small molecule compounds (90s), and dissociation in 1× binding buffer (90s). After each cycle, the superstreptavidin biosensor was washed in 1× binding buffer for 120s before entering the next cycle. At the same time, a group of superstreptavidin biosensors without RNA coupling were used as controls and the same operation was performed.

[0084] The experimental data were corrected by first subtracting the response of the uncoupled RNA sensor and then subtracting the response of the sensor coupled to RNA but without the addition of the secondary screening small molecule compound. Data analysis and curve fitting were performed using Octet DataAnalysis HT software (version 12.0). The experimental data were fitted using a 2:1 heterogeneous ligand (HL) model. Assuming reversible binding, the data were globally analyzed using nonlinear least squares for different compound concentrations. The calculation formula is:

[0085]

[0086] K D The value was calculated by steady-state kinetic analysis, and the equilibrium response value (Req ), where [Compound] represents the concentration of the compound, R eq is the estimated equilibrium response value, k on is the binding rate constant, k off is the dissociation rate constant.

[0087] The high affinity of biotin and streptavidin is used to immobilize the Tetrahymena ribozyme RNA on the SSA sensor dedicated to the Octet instrument, and then a secondary screening test of the binding of small molecule compounds to the Tetrahymena ribozyme RNA is performed.

[0088] (4) Cryo-electron microscopy structure analysis: The complex structure of small molecule compounds and Tetrahymena ribozyme is screened by cryo-electron microscopy analysis. The conformation with the best complex effect is screened out from the complex structure, and the small molecule compound that regulates the catalytic activity of Tetrahymena ribozyme is obtained.

[0089] Sample preparation: The renatured Tetrahymena ribozyme RNA was mixed with the small molecule compound screened in the secondary screening to obtain a complex sample with a final concentration of 2 μM Tetrahymena ribozyme RNA and 2 mM small molecule compound in a total volume of 10 μl. The mixture was incubated at room temperature for 15 minutes and then cooled on ice for later use. 3 μl of the complex sample was dropped onto a 200-mesh R2-1 Quantifoil copper grid that had been glow-discharged (45 seconds). Under 100% humidity and 4°C conditions, the grid was blotted dry with filter paper for 2.5 seconds, then quickly frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher), and then transferred to liquid nitrogen for "pressing", that is, fixing the grid to an O / C ring, and finally transferred to liquid nitrogen for storage or directly applied to the machine.

[0090] Data collection: The frozen copper grid was loaded into a Titan Krios electron microscope (Thermo Fisher) at 300 kV, a condenser aperture of 50 μm, a spot size of 6, and a parallel beam illumination area with a diameter of 1.03 μm. The microscope magnification was set to 165,000 times (corresponding to a calibration sampling rate of / pixel), and data were collected automatically using EPU software on a K2 direct electron camera. The camera was equipped with a Bioquantum energy filter and operated in count mode with a recording rate of 5 frames per second, a total exposure time of 6 seconds, and 30 frames per image. The preset defocus value range was -1.0 to -1.5 μm, and the total electron count was approximately

[0091] Data processing:

[0092] 1) The collected photos were first subjected to displacement correction using MotionCorr2, and then Fourier transform analysis was performed using CTFFIND4 to remove photos of poor quality;

[0093] 2) The remaining photos are sequentially fed into EMAN2 for neural network particle selection, followed by two-dimensional particle classification (2D classification) in Relion4. In this step, invalid particles are removed to obtain valid particles.

[0094] 3) Use CryoSPARC to further perform 3D classification on the valid particles to obtain a complex density map. At this point, the structure of Tetrahymena ribozyme L-16 has been resolved and can be used as a reference for 3D classification. From the obtained 3D classification results, select one or more categories with the most obvious RNA features, the highest resolution, and the largest number of particles for further optimization to obtain the complex structure density after resolution optimization. This is the optimal resolution. Resolution optimization generally uses refine 3D (Relion4) and homogeneous / local / non-uniform refinement (cryoSPARC).

[0095] 4) Perform density-atomic model differential analysis on the complex density after resolution optimization. Check whether the obtained structure contains compound density by subtracting the atomic model from the structural density to identify small molecule compounds that regulate the Tetrahymena ribozyme.

[0096] Through the HitSTARS screening method of the present invention, the present application successfully screened out compounds ZPT01 and ZPT70, which bind to site 1 and site 2 of the Tetrahymena ribozyme, respectively (refer to Figure 2 c), their inhibitory activities were 9.8 μM and 230 μM, and their binding activities were 25 μM and 190 μM, respectively (instructions for use). Figure 2 d) Compared with previously reported inhibitors, ZPT01 and ZPT70 exhibited significant inhibitory effects. For example, the guanosine-competitive inhibitor ddG was unable to completely inhibit the cleavage activity of the Tetrahymena ribozyme even at a concentration of 8 mM. Furthermore, the binding sites of ZPT01 and ZPT70 differ from those of endogenous substances such as guanosine and substrate analogs, suggesting that they may possess higher binding specificity.

[0097] To further elucidate the inhibitory mechanism of the compound, the structure of the compound-RNA complex was analyzed using cryo-electron microscopy, and the inhibitory mechanism of ZPT70 was explained in detail. During the structural analysis, it was found that the addition of ZPT70 induced a conformational change in the Tetrahymena ribozyme, and this new conformation was named the Inhibited State (I state) (refer to 3c), while the wild-type conformation was called the Natural State (N state). In the I state, the peripheral region of the ribozyme (including P9.2, P9.1, P9) and P7 of the catalytic core all shifted away from the core. Among them, the displacement of P7 resulted in an increase in the distance between the exogenous guanosine (exoG) bound to P7 and the catalyzed site (P1), thereby hindering the catalytic reaction. In addition, in the N state, ZPT70 was observed to bind to the binding pocket composed of P5a, P5c, and P14 of the Tetrahymena ribozyme, and formed a stable interaction with bases A184, A183, C170, C165 and the metal ion M5 (refer to Figure 3 a, b). At the same time, P5 near the binding pocket in the I state shifts, destroying the interaction between P5 and L9. P9 cannot maintain its conformation, which leads to the displacement of P9.2, P9.1, and P9. Figure 3 d.

[0098] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes, characterized in that: The following steps are involved: S1, obtain all the docking binding sites of Tetrahymena ribozyme, perform virtual screening of the entity compounds in the CSTAR compound library through Dock6 molecular docking, and obtain the top-ranked small molecule compounds to be screened; S2, designing a fluorescent group substrate Cy3-CCCUCUAAACC-FAM, and detecting the activity of the small molecule compounds to be screened by fluorescence resonance energy transfer. First, a dual-concentration inhibition rate test was performed, and then a concentration-inhibition rate dependence test was performed to analyze the inhibitory effect of the small molecule compounds to be screened, and obtain the secondary screening small molecule compounds; S3, using biomembrane interferometry to analyze the affinity between the secondary screening small molecule compounds and the Tetrahymena nuclear membrane loaded on the sensor, and fitting and calculating the affinity constant K D ; S4, through cryo-electron microscopy analysis of the complex structure of secondary screened small molecule compounds with both inhibitory activity and affinity activity and Tetrahymena ribozyme, the inhibitory mechanism of the compound was analyzed based on the complex structure, and the small molecule compound that regulates the catalytic activity of Tetrahymena ribozyme was obtained.

2. The method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes according to claim 1, characterized in that: Step S1 includes the following steps: S10, convert the entity compounds in the CSTAR compound library into Mol2 format, and remove the non-main body parts in the entity compounds using Open Babel 3.1.1; S11, removing the interfering docking region of the Tetrahymena ribozyme model in the protein database to obtain the modified Tetrahymena ribozyme model; S12, adding hydrogen atoms to the modified Tetrahymena ribozyme model and assigning AM1-BCC charges to generate the binding site; S13, visual inspection was used to exclude potential nonspecific docking regions and the size was obtained. The grid is used for Dock6 molecular docking. The docking results are ranked according to the grid scores and screened using molecular visualization tools to obtain small molecule compounds to be screened.

3. The method for screening small molecule compounds for regulating the catalytic activity of Tetrahymena ribozyme according to claim 1, characterized in that: Step S2 includes the following steps: S20, extracting Tetrahymena ribozyme RNA, denaturing and renaturing it, and then adding GTP and shearing buffer to obtain a reaction mixture; S21, dissolving the small molecule compound to be screened, adding it to the reaction mixture, and detecting the fluorescence interference of the compound I int , while detecting the fluorescence background I0 in the wells without compound addition; S22, after adding the fluorescent group substrate, the mixture was incubated at 25°C for 15 minutes, and the fluorescence intensity I in this state was detected. cpd ; Among them, the wells without Tetrahymena ribozyme RNA were used as positive controls. pos ; Add Tetrahymena ribozyme RNA to the well as a negative control 1 neg ; The activity of the small molecule compounds to be screened is expressed by the relative FAM fluorescence intensity value; S23, obtain secondary screening small molecule compounds with concentration-dependent effects on Tetrahymena ribozyme RNA.

4. The method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes according to claim 3, characterized in that: The calculation formula for the relative FAM fluorescence intensity of the small molecule compounds to be screened is:

5. The method for screening small molecule compounds for regulating the catalytic activity of Tetrahymena ribozyme according to claim 4, characterized in that: A dose-response experiment was performed on small molecule compounds to be screened for inhibition of Tetrahymena ribozyme by more than 60% at a concentration of 1000 μM. Each compound was tested at 6 concentrations, ranging from 1000 μM to 31 μM. The inhibition rate at each concentration was calculated as follows: IC 50 Values were calculated by dose-response inhibition analysis using the following formula: Among them, Top and Bottom represent the platform values of the Y-axis unit, and HillSlope describes the steepness of the curve family.

6. The method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes according to claim 4, characterized in that: Step S3 includes the following steps: S30, Tetrahymena ribozyme RNA was labeled with biotin and coupled to the surface of the superstreptavidin biosensor; S31, analysis cycle: including obtaining a baseline, binding of the superstreptavidin biosensor to the secondary screening small molecule compound, and dissociation in the binding buffer; after the superstreptavidin biosensor is cleaned, the next analysis cycle is entered; S32, affinity constant K was calculated by fitting using Octet Data Analysis HT software D .

7. The method for screening small molecule compounds that regulate the catalytic activity of Tetrahymena ribozymes according to claim 6, wherein the calculation formula is: in, K D The value is calculated by steady-state kinetic analysis, [Compound] represents the concentration of the compound, R eq is the estimated equilibrium response value, k on is the binding rate constant, k off is the dissociation rate constant, R max Maximum binding of Tetrahymena ribozyme RNA to secondary screening small molecule compounds.

8. The method for screening small molecule compounds that modulate the catalytic activity of Tetrahymena ribozymes according to claim 6, wherein step S4 comprises the following steps: S40, the renatured Tetrahymena ribozyme RNA was mixed with the secondary screening small molecule compound and loaded onto a copper grid. The frozen copper grid was loaded into a Titan Krios electron microscope and images were collected. S41, MotionCorr2 was used to perform displacement correction on the collected photos, and then Fourier transform analysis was performed through CTFFIND4 to remove photos of poor quality; S42, the remaining photos are sequentially input into EMAN2 and Relion4 for neural network particle selection and two-dimensional classification of particles, respectively, to remove invalid particles and obtain valid particles; S43, the effective particles were three-dimensionally classified using CryoSPARC to obtain a complex density map; S44, the complex density map was optimized by Relion4 and CryoSPARC to obtain the complex structure density after resolution optimization; S45, perform density-atomic model differential analysis on the complex structure density after resolution optimization to identify small molecule compounds that regulate the Tetrahymena ribozyme.

9. A small molecule compound that regulates the catalytic activity of Tetrahymena ribozyme, characterized in that: The screening method for screening small molecule compounds for regulating the catalytic activity of Tetrahymena ribozyme according to any one of claims 1 to 8 comprises ZPT01 and ZPT70, wherein the binding site of ZPT01 is site 1 of Tetrahymena ribozyme, the inhibitory activity is 9.8 μM, and the binding activity is 25 μM; The binding site of ZPT70 is site 2 of Tetrahymena ribozyme, with an inhibitory activity of 230 μM and a binding activity of 190 μM.

10. Use of the small molecule compound for regulating the catalytic activity of Tetrahymena ribozyme according to claim 9 in inhibiting the cleavage activity of Tetrahymena ribozyme.

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