A monkeypox virus n7-methyltransferase inhibitor high-throughput screening method based on fluorescence polarization technology and application

Abstract

This invention discloses a high-throughput screening method for monkeypox virus (MPXV) N7-methyltransferase (N7-MTase) inhibitors based on fluorescence polarization technology and its application. This method uses MPXV E1... CTD Using the / E12 protein complex as a target, and leveraging the specific binding characteristic of the fluorescent probe FL-NAH to the target catalytic center, small molecule inhibitors that competitively bind to the SAM binding site are screened by monitoring changes in fluorescence polarization signals. This invention yielded a series of candidate compounds with significant inhibitory activity. Experiments demonstrated that these inhibitors exhibit strong inhibitory effects on MPXV N7-MTase, with a biochemical IC50 level of [missing information]. 50 Preferably, the concentration can reach 4.65 μM; at the cellular level, the compound exhibits a clear attenuation effect against poxviruses, with an EC50 value of [missing value]. 50 The range is up to 46.75 μM, and it has low cytotoxicity (CC). 50 >200 μM).

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antiviral drug development, specifically to a method for high-throughput screening of monkeypox virus (MPXV) N7-methyltransferase (N7-MTase) inhibitors based on fluorescence polarization (FP) technology, as well as the inhibitors obtained by this method and their applications. Background Technology

[0002] Orthopodoviruses belong to the Poxviridae family and are enveloped double-stranded DNA viruses. They include various viral members that can infect humans and cause disease, posing a persistent threat to global public health. Currently, the main orthopodoviruses known to infect humans include smallpox virus (VARV), monkeypox virus (MPXV), vaccinia virus (CPXV), and vaccinia virus (VACV). Existing antipodovirus drugs, such as cidofovir and tecovirimat, have drawbacks such as high toxicity and limited applicability, making the development of novel and effective drugs urgently needed.

[0003] Monkeypox virus (MPXV) encodes E1 CTD The / E12 complex possesses N7-methyltransferase activity, responsible for catalyzing the N7-methylation of the 5′ cap structure of viral mRNA. This process is crucial for viral RNA replication, maturation, and evasion of the host's innate immunity. Furthermore, its catalytic pocket differs structurally from human methyltransferases, making it an ideal antiviral target. However, the development of inhibitors targeting MPXV N7-MTase is still in its early stages, with the core bottleneck being the lack of efficient and specific high-throughput screening platforms.

[0004] Traditional screening methods are complex and have low throughput, while fluorescence polarization (FLP) technology offers advantages such as ease of operation, rapid detection, and no need for separation steps. It has been successfully applied to the screening of methyltransferase inhibitors, as illustrated in invention patents CN118255841A and CN113699212A. The fluorescent probe FL-NAH, as a SAM (S-adenosylmethionine) analog, can specifically bind to the SAM pocket of viral methyltransferases, but its application in MPXV N7-MTase screening has not yet been reported. Therefore, establishing a high-throughput FL-NAH-based FP screening method for the rapid discovery of novel anti-MPXV inhibitors is of great significance for the prevention and treatment of orthopoxvirus infections. Summary of the Invention

[0005] To address the problems of existing methods for screening monkeypox virus (MPXV) inhibitors, such as complex operation, low throughput, and lack of specific evaluation systems for N7-methyltransferase (N7-MTase), this invention provides a high-throughput screening platform for MPXV N7-MTase inhibitors based on fluorescence polarization (FP) technology. This invention utilizes this platform to screen orthopoxvirus inhibitors with significant biochemical inhibitory activity and antiviral effects from a library of tens of thousands of small molecules, providing lead compounds and technical support for the prevention and treatment of monkeypox and related orthopoxvirus infectious diseases. The technical solution of this invention is as follows: In a first aspect, the present invention provides a group of vaccinia virus inhibitors, said inhibitor being at least one selected from the following compounds: LX-01: The IUPAC name is 5-[(5-bromofuran-2-yl)methylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione; LX-02: The IUPAC name is 4-[2-(4-hydroxy-3,5-dinitrophenyl)propan-2-yl]-2,6-dinitrophenol; LX-03: IUPAC name is 3-[(9,10-dioxoanthracen-2-yl)sulfonylamino]benzoic acid; LX-05: IUPAC name is 4-(4-oxo-2-sulfanylidene-1) H -thieno[3,2-d]pyrimidin-3-yl)benzoic acid; LX-06: The IUPAC name is ( NE )-4-ethoxy- N -(4-oxonaphthalen-1-ylidene)benzenesulfonamide; LX-07: The IUPAC name is ( NE )- N -(3-bromo-4-oxocyclohexa-2,5-dien-1-ylidene)-2,5-dimethylbenzenesulfonamide; LX-08: IUPAC name is 3-chloro- N -[3-(furan-3-yl)-1,2-oxazol-5-yl]-4-methoxybenzenesulfonamide; LX-09: The IUPAC name is 4-[2-(cyclohexen-1-yl)ethylamino]-3,5-dinitrobenzoic acid.

[0006] The structural formulas are as follows: This invention also provides the application of the aforementioned ornithopox virus inhibitor in the preparation of reagents for targeted intervention of ornithopox virus N7-methyltransferase activity, wherein the ornithopox virus is monkeypox virus and the N7-methyltransferase is E1. CTD / E12 protein complex. The E1 CTD E1 in the E12 protein complex CTD The amino acid sequences of the protein and the E12 protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0007] Furthermore, the orthopoxvirus inhibitor exhibits inhibitory activity against monkeypoxvirus N7-methyltransferase. The inhibitor exerts its effect by competitively binding to the SAM binding pocket of N7-MTase. The half-maximal inhibitory concentration (IC50) of the inhibitor against monkeypoxvirus N7-methyltransferase activity is [not specified in the original text]. 50 The concentration was less than 250 μM. At the biochemical level, the inhibitor exhibited significant concentration-dependent inhibitory activity against MPXV N7-MTase, with LX-03 showing an IC50 value less than 250 μM. 50 < 5 μM.

[0008] Furthermore, the inhibitor exhibits significant antiviral activity against vaccinia virus (VACV) infection at the cellular level. Preferably, the inhibitor LX-07 inhibits VACV at the 50% effective concentration (EC50) at the cellular level. 50 <70 μM, EC50 of the inhibitor LX-09 50 <50 μM. Meanwhile, the LX-07 and LX-09 inhibitors exhibited low cytotoxicity, with a CC value of <50 μM. 50 >200μM.

[0009] On the other hand, the present invention also provides the use of the aforementioned monkeypox virus inhibitor in the preparation of medicaments for treating orthopoxvirus infectious diseases. The orthopoxvirus includes, but is not limited to, monkeypox virus (MPXV), smallpox virus (VARV), vaccinia virus (CPXV), and vaccinia virus (VACV).

[0010] Furthermore, this invention also provides a high-throughput screening method for monkeypox virus N7-methyltransferase inhibitors based on fluorescence polarization technology, comprising the following core steps: (1) Preparation of target protein: MPXV E1 containing N-terminal His6-SUMO tag was constructed.CTD The / E12 recombinant expression vector was induced to express and purified in a prokaryotic system to obtain a high-purity (≥95%) and tag-free natural active complex.

[0011] (2) Establishment and optimization of the fluorescence polarization screening system: Using FL-NAH as a fluorescent probe, the dissociation constant (K0) between FL-NAH and the target protein was determined. D The reaction system was optimized as follows: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20 and 1 mM DTT; the probe concentration was set at 10–100 nM (preferably 30 nM), the protein concentration at 20–200 nM (preferably 50 nM), and the incubation time was 30 min.

[0012] (3) High-throughput screening: The test compound is pre-incubated with the target protein and then the probe is added. Positive compounds are screened by monitoring the decrease in fluorescence polarization signal. Preferably, the Z′ factor of the screening system is ≥0.87 and the coefficient of variation (CV) is ≤3.5%.

[0013] (4) Multidimensional verification of positive compounds: Activity verification: The inhibitory effect of the compound on the catalytic function of N7-methyltransferase was determined using the MTase-Glo kit. Thermal stability verification: The specific binding signal of the target protein denaturation temperature change was detected using differential scanning fluorescence (DSF). Biophysical verification: K was determined using surface plasmon resonance (SPR). D Values ​​were used to evaluate affinity. Molecular docking: using VCV D1 CTD The high-resolution crystal structure of / D12 (PDB:2VDW) simulates the binding mode of the compound at the SAM site.

[0014] (5) Evaluation of antiviral activity: The inhibitory effect of positive compounds on VCV was detected at the cellular level by plaque reduction assay and flow cytometry, and the half-maximal effective concentration (EC50) was determined. 50 ) and half-maximal cytotoxic concentration (CC) 50 Preferably, the cell model includes Vero cells and HeLa cells, and the antiviral effect is evaluated by plaque counting and GFP fluorescence detection.

[0015] Furthermore, the purified recombinant E1 in step (1) CTD The / E12 complex needs to have its His6-SUMO tag removed to ensure the native activity of the target protein.

[0016] Furthermore, in step (3), the final concentration of the compounds to be screened is 50 μM. Negative and positive controls need to be set up during the screening process of the compound library to improve the reliability of the screening results.

[0017] Furthermore, in step (5), the cytotoxicity evaluation was performed using the CCK-8 method, and the multiplicity of infection (MOI) in the antiviral activity assay was set to 0.5~1.0, which was adjusted according to the cell type and virus characteristics.

[0018] The monkeypox virus inhibitors obtained by the above method are the inhibitors described above.

[0019] The present invention also provides a pharmaceutical composition for the prevention or treatment of orthopoxvirus infection, wherein the active ingredient of the pharmaceutical composition comprises the orthopoxvirus inhibitor described above or a monkeypoxvirus inhibitor obtained by screening by the above methods, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0020] The beneficial effects of this invention are: This invention targets N7-MTase, a core enzyme in the capping process of monkeypox virus RNA, blocking viral protein translation and immune escape, demonstrating strong potential for broad-spectrum intervention against orthopoxvirus. The high-throughput screening method based on fluorescence polarization technology developed in this invention features no separation required, rapid detection, and high sensitivity, with a Z′ factor of 0.87, far exceeding the acceptable standard of ≥0.5, and exhibiting good DMSO tolerance and signal stability. The LX series inhibitors obtained through screening have a clear mechanism of action: they competitively bind to the SAM binding pocket of monkeypox virus N7-methyltransferase, thereby blocking the catalytic activity of N7-MTase. This series of inhibitors exhibits clear antiviral activity in cell models, effectively inhibiting vaccinia virus infection and demonstrating good cellular safety, providing a foundation for the development of antiviral drugs.

[0021] This invention provides a standardized technical platform for high-throughput screening of monkeypox virus N7-methyltransferase inhibitors and also provides a reference for the development of methyltransferase inhibitors for other viruses. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 MPXV E1 in Example 1 CTD / E12 and VCV D1 CTD Purification results of the / D12 complex and SDS-PAGE purity verification image, where AB represents E1. CTD / E12 protein purification volume-absorbance curve, C is SDS-PAGE verification of E1CTD / E12 complex correctness and purity; DE is D1 CTD / D12 protein purification volume-absorbance curve, F is SDS-PAGE verification of D1 CTD Correctness and purity of the D12 complex.

[0024] Figure 2 The results of the establishment and optimization verification of the fluorescence polarization screening system in Example 2 are shown, where A is a schematic diagram of the FL-NAH probe structure, and B is the MPXV E1. CTD Determining the optimal reaction concentration of / E12, C is VAC / V D1 CTD / D12 Determination of the optimal reaction concentration, D is the determination of the DMSO tolerance of the reaction system, E is the verification of the competitive polarization experiment of SAM and GpppG, and F is the dose-dependent substitution effect of sinefungin on FL-NAH. Figure 3 The results of the preliminary screening of the FDA-approved drug library using the FL-NAH fluorescence polarization assay in Example 3 are shown in Figure A, which is a scatter plot of fluorescence polarization inhibition values ​​obtained by screening 2960 compounds at a final concentration of 50 μM (gray dots: library compounds; blue dots: negative control; red dots: positive control; dashed horizontal line represents the 75% inhibition cutoff value used to select active compounds); Figure B shows the determination results of Z′ factor and coefficient of variation (CV) in the preliminary screening; Figure C compares the fluorescence polarization inhibition rates of active compounds in the primary and secondary screening (results were standardized with positive control); Figure D shows the dose-dependent displacement effect of SAM, α-lipoic acid, and suramin on FL-NAH in the library stock solution.

[0025] Figure 4 Example 4 shows the results of high-throughput screening of a structurally diverse compound library using FL-NAH fluorescence polarization assays. A is a scatter plot of fluorescence polarization inhibition values ​​obtained from screening 10,000 compounds at a concentration of 50 μM (gray dots: library compounds; blue dots: negative control; red dots: positive control; dashed line represents the 50% inhibition cutoff value used for selection); B shows the determination results of Z′ factor and CV in the high-throughput screening; C is a comparison of the fluorescence polarization inhibition rates of 12 active compounds in the primary and secondary screenings (results were standardized with positive controls); D shows the resistance of candidate compounds to MPXV E1 as determined by MTase-Glo. CTD / E12 complex enzyme activity inhibition effect; E represents the comparison of the inhibitory effects of 12 active compounds on N7-methyltransferase activity (comparison of the activity of the stock sample and the reordered compound sample, with the results standardized as positive controls).

[0026] Figure 5The target compound in Example 5 is MPXV E1. CTD Dose-dependent inhibition curves of the / E12 complex enzyme activity.

[0027] Figure 6 This is an evaluation of the binding specificity of the positive compound in Example 5 to MPXV N7-MTase, where A represents the result of differential scanning fluorometry analysis (MPXV E1 in the presence of the candidate compound). CTD The melting temperature of the / E12 complex shifted (SAH was used as a positive control); B represents the surface plasmon resonance (SPR) analysis of the compound and MPXV E1. CTD The binding status of the / E12 complex.

[0028] Figure 7 MPXV E1 in Example 6 CTD Interaction analysis of the / E12 complex inhibitor with the target, AC represents the substrate competition assay for sinefungin, LX-03, and LX-05 (showing IC50 under increasing SAM concentration). 50 (dose-dependent changes in value); D represents LX-03 and VAV D1 CTD Molecular docking model of the D12 complex (LX-03 is shown as a yellow ball-and-stick model; D1 CTD E is a light orange cartoon model, and D12 is a light green cartoon model; E is an enlarged view of the binding mode of LX-03 in the catalytic pocket (LX-03 is shown as a yellow stick model, and SAH as a green stick model); F shows the interaction between LX-03 and D1. CTD Key residues for the / D12 complex to bind (dashed lines represent hydrogen bond interactions).

[0029] Figure 8 The following is an evaluation of the antiviral activity of the candidate compounds in Example 7 against VACV in a cell culture model. A represents the plaque reduction experiment using the VACV Tian Tan strain (each compound was tested at 100 μM or 50 μM concentration); B represents the quantitative analysis of the infection rate of Vero cells treated with the candidate compounds; CE represents the dose-dependent inhibitory effects of LX-07, LX-09, and Tecovirimat on VACV infection in Vero cells (cytotoxicity was determined using the CCK-8 assay); F represents the quantitative analysis of the VACV-GFP infection rate in Vero cells treated with LX-07 or LX-09; and G represents representative fluorescence images of HeLa cells infected with VACV-EGFP after treatment with different concentrations of LX-07 or LX-09. Detailed Implementation

[0030] Example 1 Target protein E1 CTD / E12 and homologous protein vaccinia virus (VACV) D1 CTD Preparation and purification of / D12 This embodiment provides a high-purity, high-activity monkeypox virus (MPXV) N7-methyltransferase complex (E1) suitable for subsequent high-throughput screening and biochemical validation. CTD / E12) protein, the specific steps are as follows: (1) Gene cloning and vector construction: based on MPXV E1 CTD The gene sequence of E12 was obtained, and the E. coli codon optimization and whole-gene synthesis were performed by Sangon Biotech Co., Ltd. Preferably, the gene fragment was inserted into the multiple cloning site of the pRSFDuet-1 vector to construct the recombinant expression vector pRSFDuet-E1. CTD / E12, and the sequence correctness was verified by DNA sequencing.

[0031] (2) Protein induction expression: The verified recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells; a single colony was picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37℃ and 220 rpm with shaking to obtain the seed culture; the seed culture was transferred to fresh medium at a volume ratio of 1:100 and cultured under the same conditions until OD. 600 The concentration was increased to 0.6-0.8. Subsequently, the culture temperature was rapidly reduced to 18°C, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM for induction, and expression was continued for 16-18 hours.

[0032] (3) Protein purification and tag removal: After induction, the bacterial cells were collected by centrifugation at 4℃ and 3500×g for 30 minutes; the bacterial cells were resuspended in pre-cooled lysis buffer (2×PBS, 20mM imidazole) (bacterial resuspension concentration was 1L bacterial cells / 20mL lysis buffer), and PMSF was added to a final concentration of 1mM, and lysozyme was added to a final concentration of 0.1mg / mL, followed by sonication (power 500W, working for 3 seconds, intermittent for 5 seconds, total duration 30 minutes); the lysate was centrifuged at 4℃ and 17000×g for 30 minutes, and the supernatant was collected; the supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with lysis buffer, and impurities were removed using washing buffer (2×PBS, 50mM imidazole), and eluted with elution buffer containing 300mM imidazole (20mM Tris-HCl pH 7.5, 300mM NaCl, 30 ... NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl, 300mM NaCl Elution was performed using 100mM imidazole, and the target protein eluent was collected. The collected protein solution was transferred to a dialysis bag with a molecular weight cutoff of 10kDa and placed in dialysis buffer containing ULP1 protease (20mM Tris-HCl pH 7.5, 150mM NaCl, 20mM imidazole, 5mM β-mercaptoethanol) and dialyzed overnight at 4°C to remove the N-terminal His6-SUMO tag. The dialyzed sample was then loaded back onto a Ni-NTA column, and the flow-through containing the target protein was collected. Elution was performed using ULP1 elution buffer (2×PBS, 100mM imidazole), and the non-specific target protein solution remaining on the Ni-NTA column was collected. This flow-through and the target protein solution were then loaded onto a HiTrap™ Heparin HP heparin affinity chromatography column and eluted using a linear gradient buffer (20mM Tris-HCl pH 7.5) containing 50mM to 1M NaCl, and the main peak was collected. Finally, the sample was analyzed using Superdex™... A 200Increase 10 / 300 GL gel filtration chromatography column was used for final purification with storage buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM DTT), and the main peak with good monodispersity was collected. The sample was analyzed by SDS-PAGE, and the protein concentration was determined using a NanoDrop™ One / OneC micro spectrophotometer. The samples were then aliquoted and stored at -80°C for later use.

[0033] The results show that E1 CTD / E12 protein purification volume-absorbance curve as shown in the figure Figure 1 As shown in AB, Figure 1 As shown in C, MPXV E1 under Coomassie Brilliant Blue staining CTDBoth E12 and E12 exhibited single, clear bands; grayscale analysis using ImageJ software showed that the purity of both was no less than 95%. This preparation method is stable and reproducible, and the resulting proteins meet the requirements for target uniformity and activity in high-throughput screening. Homologous protein VCV D1 CTD / D12 (where D1) CTD The amino acid sequences of the protein and D12 protein are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The purification method is the same as that of MPXV E1. CTD / E12 (where E1) CTD The amino acid sequences of the D11 protein and the E12 protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and their nucleotide sequences are the same as those in SEQ ID NO.5 and SEQ ID NO.6, respectively. CTD / D12 protein purification volume-absorbance curve as shown in the figure Figure 1 As shown in DE, Figure 1 SDS-PAGE in F displays D1 CTD It has a single D12 band and a purity of over 95%.

[0034] Example 2: Establishment and optimization of a high-throughput fluorescence polarization (FP) screening system This embodiment aims to establish a stable, reliable, and highly specific fluorescence polarization detection platform, providing a basis for utilizing MPXVE1. CTD The SAM binding pocket of / E12 lays the foundation for high-throughput inhibitor screening. The specific steps are as follows: (1) Probe-target affinity determination (K D In a 384-well black low-adsorption microplate, a series of 20 μL reaction systems were prepared: each system contained a fixed concentration of 30 nM of the fluorescent probe FL-NAH (structural formula shown). Figure 2 As shown in A), and E1 diluted at different concentrations. CTD / E12; The reaction buffer consisted of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v / v) Tween-20, and 1 mM DTT. After incubation at room temperature in the dark for 30 minutes, the fluorescence polarization value (mP) of each well was detected using a TECAN Infinite 200 PRO multi-mode microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The curve of mP value versus protein concentration was fitted nonlinearly using the “One site-Specific binding” model in GraphPad Prism software. Figure 2 B displays FL-NAH and E1CTD / E12 equilibrium dissociation constant K D =32 nM, indicating that the probe has a high affinity for the target protein and is suitable for competition-based detection; according to K D The optimal protein concentration for detection was determined to be 1.5 times K. D E1 CTD / E12 is 50 nM. Similarly, the determination of FL-NAH and D1... CTD / D12 equilibrium dissociation constant K D =114 nM ( Figure 2 C), determining the optimal protein concentration for detection to be 170 nM.

[0035] (2) DMSO tolerance test: To evaluate the system's compatibility with the compound solvent, DMSO tolerance was tested at fixed concentrations of FL-NAH (30 nM) and E1. CTD In a system with a concentration of / E12 (50 nM), DMSO was added to final concentrations of 0%, 2%, 5%, and 10% (v / v), respectively, and the apparent K was re-measured and calculated. D value; Figure 2 D shows the apparent K measured within the 0-10% DMSO concentration range. D The values ​​fluctuated within the range of 40 ± 8 nM with minimal fluctuation, demonstrating that the FP system has excellent tolerance to DMSO. To ensure the optimal signal-to-noise ratio and compatibility with the storage conditions of conventional compound libraries, 5% DMSO was selected as the standard final concentration for subsequent screening.

[0036] (3) Specificity verification of signal: To confirm that the change in FP signal is solely due to competition for SAM binding pocket, a substitution experiment was conducted: E1 was fixed CTD / E12 (50 nM), add gradient concentrations of the natural substrate SAM (0.1-10 μM), the RNA cap analog GpppG (0.1-10 μM), or the known inhibitor sinefungin (0.001-1 μM), incubate for 30 minutes, then add FL-NAH (30 nM), incubate at room temperature in the dark for 30 minutes, and then detect; if Figure 2 E and Figure 2 As shown in Figure F, both SAM and sinefungin can dose-dependently reduce the fluorescence polarization mP value, and their IC50 values ​​are significantly higher than those of SAM and Sinefungin. 50 The values ​​were 2.34 μM and 275 nM, respectively; and even the highest concentration (10 μM) of GpppG did not cause a significant change in the mP value (the change range was <5%); this result confirms that the signal specificity of the FP detection method of the present invention originates from the interaction between the ligand and the SAM binding pocket, and is not affected by the RNA cap binding site.

[0037] Example 3: Screening and Validation and Lead Compound Discovery Based on an FDA-Approved Drug Library The FP high-throughput screening platform established in Example 2 was used to screen a library of FDA-approved drugs with known structures to verify the practicality of the method and to identify active molecules in the drugs. The specific steps are as follows: (1) Primary high-throughput screening: An FDA-approved drug library (a total of 2960 compounds) was used. All compounds were stored in 100% DMSO at a concentration of 2 mM. In a 384-well black low-adsorption microplate, 9 μL of reaction buffer was added to each well, followed by 1 μL of compound stock solution, and then 5 μL of E1 at a concentration of 200 nM. CTD / E12 protein solution (diluted with reaction buffer) to achieve a compound concentration of 50 μM in the pre-incubation step; after incubation at room temperature for 30 minutes, add 5 μL of 120 nM FL-NAH probe solution to each well to make a final reaction volume of 20 μL (final concentration: E1). CTD / E12 50 nM, FL-NAH 30 nM, test compound 50 μM, DMSO 5%); after incubation at room temperature in the dark for 30 minutes, fluorescence polarization detection was performed; the average mP value of the positive control well (25 μM SAH) and negative control well (5% DMSO) set in each plate was defined as 100% inhibition and 0% inhibition, respectively, and the inhibition rate of each test compound was calculated.

[0038] See the screening results Figure 3 For compounds A and B, the vast majority (>99%) in the library had inhibition rates below 25%. The average Z′ factor in this screening was 0.87, and the coefficient of variation (CV) was 3.2%. Using an inhibition rate ≥75% as the initial screening criterion, a total of 8 compounds were obtained, resulting in an initial screening hit rate of 0.27%. For these 8 compounds, their autofluorescence spectra were first measured to exclude 5 compounds exhibiting strong fluorescence emission or quenching effects near the detection wavelength. The remaining 3 compounds (S-adenosylmethionine (SAM), α-lipoic acid, and suramin) underwent manual rescreening. Figure 3 C) and complete dose response experiments (concentration range: 0.01-100 μM).

[0039] Literature reports that α-lipoic acid can regulate or inhibit various methyltransferases in vitro, while suramin is a known broad-spectrum inhibitor of multiple SAM-dependent methyltransferases; further dose-response curves are shown below. Figure 3 D showed that the two positive compounds, α-lipoic acid and suramin, could effectively competitively replace FL-NAH, with an IC50 value of [missing information]. 50 The values ​​were 9.53 μM and 0.28 μM, respectively; SAM, as an endogenous substrate, also showed the expected competitive activity, IC50... 50The value is 1.84 μM.

[0040] Example 4: High-throughput screening for large and diverse compound libraries The following steps were taken to verify the ability of the screening platform of this invention to process ultra-large-scale compound libraries and to discover novel inhibitor lead compounds: (1) Large-scale screening was performed: a “GoldenScaffold” library containing 10,000 structurally diverse small molecules was used; the screening process, reaction conditions and compound concentration settings were exactly the same as in Example 3, and a total of about 55 384-well plates were processed.

[0041] like Figure 4 As shown in A and B, the average Z′ factor of this large-scale screening was 0.88, the inter-plate signal was stable, and the coefficient of variation (CV) remained at around 2.7%, further demonstrating the excellent robustness of this method in large-scale, automated applications. Using an inhibition rate of ≥50% as the cutoff value, a total of 28 compounds were obtained from the initial screening, with an overall hit rate of 0.28%, which is very close to the hit rate of the FDA drug database, indicating that the screening conditions were set reasonably. Preliminary cheminformatics analysis was performed on the 28 initial screening compounds. After excluding compounds with strong fluorescence emission or quenching effects near the detection wavelength, 12 representative compounds were selected for manual rescreening and dose response testing.

[0042] Figure 4 CE results showed that 8 out of 12 compounds exhibited reproducible inhibitory activity during secondary screening; single-point enzyme activity assays revealed that multiple compounds showed high inhibitory effects at a concentration of 50 μM, exhibiting significant hierarchical characteristics. This embodiment fully demonstrates that the FP high-throughput screening platform established in this invention possesses the powerful capability to efficiently and accurately process compound libraries of tens of thousands of compounds, and can reliably discover N7-MTase inhibitor lead molecules with novel structures.

[0043] Example 5: Verification of in vitro enzyme activity inhibition and target affinity of positive compounds The inhibitory effect of the positive compounds obtained from FP screening on the catalytic activity of N7-MTase was verified by direct functional experiments. The specific steps are as follows: (1) Methyltransferase activity inhibition assay (ICP-C) 50 (Determination): In a white, opaque 96-well plate, a 12 μL reaction system was established, containing: 150 nM E1 CTDThe reagents included E12, 1 μM SAM, 1 μM GpppG, and a series of analyte compounds at varying concentrations. The reaction buffer consisted of 50 mM Tris-HCl pH 8.0, 6 mM KCl, 1 mM DTT, 1.25 mM MgCl2, and 5% DMSO. After incubation at 37°C for 30 minutes, 1 μL of MTase-Glo™ 10× Reagent was added to each well, and the mixture was incubated at room temperature in the dark for 30 minutes. Then, 5 μL of the detection solution was added, and after incubation for 5 minutes, the chemiluminescence signal was detected using a TECAN Infinite 200 PRO. Taking compounds LX-03 and LX-05 as examples, the results showed that they could inhibit SAH formation in a dose-dependent manner. The effect of LX-03 on E12 was calculated using GraphPad Prism software. CTD IC50 of E12 enzyme activity 50 The IC of LX-05 is 4.65 μM. 50 21.80 μM ( Figure 5 ).

[0044] (2) Differential scanning fluorescence (DSF) verification of direct binding: In a 25 μL reaction system, 5 μM E1 was mixed CTD / E12 protein, 5×SYPRO Orange dye, and gradient concentrations of the test compound (0, 20, 100, 500 μM); using a CFXConnect™ real-time quantitative PCR instrument, the temperature was increased from 20℃ to 80℃ at a rate of 0.2℃ / 5 seconds, and the fluorescence intensity was continuously monitored. The protein melting point temperature (Tm) was determined by analyzing the fluorescence inflection point. Taking compounds LX-03 and LX-05 as examples, at a concentration of 500 μM, LX-05 caused E1 CTD The Tm value of the / E12 protein increased by 4.4°C. At a concentration of 100 μM, LX-03 induced E1 CTD The Tm value of the / E12 protein increased by 2.8°C; the significant increase in Tm confirms that the compound has a direct and specific binding to the target protein. Figure 6 A).

[0045] SPR validation directly combines: MPXV E1 CTD The / E12 complex was immobilized on the CM5 sensor chip via amino coupling; for each compound, a concentration gradient was prepared using running buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5% DMSO, 0.05% Tween-20), in 30 μL·min⁻¹. -1The sample flow rate was increased to flow across the chip surface; a multi-cycle kinetic analysis mode was employed, with binding and dissociation phases set to 180 seconds and 120 seconds, respectively; solvent correction was performed using a buffer solution with DMSO concentrations ranging from 0.45% to 0.58%; sensor data for all compounds were globally fitted using a 1:1 interaction model with Biacore Insight Evaluation Software (Cytiva) to determine the equilibrium dissociation constant (K0). D Finally, the sensor map was drawn using GraphPad Prism software. Surface plasmon resonance analysis, such as Figure 6 B indicates that multiple hitting compounds can bind to monkeypox virus E1 with micromolar affinity. CTD The / E12 complex has an equilibrium dissociation constant of 34.85 μM for LX-03, 9.97 μM for LX-05, and 58.39 μM for LX-09.

[0046] Example 6: Inhibitor Binding Mechanism and Molecular Simulation Study The inhibition kinetics mechanism of positive compounds was elucidated, and their atomic-level binding modes were explored through computational simulations. The specific steps are as follows: Substrate competition assay: Using the enzyme activity assay method described in Example 5, under a fixed GpppG concentration (1.0 μM), a series of different SAM concentrations (1.0, 5.0, 25.0 μM) were set, and the IC50 values ​​of compounds LX-03 and LX-05 at each SAM concentration were measured. 50 value; like Figure 7 As shown in AC, the IC50 of sinefungin decreases with increasing SAM concentration. 50 As the concentration increased from 0.62 μM to 4.53 μM, the inhibition curve showed a dose-dependent rightward shift, exhibiting typical SAM competitive characteristics. Similar substrate competition analyses were performed on the two compounds with the strongest biochemical inhibitory activity, LX-03 and LX-05. Similar to sinefungin, LX-03 showed a significant IC50 value. 50 Value offset, its IC 50 The value increased from 6.72 μM to 41.35 μM with increasing SAM concentration; LX-05 also showed a similar trend, with its IC50 value increasing from 6.72 μM to 41.35 μM. 50 The value shifted from 12.33 μM to 124.70 μM.

[0047] Molecular docking simulations: MPXV and VCV's N7-MTase are highly similar, given the current MPXV E1... CTD The / E12 structure has low resolution, therefore VCV D1 is used. CTDMolecular docking was performed using / D12 with the publicly available VCV D1 CTD Using the / D12 crystal structure (PDB:2VDW) as a template, the active compound LX-03 was coupled to E1 using AutoDock Vina software. CTD Simulations were performed in the SAM binding pocket of / E12; molecular docking results showed that LX-03 occupied the SAM / SAH binding pocket ( Figure 7 D, and has extensive spatial overlap with the SAH molecule at the catalytic center ( Figure 7 E); The terminal carboxyl group of LX-03 forms hydrogen bonds with the side chains of Arg548 and Asn550, and its benzene ring undergoes π-π stacking with Tyr683; its anthraquinone core skeleton further interacts with Phe556 and Phe679 in a π-π manner, while a carbonyl oxygen atom forms a hydrogen bond with the side chain of Lys573 ( Figure 7 F).

[0048] Example 7: Evaluation of antiviral activity and safety at the cellular level The antiviral efficacy and cytotoxicity of the lead compound were evaluated in cell models, and the specific steps are as follows: (1) Preliminary assessment of plaque reduction experiment: To evaluate the antiviral potential of N7-MTase candidate inhibitors, plaque reduction experiment was conducted using the Tian Tan strain of vaccinia virus as a representative orthopoxvirus model; Vero cells were inoculated with 50 plaque-forming units of VCV virus for 1 hour, and then replaced with DMEM medium containing 1.2% microcrystalline cellulose, 2% fetal bovine serum and 50 or 100 μM of the test compound; the plaques were quantified after 48 hours; among the seven hit compounds, with DMSO as a negative control, LX-07 and LX-09 showed the strongest antiviral effect, reducing the number of plaques by more than 70% at a concentration of 100 μM; while LX-01, LX-03 and LX-08 produced moderate but significant inhibitory effects ( Figure 8 Based on these results, LX-07 and LX-09 were selected for detailed antiviral evaluation (AB).

[0049] (2) Plaque reduction experiment to determine the half-maximal effective concentration (EC50) 50 The plaque reduction experiment was conducted using the Tian Tan strain of vaccinia virus as a representative orthopoxvirus model. Vero cells were inoculated with 50 plaque-forming units of VAV virus for 1 hour, then replaced with DMEM medium containing 1.2% microcrystalline cellulose, 2% fetal bovine serum, and serially diluted test compounds. Plaque reduction was quantified after 48 hours. The EC50 of compound LX-07 was... 50 <70 µM ( Figure 8 C), LX-09's EC 50 < 50 µM ( Figure 8 D), EC of the positive control compound Tecovirimat 50 <30 nM ( Figure 8 E).

[0050] (3) Cytotoxicity assay (CC) 50 Vero cells were packed at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates and cultured overnight. Subsequently, cells were treated with different concentrations of compound LX-07 or LX-09 diluted in DMEM medium containing 2% fetal bovine serum. After incubation at 37°C for 24 hours, cell viability was assessed using CCK-8 solution according to the manufacturer's instructions. The results showed that compounds LX-07 and LX-09 did not exhibit significant cytotoxicity on Vero cells. LX-07 showed lower cytotoxicity, with a half-maximal cytotoxicity concentration greater than 200 μM. Figure 8 C); while LX-09 showed lower cytotoxicity, greater than 320 μM ( Figure 8 D).

[0051] (4) Antiviral activity assay based on VAV-GFP virus strain: Vero cells were seeded in 96-well plates and cultured overnight. VAV-GFP expressing green fluorescent protein was then infected with multiplicity of infection (MOI) = 1.0. One hour after infection, the medium was replaced with DMEM containing 2% fetal bovine serum and different concentrations of small molecule compounds. After 24 hours of culture, the cells were collected and the proportion of GFP-positive cells was detected by flow cytometry. Similarly, VAV-GFP was seeded in HeLa cells with the same MOI. One hour after infection, the medium was replaced with DMEM containing 2% fetal bovine serum and different concentrations of small molecule compounds. After 24 hours of culture, the cells were observed and images were taken using a fluorescence microscope. The results showed that as the compound concentration decreased, the number of GFP-positive cells increased accordingly, which was consistent with the dose-dependent decrease in antiviral efficacy. Figure 8 F): At a concentration of 120 μM, LX-07 and LX-09 reduced the VCV-GFP infection rate to 20.8% and 15.1%, respectively; while at a concentration of 40 μM, the infection rate increased to 59.7% and 61.6%. Figure 8 F); further investigation was conducted on the effects of these compounds on vaccine virus infection in human cell lines. Fluorescence imaging results showed that LX-07 and LX-09 also effectively inhibited VCV-GFP infection in HeLa cells. Figure 8 G).

[0052] This invention revolves around monkeypox virus N7-methyltransferase (E1) CTDFor the key target (E12 complex), a standardized and efficient high-throughput screening system based on fluorescence polarization (FP) technology was established, providing core technical support for the rapid discovery of inhibitors against monkeypox virus.

[0053] Regarding the establishment and optimization of the FP screening method, this invention uses the SAM analog FL-NAH as a fluorescent probe, and determines the optimal screening conditions through systematic optimization: 30 nM FL-NAH probe, 50 nM E1 CTD / E12 complex (or 170 nM VACVD1) CTD The system utilizes a D12 homologous control complex and a 30-minute incubation time, exhibiting excellent tolerance to DMSO (≤5%), effectively meeting the screening requirements of large-scale compound libraries. The system demonstrates outstanding signal stability and specificity, with a Z′-factor exceeding 0.87 and a coefficient of variation (CV) ≤3.5%. SAM competitive validation and GpppG non-competitive validation confirmed that FP signal changes originate solely from the ligand's SAM-binding pocket-specific interaction with N7-MTase, unaffected by RNA cap binding sites, ensuring reliable screening results.

[0054] In compound library screening applications, this FP method demonstrated highly efficient screening capabilities: For an FDA-approved drug library containing 2960 compounds, with an inhibition rate ≥75% as the standard, the initial screening hit rate was 0.27%. After secondary screening and artifact removal, three compounds with stable inhibitory activity—SAM, α-lipoic acid, and suramin—were obtained, with IC50 values ​​of [missing information]. 50 The values ​​were 1.8 μM, 9.53 μM, and 0.28 μM, respectively. For the "Golden Scaffold" structural diversity library containing 10,000 compounds, with an inhibition rate of ≥50% as the standard, the initial screening hit rate was 0.28%. After secondary screening, 8 lead compounds with repeatable inhibitory activity were obtained, among which several compounds showed significant N7-MTase enzyme activity inhibition effects at a concentration of 50 μM.

[0055] Multidimensional validation of positive compounds further confirmed the effectiveness of the FP screening method: using the MTase-Glo kit, compounds such as LX-03 and LX-05 showed positive results for E1. CTD IC50 of E12 enzyme activity 50 The concentrations were as low as 4.65 μM and 21.80 μM, respectively; differential scanning fluorescence (DSF) analysis showed that LX-03 and LX-05 significantly increased the melting temperature (Tm) of the target protein (maximum ΔTm = 4.4℃), confirming their direct and specific binding to the target; surface plasmon resonance (SPR) experiments showed that compounds such as LX-03 and LX-05 interacted with E1... CTDThe equilibrium dissociation constant (K) of the / E12 complex D All values ​​were in the micromolar range, further verifying the binding affinity; molecular docking and substrate competition experiments clarified the mechanism of action of the inhibitor, namely, by occupying the SAM binding pocket, it blocks methyltransferase activity in a competitive inhibition mode.

[0056] In antiviral activity evaluation, inhibitors such as LX-07 and LX-09, screened using the FP method, showed excellent application potential: in Vero and HeLa cell models, they significantly inhibited VACV replication and ECMO. 50 The effective concentration ranged from 46.75 μM to 62.39 μM; it exhibited low cytotoxicity, with a CC value of [missing value]. 50 All >200 μM.

[0057] In summary, the FP high-throughput screening method established in this invention combines the advantages of high specificity, high stability, and high throughput, effectively solving the pain points of traditional screening methods such as complex operation, low throughput, and insufficient specificity. It successfully screened a batch of low-toxicity, highly efficient, and mechanism-of-action monkeypox virus N7-MTase inhibitor lead compounds. This method not only provides a standardized technical platform for the development of monkeypox virus inhibitors but also provides an important reference for the screening of other viral methyltransferase-targeting drugs, demonstrating broad application prospects and clinical translational value.

[0058] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A vaccinia virus inhibitor, characterized in that, The inhibitor is selected from the following compounds: LX-01: The IUPAC name is 5-[(5-bromofuran-2-yl)methylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione; LX-02: The IUPAC name is 4-[2-(4-hydroxy-3,5-dinitrophenyl)propan-2-yl]-2,6-dinitrophenol; LX-03: The IUPAC name is 3-[(9,10-dioxoanthracen-2-yl)sulfonylamino]benzoic acid; LX-05: IUPAC name is 4-(4-oxo-2-sulfanylidene-1) H -thieno[3,2-d]pyrimidin-3-yl)benzoic acid; LX-06: The IUPAC name is ( NE )-4-ethoxy- N -(4-oxonaphthalen-1-ylidene)benzenesulfonamide; LX-07: The IUPAC name is ( NE )- N -(3-bromo-4-oxocyclohexa-2,5-dien-1-ylidene)-2,5-dimethylbenzenesulfonamide; LX-08: The IUPAC name is 3-chloro- N -[3-(furan-3-yl)-1,2-oxazol-5-yl]-4-methoxybenzenesulfonamide; LX-09: The IUPAC name is 4-[2-(cyclohexen-1-yl)ethylamino]-3,5-dinitrobenzoic acid.

2. The application of the orthopoxvirus inhibitor according to claim 1 in the preparation of reagents for targeting orthopoxvirus N7-methyltransferase activity, characterized in that, The orthopoxvirus is monkeypoxvirus, and the N7-methyltransferase is E1. CTD / E12 protein complex; the E1 CTD E1 in the E12 protein complex CTD The amino acid sequences of the protein and the E12 protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. The use of the orthopoxvirus inhibitor according to claim 1 in the preparation of a medicament for the prevention or treatment of orthopoxvirus infectious diseases, characterized in that, The orthopox virus is selected from monkeypox virus, vaccinia virus, smallpox virus, or cowpox virus.

4. A high-throughput screening method for monkeypox virus N7-methyltransferase inhibitors based on fluorescence polarization technology, characterized in that, Includes the following steps: S1. Constructing a fluorescence polarization screening system: using FL-NAH as the fluorescent probe and monkeypox virus E1 as the target. CTD A fluorescence polarization screening system was established using the / E12 protein complex as the target protein. S2. Screening process: The test compound is pre-incubated with the target protein, the fluorescent probe is added, and the compound that can bind to the target protein is screened by detecting the competitive change of the fluorescence polarization signal.

5. The high-throughput screening method according to claim 4, characterized in that, In step S1, E1 CTD The / E12 protein complex was prepared by cloning monkeypox virus E1. CTD A recombinant expression vector was constructed using the E12 gene and induced for expression in a prokaryotic expression system, followed by purification. Among them, E1 CTD E1 in the E12 protein complex CTD The amino acid sequences of the protein and the E12 protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

6. The high-throughput screening method according to claim 5, characterized in that, The reaction buffer for establishing the fluorescence polarization screening system consisted of: 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20, and 1 mM DTT; the concentration of the FL-NAH probe was 10–100 nM, E1 CTD The concentration of the / E12 complex is 20–200 nM.

7. The monkeypox virus inhibitor obtained by screening according to any one of claims 4 to 6, characterized in that, The monkeypox virus inhibitor is the orthopox virus inhibitor as described in claim 1.

8. A pharmaceutical composition for the prevention or treatment of poxvirus infection, characterized in that, The active ingredient of the pharmaceutical composition comprises the orthopoxvirus inhibitor of claims 1 to 3 or the monkeypoxvirus inhibitor of claim 7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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