A method for high-throughput screening of novel coronavirus membrane fusion inhibitors in complex systems and its application

Abstract

This invention provides a method for high-throughput screening of SARS-CoV-2 membrane fusion inhibitors and its application. The method disclosed in this invention, based on fluorescence polarization technology combined with a high-resolution activity profile analysis platform, is applied to the screening of SARS-CoV-2 membrane fusion inhibitors from honeysuckle extract. Previously, no research had directly screened SARS-CoV-2 membrane fusion inhibitor monomers from natural products using high-throughput screening. This technique, by combining fluorescence polarization technology compatible with 384-well plates with a high-resolution activity profile analysis platform, for the first time rapidly screened 19 active ingredients from honeysuckle. Further screening and identification of the main active fractions were conducted using semi-preparative liquid chromatography and chromatographic columns with different separation mechanisms. The in vitro inhibitory activities of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin, as well as their anti-SARS-CoV-2 pseudovirus activity, were accurately identified and verified.

Description

Technical Field

[0001] This invention relates to the field of high-throughput detection, and in particular to a method for high-throughput screening of membrane fusion inhibitors of the novel coronavirus and its application. Background Technology

[0002] The key to the fusion of the SARS-CoV-2 spike protein (S protein) with the human host cell membrane lies in the formation of the six-helix bundle (6-HB) structure. Its highly conserved sequence and low variability make it one of the most ideal targets for anti-COVID-19 drug design. Currently, several SARS-CoV-2 membrane fusion inhibitors based on small molecules or lipopeptides have been reported, but clinical trials are insufficient, and their safety profile remains unclear. Therefore, the development of novel SARS-CoV-2 membrane fusion inhibitors is essential.

[0003] Honeysuckle is the dried flower bud or newly opened flower of *Lonicera japonica* Thunb., a plant in the Caprifoliaceae family. Current research indicates that honeysuckle and its compound preparations can alleviate symptoms of COVID-19 such as fever, cough, fatigue, and sore throat, promote the recovery of abnormal chest imaging findings, and effectively inhibit inflammation. However, honeysuckle extract has a complex mechanism of action involving multiple components and multiple targets, making the rapid and high-throughput screening and identification of targeted active ingredients extremely challenging.

[0004] In today's screening environment, screening techniques for SARS-CoV-2 membrane fusion inhibitors mainly rely on viral activity testing and biomembrane fusion experiments using known monomeric compound libraries to determine the membrane fusion inhibitory activity of known compounds. However, existing methods are not suitable for screening unknown compounds in complex systems. Overcoming the limitations of current technologies to directly test and identify novel, bioactive membrane fusion inhibitors in complex systems is of significant practical importance. In recent years, high-resolution biomarker analysis has broken through the limitations of traditional screening methods, highly integrating liquid phase separation, mass spectrometry, and high-throughput bioactivity evaluation systems. This effectively enables simultaneous comparison of chromatograms, mass spectra, and bioactivity spectra in complex systems, revealing the "spectrum-activity relationship" of bioactive components and improving screening efficiency. It has become one of the ideal methods for efficient and high-throughput screening of bioactive components from natural products. However, the development of high-throughput screening technologies for membrane fusion proteins is difficult, mainly due to their allosteric characteristics and cumbersome multi-step experimental procedures. Therefore, we propose a high-resolution activity profile analysis platform based on fluorescence polarization technology, which is expected to overcome the shortcomings and deficiencies of existing technologies, accelerate the development of novel SARS-CoV-2 membrane fusion inhibitors, and provide a universally applicable screening mode for screening protein inhibitors with allosteric characteristics. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for high-throughput screening of novel coronavirus membrane fusion inhibitors.

[0006] Another object of the present invention is to provide the application of the above method in screening membrane fusion protein inhibitors in natural products.

[0007] Another object of the present invention is to provide the application of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin in the preparation of 6-HB protein fusion inhibitors.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for high-throughput screening of membrane fusion inhibitors of the novel coronavirus, comprising the following steps:

[0010] (1) Extract and simplify the sample;

[0011] (2) The sample obtained after extraction and simplification is injected into the high-resolution active profile analysis platform. After the sample is separated by the liquid chromatography column in the platform, the post-column fraction is divided into two. One part of the fraction enters the diode array detector and mass spectrometer, and the remaining fraction enters the micro-fraction collection device. The micro-fraction collection device collects the fractions sequentially in the microplate according to the set program.

[0012] (3) After the fraction in the microplate is vacuum dried, NusA-5-HB protein solution is added using an electronic pipette and incubated. Then, HR2-FL solution is added and incubated for a longer period. After incubation, the fluorescence polarization intensity is measured. Combined with the detection results of liquid chromatography / mass spectrometry, the active ingredients can be screened and their structures identified in real time.

[0013] The sample mentioned in step (1) is a sample containing a potential inhibitor of novel coronavirus membrane fusion; preferably a natural plant sample.

[0014] The specific steps of extraction described in step (1) are as follows:

[0015] Take 30.0g of sample powder and extract it three times with 300mL of 70% ethanol at 50℃ (40kHz, 100W) for 10min each time. After the ultrasonic extraction, combine all the extracts and evaporate the solvent at 150rpm at 45℃ using a rotary evaporator to obtain a concentrated solution without alcohol odor. Then, add 150mL of pure water to suspend the concentrated solution and extract it with petroleum ether and ethyl acetate in equal proportions. After concentration under reduced pressure, obtain the ethyl acetate layer extract.

[0016] The specific steps of the simplification described in step (1) are as follows:

[0017] Prepare 50 mg / mL solution using 70% MeOH (containing 25% DMSO).-1 After extracting the sample with ethyl acetate, the sample was filtered through a 0.22 μm organic filter membrane. 200 μL of sample was injected into a semi-preparative liquid phase for separation and fractional collection. The chromatographic column was a Zhongpu Technology RD-C18 column (5 μm, 10.0 × 250 mm). Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol. The injection volume was 200 μL, and the flow rate was 2.5 mL / min. -1 The column temperature was 30℃; the detection wavelength was 254nm; the gradient conditions were: 0min-20%B, 5min-30%B, 35min-51%B, 50min-95%B, 70min-98%B; the elution buffer was taken for 30-40min to obtain the simplified sample.

[0018] The high-resolution activity profile analysis platform described in step (2) achieves the collection of post-column microfractions and real-time monitoring of UV / MS spectrometry by modifying the post-column flow path of the Agilent 1100 liquid chromatography system. The natural product extract is injected via a Cheminert two-position four-way injection valve and stored in a 20 μL quantitative loop. After valve disconnection, the sample is pumped into a 250 μm inner diameter Peek tube by the mobile phase in a quaternary pump, and flows through the liquid chromatography column and a three-way splitter. The fraction after splitting through the three-way splitter enters the diode array detector (DAD) and Orbitrap Exploris detector at 1 / 3 of their respective flow rates via two Peek tubes (120 μm ID) of different lengths. TM A 120 mass spectrometer (MS) was used, and the remaining fraction was collected in a microfluidic collection device (modified from the CTC HTS PAL automated sample introduction system).

[0019] The microplate mentioned in step (2) is a 384-well microplate.

[0020] The collection frequency of the microfluidic collection device described in step (2) is 10 s / well.

[0021] The liquid chromatography column mentioned in step (2) is at least one of the following: small particle size RD-C18 column (3μm, 10.0×250mm), large particle size RD-C18 column (5μm, 4.6×250mm), Accucore HILIC Dim column (5μm, 4.6×250mm), AQ-C18 column (250×4.6mm, 5μm), or RD-Phenyl column (250×4.6mm, 5μm); preferably, small particle size RD-C18 column, AQ-C18 column, and RD-Phenyl column are used respectively.

[0022] The liquid phase conditions for the small-particle-size RD-C18 column and the large-particle-size RD-C18 column are as follows:

[0023] Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; injection volume was 20 μL; flow rate was 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradients 0min-20%B, 5min-30%B, 35min-51%B, 50min-95%B, 70min-98%B.

[0024] The liquid phase conditions for the Accucore HILIC Dim column are as follows:

[0025] Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile; injection volume was 20 μL; flow rate was 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradient conditions: 0min-80%B, 10min-50%B, 20min-50%B;

[0026] The liquid phase conditions for the AQ-C18 column are as follows:

[0027] Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile; injection volume was 20 μL; flow rate was 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradient 0min-10%B, 20min-25%B, 35min-25%B, 60min-28%B.

[0028] The liquid phase conditions for the RD-Phenyl column are as follows:

[0029] Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; injection volume was 20 μL; flow rate was 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradient 0min-30%B, 10min-40%B, 50min-45%B, 65min-95%B, 70min-98%B.

[0030] The mass spectrometry conditions described in step (2) are as follows:

[0031] Mass spectrometry detection was performed simultaneously in positive and negative ion modes. The ion source type was H-ESI, and the analyzer was a quadrupole-electrostatic track trap mass analyzer. The main parameters of the first-stage / second-stage mass spectrometry are as follows: first-stage / second-stage resolution: 60000 / 15000; sheath gas: 50 Arb; auxiliary gas: 10 Arb; sweep gas: 1 Arb; ion source temperature: 400℃; scan voltage: +3500V or -3000V; scan range: 100-1000 Da; scan time: 0.15 s; HCD collision energies: 20%, 50%, and 80%.

[0032] The NusA-5-HB and HR2-FL mentioned in step (3) were kindly provided by Professor Gao's research group at the School of Marine Sciences, Sun Yat-sen University. For specific preparation methods and verification processes, please refer to the literature Yin X, Chen L, Yuan S, et al. A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors[J]. Bioorganic Chemistry, 2021.DOI:10.1101 / 2021.06.17.448891.

[0033] The NusA-5-HB protein solution described in step (3) was diluted with phosphate buffer to a final concentration of 33.33 nmol·L⁻¹. -1 .

[0034] The HR2-FL solution described in step (3) was diluted with phosphate buffer to a final concentration of 25 nmol·L⁻¹. -1 .

[0035] The phosphate buffer solution has a salt concentration of 20 mmol·L⁻¹. -1 It contains 0.125% NP-40 and adjusts the pH to 7.4.

[0036] The amount of NusA-5-HB protein solution added in step (3) is 30 μL per well.

[0037] The amount of HR2-FL solution added in step (3) is 20 μL per well.

[0038] The incubation conditions described in step (3) are: incubation at 37°C and 150 rpm for 30 min.

[0039] The fluorescence polarization intensity mentioned in step (3) is measured using an ELISA reader; preferably, it is measured using Biotek Synergy. TM H1 multifunctional enzyme-linked immunosorbent assay (ELISA) was used for measurement.

[0040] The parameters of the microplate reader are set as follows: temperature 37℃, light source is xenon flash lamp, excitation wavelength of filter group is 485 / 20nm, emission wavelength is 528 / 20nm, gain is 50, and detection height is 10.5mm.

[0041] The specific steps for real-time screening and structural identification of the active ingredients described in step (3) are as follows:

[0042] ① After microplates are tested using an ELISA reader, the fluorescence polarization intensity endpoint value is exported and used as the ordinate (unit: mP) of the bioactivity spectrum. Subsequently, according to the order in which the post-column fractions are collected in the microplates, the single-well collection time (i.e., total collection time / number of collection wells) is obtained based on the total collection time and the number of collection wells. This time is then converted into single-well time points corresponding to the retention times of the liquid chromatogram / mass spectrometry and used as the abscissa (unit: min). The bioactivity spectrum can be plotted using the data from the above-processed abscissa and ordinate. Among them, negative peaks in the bioactivity spectrum indicate that the fluorescence polarization intensity at the location of the well is low, which may contain active ingredients that can inhibit the binding of NusA-5-HB protein to the HR2-FL peptide probe. The stronger the inhibition, the more obvious the negative peak. Among them, the fluorescence polarization endpoint value in a single well that is more than 30% lower than the average value of the negative control is considered to have potential active ingredients and is also one of the activity data points composed of negative peaks.

[0043] ② Based on the bioactivity spectrum obtained in ①, combined with the chromatogram of the corresponding diode array detector of the chromatographic column, the liquid chromatographic peak corresponding to the time of the negative peak in the bioactivity spectrum is identified as a potential active ingredient; however, in some cases, insufficient chromatographic resolution may cause multiple components to co-elute at the same time, making it impossible to accurately identify the specific active ingredient. Therefore, chromatographic columns of different types / separation mechanisms are used to achieve the separation of co-elute components. If the same component is detected at the position corresponding to the negative peak under different chromatographic column separation (using mass spectrometry secondary fragment information as the judgment index), it is identified as an active ingredient.

[0044] ③ Based on the retention time of the potential active ingredients separated by the chromatographic column, combined with the mass spectrometry results and standard data at the corresponding time, the chemical structure of the potential novel coronavirus membrane fusion inhibitor was obtained through analysis.

[0045] The application of the high-throughput screening method for novel coronavirus membrane fusion inhibitors in the screening of natural products.

[0046] Application of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin in the preparation of 6-HB protein fusion inhibitors.

[0047] Application of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin in the preparation of drugs for the treatment of novel coronavirus.

[0048] Application of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin in the in vitro inhibition of the formation of viral 6-HB fusion structures.

[0049] The present invention has the following advantages and effects compared with the prior art:

[0050] (1) The purpose of this invention is to provide a high-throughput screening method for novel coronavirus membrane fusion inhibitors based on fluorescence polarization technology combined with a high-resolution activity profile analysis platform. The main research focuses on novel coronavirus membrane fusion inhibitors derived from honeysuckle and their antiviral applications. It was found that isochlorogenic acid A, isochlorogenic acid C, hyperoside and luteolin have SARS-CoV-2 membrane fusion inhibitory effects, and are expected to be promoted and used as a new type of SARS-CoV-2 membrane fusion inhibitor anti-COVID-19 drug.

[0051] (2) The method disclosed in this invention, based on fluorescence polarization technology combined with a high-resolution activity profile analysis platform, was applied to the screening of novel coronavirus membrane fusion inhibitors in honeysuckle extract. Previously, no research had directly screened novel coronavirus membrane fusion inhibitor monomers from natural products using high-throughput methods; instead, single active ingredients were explored using known standard compound libraries or virtual screening techniques. This technique, by combining fluorescence polarization technology compatible with 384-well plates with a high-resolution activity profile analysis platform, for the first time rapidly screened 19 active ingredients from honeysuckle. Further screening and identification of the main active fractions were conducted using semi-preparative liquid chromatography and chromatographic columns with different separation mechanisms. The in vitro inhibitory activities of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin, as well as their anti-SARS-CoV-2 pseudovirus activity, were accurately identified and verified. Experiments showed that isochlorogenic acid A, isochlorogenic acid C, and hyperoside had no significant cytotoxicity, and the half-maximal inhibitory concentration (IC50) against 6-HB was [not specified]. 50 The values ​​were 4.9291 ± 2.0892 μmol·L⁻¹. -1 6.1935±1.0073μmol·L -1 With 4.9576±0.7146 μmol·L -1 Furthermore, the half-maximal effective concentrations (EC50) of isochlorogenic acid A, isochlorogenic acid C, and hyperoside against SARS-CoV-2 pseudovirus (wild-type strain WH-1) were determined. 50 The values ​​were 6.2098 ± 6.0369 μmol·L⁻¹. -17.5140±4.9615μmol·L -1 With 5.2284±7.5587 μmol·L -1 .

[0052] (3) The high-throughput screening method provided by this invention has found that isochlorogenic acid A, isochlorogenic acid C, hyperoside and luteolin can be used as safe and effective natural novel coronavirus membrane fusion inhibitors from honeysuckle extract. This provides a theoretical basis for the preparation of drugs to prevent and treat COVID-19 and is expected to be promoted and used as a new SARS-CoV-2 membrane fusion inhibitor anti-COVID-19 drug. Attached Figure Description

[0053] Figure 1 This is a schematic diagram illustrating the main principles of the technology of this invention;

[0054] Figure 2 In Example 1 of this invention, the activity profile of the ethyl acetate extract of honeysuckle was analyzed using a small-particle-size RD-C18 column, a phenyl column, and a polar C18 column.

[0055] Figure 3 Example 1 of the present invention uses a large-particle-size RD-C18 column and an Accucore HILIC Dim column to analyze the activity profile of the ethyl acetate extract of honeysuckle.

[0056] Figure 4 This is a graph showing the in vitro inhibitory activity of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin against 6-HB protein in Example 2 of the present invention.

[0057] Figure 5 This is a graph showing the toxicity test results of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin on Huh-7 cells in Example 3 of the present invention.

[0058] Figure 6 This is a bar chart showing the effects of isochlorogenic acid A, isochlorogenic acid C, and hyperoside on the infection of ACE2-highly expressed Huh-7 cells by SARS-CoV-2 pseudovirus (wild-type strain WH-1) in Example 4 of the present invention.

[0059] Figure 7 This is a fitting graph of the half-maximal effective concentrations (WMCs) of isochlorogenic acid A, isochlorogenic acid C, and hyperoside against SARS-CoV-2 pseudovirus (wild-type strain WH-1) in Example 4 of the present invention. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0061] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0062] Example 1: Preparation and High-Resolution Activity Profile Analysis of Honeysuckle Samples

[0063] (1) Sample preparation, fractionation and liquid chromatography-mass conditions

[0064] Take an appropriate amount of honeysuckle herbal slices (Kangmei Pharmaceutical Co., Ltd.), pulverize and sieve (200 mesh), then weigh 30.0g of honeysuckle powder. Extract three times with 300mL of 70% ethanol at 50℃ using ultrasonication (40kHz, 100W), 10min each time. After ultrasonication, combine all extracts and evaporate the solvent to dryness using a rotary evaporator at 45℃ and 150rpm to obtain a concentrated solution without alcohol odor. Then, add 150mL of pure water to suspend the concentrated solution, and extract successively with petroleum ether and ethyl acetate in equal proportions. After concentration under reduced pressure, 1.4g of ethyl acetate extract is obtained. Prepare a 20mg·mL⁻¹ solution with 70% methanol. -1 The ethyl acetate extract of honeysuckle was filtered using a 0.22 μm organic filter membrane and stored at 4°C for later use.

[0065] Liquid chromatography conditions for honeysuckle ethyl acetate extract: Spectroscopic column: Zhongpu Technology RD-C18 column (3 μm, 4.6 × 250 mm); mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-methanol; injection volume: 20 μL; flow rate: 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradient conditions: 0min-20%B, 5min-30%B, 35min-51%B, 50min-95%B, 70min-98%B.

[0066] To simplify the analysis of the ethyl acetate fraction of honeysuckle and improve resolution, this study employed a semi-preparative high-performance liquid chromatography (HPLC) solution to simplify and enrich the main active fraction (30-40 min). A 50 mg / mL solution was prepared using 70% MeOH (containing 25% DMSO). -1 After extracting the ethyl acetate extract of honeysuckle, the sample was filtered using a 0.22 μm organic filter membrane, and 200 μL of sample was injected into the semi-preparative liquid phase for separation and fractional collection.

[0067] For the main active components mentioned above, columns with different retention mechanisms were used for analysis: a small-particle-size RD-C18 column (3 μm, 10.0 × 250 mm), a phenyl column (RD-Phenyl, 5 μm, 4.6 × 250 mm), and a polar C18 column (AQ-C18, 5 μm, 4.6 × 250 mm), to better identify potential co-eluting components. The chromatographic conditions are as follows:

[0068] Small-particle-size RD-C18 column; mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-methanol; injection volume: 200 μL; flow rate: 2.5 mL / min -1 Column temperature 30℃; detection wavelength 254nm; gradient conditions: 0min-20%B, 5min-30%B, 35min-51%B, 50min-95%B, 70min-98%B.

[0069] AQ-C18 column: Mobile phase A is 0.1% formic acid-water, and mobile phase B is 0.1% formic acid-acetonitrile; injection volume 20 μL; flow rate 0.5 mL·min -1 Column temperature 30℃; detection wavelength 254nm; gradient 0min-10%B, 20min-25%B, 35min-25%B, 60min-28%B;

[0070] RD-Phenyl column: Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; injection volume was 20 μL; flow rate was 0.5 mL / min. -1 Column temperature 30℃; detection wavelength 254nm; gradient 0min-30%B, 10min-40%B, 50min-45%B, 65min-95%B, 70min-98%B.

[0071] In addition, in this study, we also attempted separation using a hydrophilic Hilic column (Accucore HILIC Dim, 5μm, 4.6×250mm, Thermo Scientific) and an RD-C18 column (5μm, 4.6×250mm, Zhongpu Technology Co., Ltd.) with larger particle size, but the resolution was unsatisfactory (see results). Figure 3 It is difficult to identify the co-eluting active ingredients, and it is not suitable for orthogonal analysis.

[0072] The optimized chromatographic conditions for the two columns are as follows:

[0073] Large-particle-size RD-C18 column (5 μm, 4.6 × 250 mm); mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-methanol; injection volume: 20 μL; flow rate: 0.5 mL / min -1Column temperature 30℃; detection wavelength 254nm; gradient conditions: 0min-20%B, 5min-30%B, 35min-51%B, 50min-95%B, 70min-98%B.

[0074] Accucore HILIC Dim column (5 μm, 4.6 × 250 mm); mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile; injection volume was 20 μL; flow rate was 0.5 mL·min -1 Column temperature 30℃; detection wavelength 254nm; gradient conditions: 0min-80%B, 10min-50%B, 20min-50%B.

[0075] Mass spectrometry conditions: Mass spectrometry detection was performed simultaneously in positive / negative ion mode. The ion source type was H-ESI, and the analyzer was a quadrupole-electrostatic track trap mass analyzer. The main parameters of the first-stage / second-stage mass spectrometry are as follows: first-stage / second-stage resolution: 60000 / 15000; sheath gas: 50 Arb; auxiliary gas: 10 Arb; sweep gas: 1 Arb; ion source temperature: 400℃; scan voltage: +3500V or -3000V; scan range: 100-1000 Da; scan time: 0.15 s; HCD collision energies: 20%, 50%, and 80%.

[0076] (2) High-resolution activity profile analysis

[0077] This experiment achieved the collection of post-column microfluidics and real-time monitoring of UV-Vis / mass spectrometry by modifying the post-column flow path of an Agilent 1100 liquid chromatography system. The basic principle of the platform is as follows: Figure 1 As shown. The natural product extract was injected via a Cheminert two-position four-way injection valve and stored in a 20 μL quantitative loop. After valve disconnection, the sample was pumped into a 250 μm inner diameter Peek tube using the mobile phase from a quaternary pump, and flowed through the liquid chromatography column and a three-way splitter. The split fraction then passed through two Peek tubes (120 μm ID) of different lengths, entering the diode array detector (DAD) and the Orbitrap Exploris at 1 / 3 of their respective flow rates. TM A 120 mass spectrometer (MS) was used, and the remaining fraction was collected in a microfluidic collection device (modified from the CTC HTS PAL automated sample introduction system).

[0078] The robotic arm of the microfluidic collection device is precisely translated along the x, y, and z axes by software code, driving the post-column fractions to collect them one by one into a black 384 microplate according to a predetermined collection path and time. The front end of the collection device is a self-assembled simple spotting needle, consisting of a two-way connector, a connector, and an approximately 8cm long polyimide-coated quartz capillary (100μm ID). By controlling the flow rate of the liquid phase system, the fractions flow out of the capillary end in a continuous droplet shape, thus achieving precise collection of microfluidics in a single well of the 384 microplate. Testing showed that the parallel movement distance of the spotting needle is accurate to 0.1mm, and the residence time in a single well is accurate to 0.1s / well.

[0079] After sample preparation, the sample was injected into a high-resolution biomarker for chromatographic separation, mass spectrometry, and microfluidic collection. Post-column samples were collected at 10 s / well in a black 384 microplate using a microfluidic collection device and then placed directly in a vacuum drying oven (35℃, -0.1 MPa) to allow the solvent to evaporate completely. Before bioactivity testing, the following working solutions need to be prepared:

[0080] a. Phosphate-buffered saline (PBS): salt concentration 20 mmol / L -1 Contains 0.125% NP-40, pH adjusted to 7.4;

[0081] b. NusA-5-HB protein solution: Dilute with PBS buffer to a final concentration of 33.33 nmol·L⁻¹ -1 ;

[0082] c. HR2-FL solution: Dilute with PBS buffer to a final concentration of 25 nmol·L⁻¹ -1 Keep away from light for later use.

[0083] NusA-5-HB and HR2-FL were kindly provided by Professor Gao's research group at the School of Marine Sciences, Sun Yat-sen University. When 5-HB binds to HR2-FL, the fluorescence polarization signal in the solution increases, while the fluorescence polarization signal decreases when a fusion inhibitor is present in the solution. This enables the testing of the activity of the novel coronavirus fusion protein and the screening of inhibitors. The purity of all proteins and the feasibility of the strategy have been verified. For specific preparation methods and verification processes, please refer to the literature Yin X, Chen L, Yuan S, et al. A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors[J]. Bioorganic Chemistry, 2021. DOI:10.1101 / 2021.06.17.448891.

[0084] After the working solution was prepared, 30 μL of NusA-5-HB protein solution was quickly and accurately added to each well of a dried 384 microplate using an electronic pipette. The plate was then sealed and incubated at 150 rpm for 30 min at 37°C using a shaker to ensure complete dissolution of the sample. Next, 20 μL of HR2-FL solution was quickly added using an electronic pipette. After sealing, the plate was incubated at 150 rpm for 30 min at 37°C using a shaker. After the reaction was complete, the plate was immediately removed and processed using Biotek Synergy. TM The fluorescence polarization endpoint value was measured using an H1 multifunctional microplate reader. The microplate reader parameters were set as follows: temperature 37℃, light source xenon flash lamp, excitation wavelength 485 / 20nm, emission wavelength 528 / 20nm, gain 50, and detection height 10.5mm.

[0085] Finally, data processing was performed to plot the bioactivity spectrum. After microplate readings were obtained using a microplate reader, the endpoint value of fluorescence polarization intensity was exported and used as the ordinate (unit: mP) of the bioactivity spectrum. Subsequently, according to the order in which the post-column fractions were collected in the microplate, the single-well collection time (i.e., total collection time / number of collection wells) was obtained based on the total collection time and the number of collection wells. This time was then converted into single-well time points corresponding to the retention times of the liquid chromatogram / mass spectrometry (LC / MS) and used as the abscissa (unit: min). The bioactivity spectrum was plotted using the data from the processed ordinate and abscissa. Negative peaks in the bioactivity spectrum indicate lower fluorescence polarization intensity at the well location, potentially indicating the presence of an active ingredient that can inhibit the binding of NusA-5-HB protein to the HR2-FL peptide probe. The stronger the inhibition, the more pronounced the negative peak. By integrating the bioactivity spectrum, chromatogram, and mass spectrometry, real-time screening and structural identification of active ingredients in complex systems can be achieved.

[0086] See results Figure 2 ,from Figure 2 At least 10 active negative peaks were found in the ethyl acetate fraction of honeysuckle. A semi-preparative high-performance liquid chromatography (HPLC) phase was used to simplify and enrich the main active fraction (30-40 min). Further separation and identification of co-eluting components were achieved using chromatographic columns with different retention mechanisms. Among them, the phenyl column, compared to the conventional RD-C18 column, has a stationary phase modified with a benzene ring structure. When the sample flows through the phenyl column, the phenyl stationary phase enhances the intermolecular interaction with planar aromatic compounds through π-π forces, thus causing aromatic compounds to exhibit a different retention mechanism and retention time than those in reversed-phase retention, improving selectivity. Although the polar AQ-C18 column is modified with polar groups, the stationary phase still maintains reversed-phase characteristics. Compared to the conventional RD-C18 column, it enhances the retention capacity of both hydrophilic and polar compounds, making it suitable for separating hydrophilic and polar compounds. Figure 2As can be seen, the phenyl column and the polar AQ-C18 column exhibit significantly different retention effects in the 30-40 min active range compared to the conventional RD-C18 column. Furthermore, the elution order of the main peak and secondary peaks in the chromatogram changes significantly. For example, the retention time of the main peak (peak 1) in the phenyl column is 29.68 min, while in the polar AQ-C18 column it is 35.06 min, and the position of the secondary peaks relative to the main peak also changes. By analyzing the retention time of the active peak and mass spectrometry information, accurate identification of co-eluting components in the conventional RD-C18 column can be achieved. For instance, it is difficult to determine whether the co-eluting compound at peak 1 is also an active ingredient based solely on the activity spectrum information from the conventional RD-C18 column. However, in the phenyl column and the polar AQ-C18 column, they are effectively separated, and the elution order changes. If this compound is an active ingredient, then corresponding negative peaks should also appear at the corresponding positions on other columns, thus enabling accurate identification of the active ingredient. Finally, nine active negative peaks were screened from the 30-40 min active range using both a phenyl column and a polar AQ-C18 column. Combined with mass spectrometry data, it was found that, except for one compound whose structure was not yet determined, all other components could be identified by the three columns with different retention mechanisms, thus eliminating interference from co-eluting components. The results are shown in Tables 1, 2, and 3.

[0087] In particular, by combining mass spectrometry data and standards, four distinct active ingredients were accurately identified: compound 1-isochlorogenic acid A, compound 2-luteolin, compound 3-hyperoside, and compound 4-isochlorogenic acid C. All of these are reported for the first time in terms of their role in inhibiting membrane fusion of the novel coronavirus.

[0088] Table 1. Mass spectrometry analysis results of a conventional RD-C18 column.

[0089]

[0090]

[0091] Table 2 Mass spectrometry analysis results of phenyl columns

[0092]

[0093]

[0094] Table 3 Mass spectrometry analysis results of polar AQ-C18 column

[0095]

[0096]

[0097] Example 2: In vitro inhibitory activity test of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin against 6-HB protein.

[0098] Standard solutions of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin were prepared using 70% methanol (containing 25% DMSO), and then diluted sequentially to obtain working concentrations of 0.01 μmol·L⁻¹. -1 0.1 μmol·L -1 1 μmol·L -1 0.01 mmol·L -1 0.1 mmol·L -1 1 mmol·L -1 10 mmol·L -1 With 100 mmol·L -1 A standard single-sample solution was prepared. Subsequently, following the steps described in Example 1, the inhibitory activity of different concentrations of the compound against 6-HB fusion was determined to obtain the half-maximal inhibitory concentration (IC50). 50 ).

[0099] See results Figure 4 The results showed that the half-maximal inhibitory concentrations (IC50) of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin for 6-HB were 4.9291 ± 2.0892 μmol·L⁻¹. -1 6.1935±1.0073μmol·L -1 4.9576±0.7146μmol·L -1 With 9.3598±2.9668 μmol·L -1 This indicates that isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin obtained from honeysuckle can strongly inhibit the fusion of 6-HB, and are expected to be further applied in research.

[0100] Example 3: Toxicity test of isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin on Huh-7 cells.

[0101] Human hepatocellular carcinoma Huh-7 cells (Shanghai CyberKang Biotechnology Co., Ltd.) with high ACE2 expression in logarithmic growth phase were harvested. Cell counts were performed using an automated cell counter, and the cell concentration was adjusted to 1×10⁻⁶ cells / cells. 4 The cells were seeded into 96-well plates and cultured overnight in a 5% CO2, 37°C incubator using the complete culture medium described above. Once the cell density reached 85%, the culture medium was discarded to allow the cells to adhere to the plate.

[0102] Prepare a complete culture medium according to the Duchenne Modified Eagle Medium (DMEM):Fetal Bovine Serum ratio of 9:1, and store at 4°C for later use. Prepare a 20 mmol / L broth using pure DMSO. -1The small molecule stock solution was then diluted with complete culture medium to working concentrations (0.01, 0.1, 1, 10, 25, 50, 100, 200 μmol·L⁻¹). -1 Cytotoxicity assays were performed using isochlorogenic acid A, isochlorogenic acid C, hyperoside, and luteolin as sample groups, with a blank control group (no sample added). The blank control group received 100 μL / well of complete culture medium; the sample groups received 100 μL / well of different concentrations of 0.01, 0.1, 1, 10, 25, 50, 100, and 200 μmol·L⁻¹, respectively. -1 The sample working solution was incubated in a 5% CO2, 37℃ incubator for 48 hours. The culture medium was then discarded, and 2 mL of PBS (20 mmol / L) was used. -1 Each well was washed three times with a solution of 10% CCK-8 (pH=7.4). Then, 100 μL of medium containing 10% CCK-8 was added to each well of the 96-well plate, and the plates were incubated for 2 hours in a 5% CO2 incubator at 37°C. Each group was repeated in triplicate. All experiments were performed on a sterile laminar flow hood.

[0103] Finally, the absorbance (OD) values ​​of each well sample at 450 nm were measured using a TECAN microplate reader. The relative cell viability and inhibition rate were calculated using the following formulas: Relative cell viability (%) = (Average OD value of experimental group - Average OD value of background group) / (Average OD value of control group - Average OD value of background group) × 100%; Relative cell inhibition rate % = 1 - Relative cell viability %, where the background group OD value is the absorbance with only CCK-8 reagent and culture medium added. The survival rates of different concentrations of samples against Huh-7 hepatocellular carcinoma cells highly expressing ACE2 were plotted using Origin Pro 9 software. Furthermore, a significance test was performed on the data, where "*" represents P < 0.05 and "**" represents P < 0.01.

[0104] See results Figure 5 25-50 μmol·L -1 Treatment with luteolin-7-O-glucoside for 48 hours exhibited certain cytotoxicity, and the relative cell viability decreased with increasing luteolin concentration. At 200 μmol·L⁻¹, the cell viability was significantly lower. -1 Cell viability dropped below 50% at that time. (200 μmol·L⁻¹) -1 Isochlorogenic A also exhibited some cytotoxicity, with a cell viability of 76.12%, while cell viability was greater than 95% at other concentrations. Furthermore, the other three compounds showed cytotoxicity at 100 μmol·L⁻¹. -1 The effect on cell viability is relatively small within a certain concentration range, at 200 μmol·L⁻¹. -1After 48 hours of treatment with high concentrations, cell viability was greater than 83%. Therefore, isochlorogenic acid A, isochlorogenic acid C, and hyperoside were subsequently selected for pseudovirus activity testing to further verify that these compounds could effectively inhibit the invasion of SARS-CoV-2 pseudovirus into human hepatocellular carcinoma Huh-7 cells that highly express ACE2.

[0105] Example 4: Inhibition of SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Huh-7 cells by isochlorogenic acid A, isochlorogenic acid C, and hyperoside.

[0106] First, add 10% fetal bovine serum (FBS), 2.5% HEPES and 1% penicillin-streptomycin antibiotic solution to DMEM high glucose medium by volume ratio, mix well, and prepare DMEM complete medium (1% penicillin-streptomycin antibiotic, 25mM HEPES, 10% FBS), and store at 4℃ for later use.

[0107] Subsequently, 250 μL of pure water was added to the edge wells of a 96-well plate to avoid edge effects. Other cell treatment procedures were the same as in Example 3, and background, negative, and experimental groups were designed. The background group consisted of DMEM complete medium and cells; the negative group consisted of DMEM complete medium, cells, and SARS-CoV-2 pseudovirus (wild-type strain WH-1); and the experimental group consisted of a series of concentration gradient samples co-incubated with DMEM complete medium, cells, and SARS-CoV-2 pseudovirus (wild-type strain WH-1), with a final pseudovirus concentration of 650 TCID in each well. 50 The number of cells per well is 3 × 10⁻⁶ 4 The tested samples included salvianolic acid A (positive), isochlorogenic acid A, isochlorogenic acid C, and hyperoside, with final concentrations of 100, 33.3, 11.1, 3.7, 1.2, and 0.4 μmol·L⁻¹, respectively. -1 .

[0108] Next, the 96-well plates with added samples were transferred to an incubator (5% CO2, 37°C) and incubated for 24 hours. After removing 150 μL of supernatant from each well using a multichannel electronic pipette, 100 μL of luciferase assay reagent was added, and the plates were incubated at room temperature in the dark for 2 minutes. Each group was repeated in triplicate, and all experiments were performed on a sterile laminar flow hood.

[0109] After the reaction was complete, the liquid in the reaction wells was repeatedly blown and aspirated 8 times using a multichannel electronic pipette to ensure complete cell lysis. 150 μL of liquid was aspirated from each well and transferred to the corresponding 96-well chemiluminescence assay plate. The chemiluminescence values ​​were then read using a multi-mode microplate reader (PE, Ensight). The virus infection rate was calculated based on the correlation between the chemiluminescence value of each well and the sample concentration. The formula was: Virus infection rate (%) = (Average luminescence intensity of experimental group - Average luminescence intensity of background group) / (Average luminescence intensity of negative group - Average luminescence intensity of background group) × 100%. Based on the calculation results, the effect of different concentrations of samples on the infection of SARS-CoV-2 pseudovirus (wild-type strain WH-1) in Huh-7 hepatocellular carcinoma cells expressing high ACE2 was plotted using Origin Pro 9 software, and the half-maximal effective concentration (EC50) was fitted. 50 ).

[0110] See results Figure 6 It was found that isochlorogenic A, isochlorogenic C, and hyperoside have similar inhibitory activities to salvianolic A. The EC50 of isochlorogenic A, isochlorogenic C, and hyperoside... 50 The values ​​were 6.2098 ± 6.0369 μmol·L⁻¹. -1 7.5140±4.9615μmol·L -1 and 5.2284±7.5587μmol·L -1 (see Figure 7 ), through combination Figure 2 The in vitro inhibition results of the above-mentioned active compounds on the 6-HB fusion structure suggest that the active compounds may produce pseudoviral inhibitory activity by inhibiting the formation of the fusion structure in the S protein, further demonstrating that the screened active compounds have potential anti-SARS-CoV-2 virus activity.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for high-throughput screening of membrane fusion inhibitors of the novel coronavirus, characterized in that... Includes the following steps: (1) Extract and simplify the sample; (2) The sample obtained after extraction and simplification is injected into the high-resolution active profile analysis platform. After the sample is separated by the liquid chromatography column in the platform, the post-column fraction is divided into two. One part of the fraction enters the diode array detector and mass spectrometer, and the remaining fraction enters the micro-fraction collection device. The micro-fraction collection device collects the fractions sequentially in the microplate according to the set program. (3) After the fraction in the microplate is vacuum dried, NusA-5-HB protein solution is added using an electronic pipette and incubated. Then, HR2-FL solution is added and incubated for a longer period. After incubation, the fluorescence polarization intensity is measured. Combined with the detection results of liquid chromatography / mass spectrometry, the active ingredients can be screened and their structures identified in real time. The specific steps of extraction described in step (1) are as follows: Take 30.0 g of sample powder and extract it three times with 300 mL of 70% ethanol at 50℃ for 10 min each time. After the ultrasonic extraction, combine all the extracts and evaporate the solvent at 150 rpm at 45℃ using a rotary evaporator to obtain an alcohol-free concentrate. Then, add 150 mL of pure water to suspend the concentrate and extract it with petroleum ether and ethyl acetate in equal proportions. After concentration under reduced pressure, obtain the ethyl acetate extract. The specific steps of simplification described in step (1) are as follows: Prepare 50 mg / mL solution using 70% MeOH containing 25% DMSO. -1 After extracting the sample with ethyl acetate, the sample was filtered through a 0.22 μm organic filter membrane. 200 μL of sample was injected into a semi-preparative liquid phase for separation and fractional collection. The chromatographic column was a Zhongpu Technology RD-C18 column; mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; the injection volume was 200 μL; and the flow rate was 2.5 mL / min. -1 Column temperature 30℃; detection wavelength 254 nm; gradient conditions: 0 min-20%B, 5 min-30%B, 35 min-51%B, 50 min-95%B, 70 min-98%B; take 30-40 min of elution buffer to obtain the simplified sample; The liquid chromatography column mentioned in step (2) is a small particle size RD-C18 column, an AQ-C18 column, and an RD-Phenyl column; The liquid phase conditions for the small-particle-size RD-C18 column are as follows: Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; injection volume was 20 μL; flow rate was 0.5 mL·min. -1 Column temperature 30℃; detection wavelength 254 nm; gradient 0 min-20%B, 5 min-30%B, 35 min-51%B, 50 min-95%B, 70 min-98%B; The liquid phase conditions for the AQ-C18 column are as follows: Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile; injection volume was 20 μL; flow rate was 0.5 mL·min. -1 Column temperature 30℃; detection wavelength 254 nm; gradients 0 min-10%B, 20 min-25%B, 35 min-25%B, 60 min-28%B; The liquid phase conditions for the RD-Phenyl column are as follows: Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-methanol; injection volume was 20 μL; flow rate was 0.5 mL·min. -1 Column temperature 30℃; detection wavelength 254 nm; gradient 0 min-30%B, 10 min-40%B, 50 min-45%B, 65 min-95%B, 70 min-98%B.

2. The method according to claim 1, characterized in that: The sample mentioned in step (1) is a sample containing a potential novel coronavirus membrane fusion inhibitor.

3. The method according to claim 1, characterized in that: The high-resolution activity profile analysis platform described in step (2) achieves post-column microfluidic collection and real-time monitoring of UV spectroscopy / mass spectrometry by modifying the post-column flow path of the Agilent 1100 liquid chromatography system. The natural product extract is injected through a Cheminert two-position four-way injection valve and stored in a 20 μL quantitative loop. After the valve is cut, the sample is pumped into a 250 μm inner diameter Peek tube by the mobile phase in the quaternary pump and flows through the liquid chromatography column and the three-way splitter. The fraction after the three-way split enters the diode array detector and the OrbitrapExploris™ 120 mass spectrometer respectively through two Peek tubes of different lengths at 1 / 3 of the flow rate. The remaining fraction enters the microfluidic collection device.

4. The method according to claim 1, characterized in that: The mass spectrometry conditions described in step (2) are as follows: Mass spectrometry was performed simultaneously in positive and negative ion modes. The ion source type was H-ESI, and the analyzer was a quadrupole-electrostatic track trap mass analyzer. The main parameters of the first-stage and second-stage mass spectrometry are as follows: the first-stage / second-stage resolution is 60,000 / 15,000; sheath gas, 50 Arb. Auxiliary gas, 10 Arb; Purge gas, 1 Arb; ion source temperature, 400°C; scan voltage, +3500 V or -3000 V; scan range, 100-1000 Da; scan time, 0.15 s; HCD collision energies of 20%, 50%, and 80%.

5. The method according to claim 1, characterized in that: The microplate mentioned in step (2) is a 384-well microplate; The collection frequency of the microfluidic collection device described in step (2) is 10 s / well; The NusA-5-HB protein solution described in step (3) was diluted with phosphate buffer to a final concentration of 33.33 nmol·L⁻¹. -1 ; The HR2-FL solution described in step (3) was diluted with phosphate buffer to a final concentration of 25 nmol·L⁻¹. -1 ; The phosphate buffer solution has a salt concentration of 20 mmol·L⁻¹. -1 Contains 0.125% NP-40, and adjusts the pH to 7.4; The amount of NusA-5-HB protein solution added in step (3) is 30 μL per well; The amount of HR2-FL solution added in step (3) is 20 μL per well; The incubation conditions described in step (3) are: incubation at 37°C and 150 rpm for 30 min; The fluorescence polarization intensity mentioned in step (3) is measured using an ELISA reader; The parameters of the microplate reader are set as follows: temperature 37℃, light source is xenon flash lamp, excitation wavelength of filter group is 485 / 20nm, emission wavelength is 528 / 20nm, gain is 50, and detection height is 10.5 mm.

6. The method according to claim 1, characterized in that: The specific steps for real-time screening and structural identification of the active ingredients mentioned in step (3) are as follows: ① After microplates are tested using an ELISA reader, the fluorescence polarization intensity endpoint value is exported and used as the ordinate of the bioactivity spectrum. Subsequently, according to the order in which the post-column fractions are collected in the microplates, the single-well collection time is obtained based on the total collection time and the number of collection wells, i.e., total collection time / number of collection wells. This time is then converted into single-well time points corresponding to the retention times of the liquid chromatogram / mass spectrometry and used as the abscissa. The bioactivity spectrum can be plotted using the data from the above-processed abscissa and ordinate. In the bioactivity spectrum, negative peaks indicate that the fluorescence polarization intensity at the location of the well is low, which may indicate the presence of an active ingredient that can inhibit the binding of NusA-5-HB protein to the HR2-FL peptide probe. The stronger the inhibition, the more obvious the negative peak. In particular, a single-well fluorescence polarization endpoint value that is more than 30% lower than the average value of the negative control is considered to have potential active ingredients and is also one of the activity data points composed of negative peaks. ② Based on the bioactivity spectrum obtained in ①, combined with the chromatogram of the corresponding diode array detector of the chromatographic column, the liquid chromatographic peak corresponding to the time of the negative peak in the bioactivity spectrum is identified as a potential active ingredient; however, in some cases, insufficient chromatographic resolution may cause multiple components to co-elute at the same time, making it impossible to accurately identify the specific active ingredient. Therefore, chromatographic columns of different types / separation mechanisms are used to achieve the separation of co-elute components. If, under different chromatographic column separations, the same component is detected at the position corresponding to the negative peak using mass spectrometry secondary fragment information as the judgment index, it is identified as an active ingredient. ③ Based on the retention time of the potential active ingredients separated by the chromatographic column, combined with the mass spectrometry results and standard data at the corresponding time, the chemical structure of the potential novel coronavirus membrane fusion inhibitor was obtained through analysis.

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