A method and system for live cell drug screening based on FRET two-hybrid analysis

Abstract

This invention discloses a live-cell drug screening method and system based on FRET two-hybrid analysis. Firstly, preliminary experiments are conducted on multi-well plates using cell samples transfected with FRET standard plasmids to determine experimental conditions, including cell seeding density, imaging objective, excitation light intensity, field of view analysis range, and measurement of FRET system calibration parameters. Secondly, live-cell drug screening based on FRET two-hybrid analysis is performed under the preliminary experimental conditions. Cell culture is first performed, and drug and control groups are set up, followed by FRET three-channel imaging to calculate E0. D E A and R c And draw E D -R c Figure and E A -(1 / R) c (Figure); then use E D -R c With E A -(1 / R) c The linearly separated FRET two-hybrid analysis method yielded E Dmax E Amax and N A / N D Data was screened using the saturation index sRatio; finally, the active and potential active substances in the drug group were obtained through a significant difference test. This application enables accurate and rapid quantitative FRET measurement under a wide-field microscope with low magnification, meeting high-throughput requirements and screening for active compounds in live cells.

Description

Technical Field

[0001] This invention belongs to the technical field of drug screening and fluorescence resonance energy transfer (FRET) detection, specifically relating to a live-cell drug screening method and system based on FRET two-hybrid analysis. Background Technology

[0002] Drug development is a lengthy and expensive process; it takes more than 10 years for a new drug to go from the laboratory to a pharmacy, with an average cost of $1.3 billion. Target-based drug screening is an important method in drug development, but its destructive treatment of cells (fixation, fragmentation, separation, etc.) cannot represent the true physiological environment of the target, and the target information obtained may be inaccurate. Based on Foster resonance energy transfer (FAST)... Live-cell drug screening using resonance energy transfer (FRET) technology can preserve the normal physiological activity of the target as much as possible, and can more realistically and objectively reflect the drug's effect, thus attracting much attention.

[0003] However, there are still some problems with FRET-based drug screening. For example, FLIM-FRET-based drug screening requires relatively expensive instruments (such as single-photon counters), and the characteristics of measuring fluorescence lifetime result in a certain sacrifice in screening speed; TR-FRET-based drug screening is often performed on microplate readers or fluorescence lifetime measuring instruments, which have high analysis speed, but obtain less target information and require more complex experimental materials.

[0004] FRET two-hybrid analysis based on 3-cube imaging, such as BUTZ ES, BEN-JOHNY M, SHEN M, et al. Quantifying macromolecular interactions in living cells using FRET two-hybrid assays[J]. Nature Protocols, 2016, 11(12): 2470-2498. and DU M, YANG F, MAI Z, et al. FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor[J]. Applied Physics Letters, 2018, 112(15): 153702., can be performed rapidly under a wide-field fluorescence microscope without the need for special donors and acceptors. It can not only quantitatively measure the interactions between target molecules, but also obtain stoichiometric ratios and relative affinity parameters, providing researchers with richer biological information. However, no method has yet been proposed for using FRET two-hybrid analysis for drug screening. Summary of the Invention

[0005] The main objective of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a live-cell drug screening method based on FRET two-hybrid analysis. By introducing a FRET two-hybrid analysis method that linearly fits the FRET efficiency to the donor-recipient concentration ratio, accurate and rapid quantitative FRET measurements can be performed under a wide-field microscope with low magnification objectives, meeting high-throughput requirements. Furthermore, active compounds can be screened by observing changes in the target's maximum FRET efficiency and stoichiometry.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On the one hand, a live-cell drug screening method based on FRET two-hybrid analysis is provided, comprising the following steps:

[0008] Step 1: Conduct preliminary experiments on multi-well plates using cell samples transfected with the FRET standard plasmid to determine experimental conditions.

[0009] Determine the cell seeding density;

[0010] Determine the imaging objective lens;

[0011] Determine the excitation light intensity;

[0012] Determine the scope of the field of view analysis;

[0013] Calibration parameters a, b, c, d, G, k, ε for measuring the FRET system of a wide-field fluorescence microscope. A / ε D Where a, b, c, and d are spectral crosstalk coefficients, G is the sensitization quenching conversion factor, k is the ratio of donor to acceptor fluorescence intensity at equal concentrations in the absence of FRET, and ε A / ε D The ratio of the molar extinction coefficients of the acceptor and the donor;

[0014] Step 2: Perform live-cell drug screening based on FRET two-hybrid analysis according to the experimental conditions of the preliminary experiment:

[0015] According to the cell seeding density determined in the preliminary experiment, cells transfected with gene-marked drug target protein particles were cultured in multi-well plates until the cells expressed donor and acceptor proteins. Drug groups and control groups were set up. The drug groups included cell groups treated with multiple drugs, and cell groups treated with the same drug had multiple sets of experiments. The control group also had multiple sets of experiments.

[0016] Under the experimental conditions of imaging objective, excitation light intensity, and field of view analysis range determined in the preliminary experiment, the drug group and the control group were placed under a wide-field fluorescence microscope for FRET three-channel imaging; the FRET three channels include DD channel, AA channel, and DA channel;

[0017] For FRET three-channel imaging, cell data with a signal-to-background ratio below a second signal-to-background ratio threshold in any channel were removed, and then the donor-centered FRET efficiency E was calculated. D FRET efficiency centered on receptors A donor-recipient concentration ratio R c And draw E D -R c Figure and E A -(1 / R c )picture;

[0018] Use E D -R c With E A -(1 / R c The linearly segregated FRET two-hybrid analysis method yielded the donor-centered FRET maximum efficiency E. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D ;

[0019] Calculate E D -R c Figure and E A -(1 / R cThe saturation index sRatio in the graph was used to filter the data from the drug group and the control group;

[0020] Based on the screened data, a significant difference test was performed on the drug group and the control group to obtain the active and potential active substances in the drug group.

[0021] As a preferred technical solution, the determination of experimental conditions specifically includes:

[0022] Various cell seeding densities and different ratios of FRET standard plasmid were tested on multi-well plates to analyze cell distribution and transfection efficiency. Cell seeding densities that caused cell accumulation, little or no contact, and low transfection efficiency were excluded, and cell seeding densities with sufficient contact between cells and high transfection efficiency were selected.

[0023] Evaluate cell morphology and cell count per field of view under different magnification objectives, and select imaging objectives with cell count per field of view greater than the set cell threshold and clear cell morphology.

[0024] Cell samples transfected with the FRET standard plasmid were subjected to FRET three-channel imaging under different excitation light intensities to assess the signal-to-background ratio and photobleaching degree of the three channels, estimate the single-well imaging time, and select samples where the final signal-to-background ratio of all three channels was greater than the first signal-to-background ratio threshold and the receptor photobleaching degree was x. A and donor photobleaching degree x D The light intensity that is less than the set bleaching threshold during the single-aperture imaging time is used as the excitation light intensity.

[0025] Gaussian fitting was performed on the fluorescence calibration slide images obtained by three-channel imaging. For each channel, multiple masks were created, and the uniformity of the fluorescence calibration slide images under different masks was evaluated. The mask that makes the uniformity of the fluorescence calibration slide images of the three channels greater than the set uniformity threshold was selected and a logical AND operation was performed. The intersection region was used as the field of view analysis range.

[0026] As a preferred technical solution, the signal-to-background ratio of each channel is calculated as follows:

[0027] The average signal-to-background ratio of the brightest cells in the same field of view is taken as the signal-to-background ratio of this field of view. The average signal-to-background ratio of multiple different fields of view in the same hole is taken as the current light intensity and the signal-to-background ratio of the current channel.

[0028] The method for calculating the degree of photobleaching is as follows:

[0029] The donor excitation light was continuously irradiated onto cells that were separately transfected with FRET donor plasmid and FRET receptor plasmid, and DD or AA channel images were taken at the same time intervals before and after photobleaching.

[0030] Regarding the donor photobleaching degree x D The calculation formula is: x D =(I DD -I DP ) / I DD , among which, I DD and I DP These are the fluorescence intensities of donor-only transfected cells in the donor detection channel image before and after photobleaching.

[0031] For the degree of receptor photobleaching x A The calculation formula is: x A =(I AA -I AP ) / I AA , among which, I AA and I AP These are the fluorescence intensities in the receptor detection channel image of cells transfected only before and after photobleaching.

[0032] The single-well imaging time is determined by the number of fields of view captured per well and is affected by the operator's skill, the degree of cell transfection, and the instrument's imaging speed.

[0033] As a preferred technical solution, the method for acquiring the fluorescence calibration slide image is as follows:

[0034] Locate the focal plane of cells transfected with fluorescent protein in cell samples transfected with FRET standard plasmid; replace the cell samples with fluorescent calibration slides;

[0035] Under the excitation light intensity determined in the preliminary experiment, three-channel images were captured at least three times consecutively by reducing the exposure time. The average gray value of each channel image was calculated pixel by pixel as the intensity distribution of fluorescence emitted by the fluorescence calibration slide for each channel.

[0036] Remove the fluorescence calibration slide and turn off the light source. Take three-channel images at least three times consecutively, and calculate the average gray value of each channel image pixel by pixel as the average thermal noise distribution of each channel.

[0037] The fluorescence calibration slide image for each channel is obtained by subtracting the camera's average thermal noise for each channel from the intensity distribution of fluorescence emitted by the fluorescence calibration slide pixel by pixel.

[0038] The mask manufacturing method is as follows:

[0039] Gaussian fitting was performed on the fluorescence calibration slide image of each channel. The regions with gray values ​​greater than the peak value within a certain range after fitting were marked as 1 pixel by pixel, and the remaining regions were marked as 0. The corresponding binarized images were obtained as masks for their respective channels.

[0040] The formula for calculating the uniformity of the fluorescence calibration slide image is as follows:

[0041]

[0042] Where σ is the standard deviation of the gray values ​​of all pixels on the line connecting the maximum and minimum gray values ​​of the fluorescence calibration slide image, and μ is the average gray value of all pixels on the line connecting the maximum and minimum gray values ​​of the fluorescence calibration slide image.

[0043] As a preferred technical solution, the process of culturing cells transfected with gene-marked drug target protein particles in a multi-well plate specifically involves:

[0044] Live cells were seeded into multi-well plates containing culture medium at the cell seeding density determined in the preliminary experiment and then cultured.

[0045] Cells were co-transfected with protein particles labeled with different concentrations of donor fluorescent protein and recipient fluorescent protein, and then mixed in one well at transfection concentration ratios of a:b and b:a to form a transfection complex.

[0046] As a preferred technical solution, the donor-centered FRET efficiency E D Measurements were performed using the E-FERT method;

[0047] The receptor-centered FRET efficiency E A Through 3 3 Measurements are performed using the FRET method.

[0048] As a preferred technical solution, the stoichiometry N A / N D The calculation formula is:

[0049]

[0050] Among them, E Dmax For donor-centric FRET to achieve maximum efficiency, E Amax For receptor-centered FRET to achieve maximum efficiency;

[0051] The receptor-centered FRET maximum efficiency E Amax The calculation method is as follows:

[0052] At the free donor concentration D free Much larger than the dissociation constant K d In cells, the efficiency of FRET centered on the receptor is increased. A The average value is used as the receptor-centered FRET maximum efficiency E Amax Or for E D -R cA linear fit was performed on the graph, and its slope was used as the maximum efficiency E of receptor-centered FRET. Amax ;

[0053] The donor-centered FRET maximum efficiency E Dmax The calculation method is as follows:

[0054] At free acceptor concentration A free Much larger than the dissociation constant K d In cells, the efficiency of FRET centered on the donor is increased. D The average value is used as the donor-centered FRET maximum efficiency E Dmax Or for E A -(1 / R c A linear fit was performed on the graph, and its slope was used as the maximum efficiency E of FRET centered on the donor. Dmax .

[0055] As a preferred technical solution, the saturation index sRatio refers to E D -R c Figure and E A -(1 / R c The ratio of the area below the isotonic fitting line to the area above it in the figure is calculated as follows:

[0056] Calculate E D -R c Figure and E A -(1 / R c The isotonic fitting line of the graph;

[0057] Based on the actual protein binding and the direction of the isotonic fitting line, E D -R c Figure and E A -(1 / R c The isotonic fitting data in the figure is initially screened to select a set of isotonic fitting data points used to determine the area calculation region.

[0058] Determine E D -R c Figure and E A -(1 / R c In the area calculation region shown in the figure, the midpoint of multiple points with the largest x-coordinates is found from the selected set of isotonic fitting data points. The x-coordinate of the midpoint is used as the right boundary, and the y-coordinate of the midpoint is used as the upper boundary, with E as the boundary. D -R c Figure and E A -(1 / R cIn the figure, the x-axis and y-axis are the lower boundary and the left boundary, respectively; the upper boundary, lower boundary, left boundary, and right boundary constitute a closed area calculation region; if the upper boundary is not greater than the upper boundary threshold, then the upper boundary is set to the upper boundary threshold.

[0059] In the area calculation region, let A be the area below the isotonic fitting line and B be the area above it, and calculate E. D -R c Figure and E A -(1 / R c The saturation index sRatio corresponding to the graph is A / B.

[0060] As a preferred technical solution, the step of performing a significant difference test on the drug group and the control group specifically involves:

[0061] Calculate the donor-centered FRET maximum efficiency E in multiple experiments for each drug group. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D The average value, and compared with the donor-centered maximum FRET efficiency E in multiple experiments of the control group. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D The average value was tested for significance.

[0062] If a certain drug group E Dmax The average value and E Amax The average value was simultaneously compared with the control group E Dmax The average value and E Amax If the average values ​​are significantly different, then the drugs in that drug group are active substances.

[0063] If a certain drug group E Dmax The average value or E Amax Any one of the average values ​​compared to the control group E Dmax The average value or E Amax The average values ​​showed significant differences, and the stoichiometry N of the drug groups was also significantly different. A / N D The average value and the stoichiometry ratio of the control group N A / N D If the average values ​​are significantly different, then the drugs in that drug group are potential active substances.

[0064] On the other hand, a live cell drug screening system based on FRET two-hybrid analysis is provided, which is applied to the above-mentioned live cell drug screening method based on FRET two-hybrid analysis. The system includes a detection condition determination module and a drug screening module.

[0065] The detection condition determination module is used to determine experimental conditions in a preliminary experiment using cell samples transfected with the FRET standard plasmid on a multi-well plate.

[0066] Determine the cell seeding density;

[0067] Determine the imaging objective lens;

[0068] Determine the excitation light intensity;

[0069] Determine the scope of the field of view analysis;

[0070] Calibration parameters a, b, c, d, G, k, ε for measuring the FRET system of a wide-field fluorescence microscope. A / ε D Where a, b, c, and d are spectral crosstalk coefficients, G is the sensitization quenching conversion factor, k is the ratio of donor to acceptor fluorescence intensity at equal concentrations in the absence of FRET, and ε A / ε D The ratio of the molar extinction coefficients of the acceptor and the donor;

[0071] The drug screening module performs live-cell drug screening based on FRET two-hybrid analysis according to the experimental conditions determined by the detection condition determination module.

[0072] According to the cell seeding density determined in the preliminary experiment, cells transfected with gene-marked drug target protein particles were cultured in multi-well plates until the cells expressed donor and acceptor proteins. Drug groups and control groups were set up. The drug groups included cell groups treated with multiple drugs, and cell groups treated with the same drug had multiple sets of experiments. The control group also had multiple sets of experiments.

[0073] Under the experimental conditions of imaging objective, excitation light intensity, and field of view analysis range determined in the preliminary experiment, the drug group and the control group were placed under a wide-field fluorescence microscope for FRET three-channel imaging; the FRET three channels include DD channel, AA channel, and DA channel;

[0074] For FRET three-channel imaging, cell data with a signal-to-background ratio below a second signal-to-background ratio threshold in any channel were removed, and then the donor-centered FRET efficiency E was calculated. D FRET efficiency centered on receptors A donor-recipient concentration ratio R c And draw E D -R c Figure and E A -(1 / Rc )picture;

[0075] Use E D -R c With E A -(1 / R c The linearly segregated FRET two-hybrid analysis method yielded the donor-centered FRET maximum efficiency E. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D ;

[0076] Calculate E D -R c Figure and E A -(1 / R c The saturation index sRatio in the graph was used to filter the data from the drug group and the control group;

[0077] Based on the screened data, a significant difference test was performed on the drug group and the control group to obtain the active and potential active substances in the drug group.

[0078] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0079] 1. To address the issue of stringent experimental requirements for FRET two-hybrid analysis, this invention determines the experimental conditions for drug screening through preliminary experiments to ensure the successful execution of FRET two-hybrid analysis while meeting high-throughput requirements.

[0080] 2. To address the problems of traditional FRET live-cell drug screening, this invention provides a live-cell drug screening method based on FRET two-hybrid analysis. Employing a wide-field imaging mode, and using a mixed culture mode of transfected cells with different donor and acceptor concentration ratios, cells with significantly different donor and acceptor expression levels are simultaneously cultured in a single well and subjected to FRET two-hybrid analysis. This reduces the number of imaging fields and accelerates drug screening. Then, unsaturated samples are eliminated using the saturation index sRatio, ensuring accurate ET values ​​are obtained simultaneously. Dmax and E Amax Then calculate the stoichiometric ratio N. A / N D It objectively and accurately represents the target information under the action of drugs and can serve as an indicator that specifically reflects the efficacy of drugs; finally, through the significant difference test, effective active substances and potential active substances can be screened out in living cells.

[0081] 3. This invention can realize high-throughput quantitative FRET microscopy imaging analysis of live cells, and at the same time obtain the effects of drug interaction on target molecules and their structure and function, as well as the stoichiometry of complex biological macromolecules.

[0082] 4. Compared with FLIM-FRET-based drug screening, this invention can use wide-field FRET microscopic imaging analysis, eliminating the need for expensive lifetime measurement equipment, and is cheaper and faster.

[0083] 5. Compared with TR-FRET-based drug screening, this invention does not require special long-lived donors and can use ordinary FRET fluorescent probes and fluorescence microscopy, which reduces experimental costs and complexity. Attached Figure Description

[0084] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0085] Figure 1 This is an overall flowchart of a live cell drug screening method based on FRET two-hybrid analysis in an embodiment of the present invention.

[0086] Figure 2A The images shown are bright-field and fluorescence images of live cells expressing C40Y under 10x and 20x objectives in this embodiment of the invention; the scale bar for the 10x image is 100 μm, and the scale bar for the 20x image is 50 μm.

[0087] Figure 2B The figures show the signal-to-background ratio (S / B) and photobleaching degree under different light intensities at 20x objective lens in this embodiment. The left figure shows the three-channel signal-to-background ratio of MCF-7 cells expressing C40Y under different light intensities. The middle and right figures show the photobleaching degree of MCF-7 cells expressing YFP and CFP after irradiation with 6% donor excitation light intensity for 10 minutes.

[0088] Figure 2C This is a three-channel heat map of a fluorescence calibration slide under 20x objective lens and 6% light intensity in an embodiment of the present invention, and a comparison image before and after adding an 85% mask.

[0089] Figure 3 In this embodiment of the invention, the photobleaching degree of MCF-7 cells expressing YFP and CFP was measured after irradiation with 12% donor excitation light intensity for 10 minutes; the photobleaching degree x was calculated from the AA channel of transfected YFP cells within 10 minutes. A More than 10%, the degree of photobleaching was calculated by the DD channel of transfected CFP cells. DMore than 20%.

[0090] Figure 4 The imaging analysis range under each mask in the embodiments of the present invention; under 6% light intensity and 100ms exposure time, thermal analysis of the three-channel image of the autofluorescent plastic slide is performed using a 20x objective lens; 90-mask, 85-mask and 80-mask represent areas with gray values ​​greater than 90%, 85% and 80% of the peak value of the autofluorescent plastic slide image after Gaussian fitting, respectively.

[0091] Figure 5 This is a flowchart of live cell drug screening in an embodiment of the present invention.

[0092] Figure 6 This is a schematic diagram illustrating the calculation of the saturation index sRatio in an embodiment of the present invention.

[0093] Figure 7A The image shows a fluorescence image of MCF-7 live cells co-expressing YFP-Bak and CFP-Bcl-xL in an embodiment of the present invention.

[0094] Figure 7B E in this embodiment of the invention is the untreated (control group) D -R c and E A -(1 / R c )picture.

[0095] Figure 7C E in the embodiments of the present invention was treated with A1331852 (positive drug group). D -R c and E A -(1 / R c )picture.

[0096] Figure 7D E in the embodiment of the present invention treated with S63845 (negative drug group) D -R c and E A -(1 / R c )picture.

[0097] Figure 7E E is calculated in the embodiments of the present invention. Amax E Dmax and N A / N D .

[0098] Figure 8 The E calculated for screening eight drugs (A1331852, S63845, AC, DSF / Cu, Met, REGO, SOFA, ABT199) in this embodiment of the invention. AmaxE Dmax and N A / N D And the results of the significance test.

[0099] Figure 9 This is a schematic diagram of a live cell drug screening system based on FRET two-hybrid analysis in an embodiment of the present invention. Detailed Implementation

[0100] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative effort are within the scope of protection of the present application.

[0101] In this application, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this application can be combined with other embodiments.

[0102] like Figure 1 As shown, this embodiment discloses a live-cell drug screening method based on FRET two-hybrid analysis, which mainly includes two steps:

[0103] Step 1: Conduct preliminary experiments on multi-well plates using cell samples transfected with the FRET standard plasmid to determine experimental conditions.

[0104] S1-1, Determine the cell seeding density;

[0105] S1-2, Determine the imaging objective lens;

[0106] S1-3. Determine the excitation light intensity;

[0107] S1-4. Determine the scope of the field of view analysis;

[0108] S1-5. System calibration parameters for measuring wide-field fluorescence microscopy: a, b, c, d, G, k, ε A / ε D Where a, b, c, and d are spectral crosstalk coefficients, G is the sensitization quenching conversion factor, k is the ratio of donor to acceptor fluorescence intensity at equal concentrations in the absence of FRET, and ε A / ε D The ratio of the molar extinction coefficients of the acceptor and the donor;

[0109] Step 2: Perform live-cell drug screening based on FRET two-hybrid analysis according to the experimental conditions of the preliminary experiment:

[0110] S2-1. According to the cell seeding density determined in the preliminary experiment, cells transfected with gene marker drug target protein particles are cultured in multi-well plates until the cells express donor and acceptor proteins. Drug groups and control groups are set up. The drug groups include cell groups treated with multiple drugs, and there are multiple sets of experiments for cell groups treated with the same drug. The control group also has multiple sets of experiments.

[0111] S2-2. Under the experimental conditions of imaging objective, excitation light intensity and field of view analysis range determined in the pre-experiment, the drug group and the control group were placed under a wide-field fluorescence microscope for FRET three-channel imaging; wherein, the FRET three-channel (3-cube) includes DD channel, AA channel and DA channel;

[0112] S2-3. For FRET three-channel imaging, remove cell data where the signal-to-background ratio in any channel is lower than the second signal-to-background ratio threshold, and then calculate the FRET efficiency E centered on the donor. D FRET efficiency centered on receptors A donor-recipient concentration ratio R c And draw E D -R c Figure and E A -(1 / R c )picture;

[0113] S2-4, Using E D -R c With E A -(1 / R c The linearly segregated FRET two-hybrid analysis method yielded the donor-centered FRET maximum efficiency E. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D ;

[0114] S2-5, Calculate E D -R c Figure and E A -(1 / R c The saturation index sRatio in the graph was used to filter the data from the drug group and the control group;

[0115] S2-6. Based on the screened data, a significant difference test was performed on the drug group and the control group to obtain the active substances and potential active substances in the drug group.

[0116] In one specific embodiment, this application determines the experimental conditions for live cell drug screening by conducting preliminary experiments on 96-well plates. First, the source of materials used in the preliminary experiments is explained:

[0117] 1. The donor fluorescent group is gene-encoded fluorescent protein CFP (abbreviated as C), and the acceptor is gene-encoded fluorescent protein YFP (abbreviated as Y); C4Y, C10Y, C40Y and C80Y are tandem plasmids formed by linking C and Y with peptide chains of different lengths, and the numbers refer to the number of glycine residues; CFP and YFP are linked by a certain number of glycine residues, and the FRET efficiency is different for different numbers of glycine residues. The tandem plasmids were kindly provided by Christian Wahl-Schott (BUTZ ES, BEN-JOHNY M, SHEN M, et al. Quantifying macromolecular interactions in living cells using FRET two-hybrid assays[J]. Nature Protocols, 2016, 11(12):2470-2498.). All experiments were performed on 96-well plates with a 0.9 mm thick polystyrene base (LABSELECT, China); autofluorescent plastic slides (catalog number: 92001) were purchased from CHROMA (Vermont, USA); Turbofect transfection reagent was used. TM Obtained from Thermo Fisher Scientific (USA); the Luciferase Mycoplasma Detection Kit was purchased from Beijing TransGen Biotech.

[0118] 2. In this embodiment, a multimodal FRET fully automated imaging analyzer (IX73, Olympus, Japan) is used as the wide-field fluorescence microscope in this application. This analyzer is equipped with a 10× / 0.25NA PL-L objective, a 20× / 0.45NA PL-FL objective, and a CMOS camera (Flash 4.0, Hamamatsu, Japan). The light source is a 130W mercury lamp (U-HGLGPS, Olympus, Japan), with six settings: 100%, 50%, 25%, 12%, 6%, and 3%. The motorized excitation turntable (IX3-RFACA) used in this embodiment, which is compatible with the IX73 frame, is equipped with eight fluorescence excitation mirror groups (Cube1 to Cube8). These can be used with 25mm or 32mm (diameter) filters. Each cube can simultaneously hold one excitation filter (Ex), one dichroic mirror (DM), and one emission filter. The filter (Em) is used; the switching time between two adjacent cubes is only 500ms; a CCD camera is connected to the analyzer; the excitation light intensity is adjusted by selecting different output levels of the light source and different attenuation levels of the attenuator. The donor channel (DD channel) consists of a 436 / 20nm excitation filter and a 480 / 20nm emission filter; the FRET channel (DA channel) consists of a 436 / 20nm excitation filter and a 530 / 20nm emission filter; and the acceptor channel (AA channel) consists of a 510 / 20nm excitation filter and a 530 / 20nm emission filter.

[0119] 3. In this embodiment, the cell culture and plasmid transfection methods in the preliminary experimental process are as follows:

[0120] In the preliminary experiment, the MCF-7 cell line was used as the cell sample, and the CFP-YFP dimer plasmid was used to transfect the FRET standard plasmid. The MCF-7 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin, and 100 μg / mL streptomycin. During the preliminary experiment, the cells were cultured in a cell culture incubator at a constant temperature of 37℃ and 5% CO2. All cell lines used in the experiment were early passaged, and their culture time was less than 3 months. Mycoplasma contamination of the cells used in the preliminary experiment was regularly detected using a luciferase mycoplasma detection kit. Cells were seeded in 96-well plates overnight and cultured using Turbofect. TM Transfection; during the preparation of the CFP-YFP dimer plasmid, 100 ng of plasmid was transfected into each well of cells, and each plasmid was treated with 2 μL of Turbofect. TM Transfection within 24 hours.

[0121] Therefore, the specific steps for determining experimental conditions through preliminary experiments on cell samples transfected with the FRET standard plasmid are as follows:

[0122] S1-1. Determine cell seeding density: Test various cell seeding densities and different proportions of transfected standard plasmids in 96-well plates, analyze cell distribution and transfection efficiency, exclude cell seeding densities that cause cell accumulation, little or no contact, and low transfection efficiency, and select cell seeding densities with sufficient contact between cells and high transfection efficiency.

[0123] In this embodiment, various cell seeding densities (1000, 2000, 4000, 6000, and 8000 cells / well) and different ratios of FRET standard plasmid transfection were tested. After plasmid transfection, cell distribution and transfection efficiency were analyzed. It was found that higher cell seeding densities (6000 and 8000 cells / well) led to cell accumulation and low transfection efficiency, while lower cell seeding densities (1000 and 2000 cells / well) resulted in little or no cell-to-cell contact and low transfection efficiency. Therefore, in this embodiment, 4000 cells / well was selected as the cell seeding density, at which point there was sufficient cell-to-cell contact and high transfection efficiency.

[0124] S1-2. Determine the imaging objective: Evaluate cell morphology and cell count per field of view under different magnification objectives, and select an imaging objective with a cell count per field of view greater than the set cell threshold and clear cell morphology.

[0125] This embodiment tested the imaging performance of two different magnification objectives (10× and 20×), with a cell threshold set to 100; Figure 2A As shown, under a 10x objective lens, the cell morphology is small and the signal-to-background ratio is low (data not shown); while under a 20x objective lens, the cell morphology is clearly visible, and more than 100 cells were observed; therefore, a 20x objective lens was chosen as the imaging objective lens.

[0126] S1-3. For cell samples transfected with the FRET standard plasmid, FRET three-channel (3-cube) imaging was performed under different excitation light intensities to assess the signal-to-background ratio and photobleaching degree of the three channels. The single-well imaging time was estimated, and selection was made when the final signal-to-background ratio of all three channels was greater than the first signal-to-background ratio threshold and the receptor photobleaching degree was x. A and donor photobleaching degree x D The light intensity that is less than the set bleaching threshold during the single-aperture imaging time is used as the excitation light intensity.

[0127] Furthermore, the signal-to-background ratio for each channel is calculated as follows:

[0128] The average signal-to-background ratio of the brightest cells in the same field of view is taken as the signal-to-background ratio of this field of view. Then, the average of the signal-to-background ratios of multiple different fields of view in the same aperture is taken as the current light intensity and the signal-to-background ratio of the current channel.

[0129] Furthermore, the calculation method for the degree of photobleaching is as follows:

[0130] The donor excitation light was continuously irradiated onto cells that were separately transfected with FRET donor plasmid and FRET receptor plasmid, respectively. Images of the DD or AA channels were taken before and after photobleaching at the same time intervals (DD channel images were taken for cells transfected only with FRET donor plasmid, and AA channel images were taken for cells transfected only with FRET receptor plasmid).

[0131] Regarding the donor photobleaching degree x D The calculation formula is: x D =(I DD -I DP ) / I DD , among which, I DD and I DP These are the fluorescence intensities (with background removed) of donor-only transfected cells in the donor detection channel image before and after photobleaching.

[0132] For the degree of receptor photobleaching x A The calculation formula is: x A =(I AA -I AP ) / I AA , among which, I AA and I AP These are the fluorescence intensities (with background removed) of cells transfected only with the receptor in the receptor detection channel image before and after photobleaching.

[0133] Furthermore, the single-well imaging time is determined by the number of fields of view captured per well and is affected by the operator's skill, the degree of cell transfection, and the instrument's imaging speed.

[0134] In this embodiment, the first signal-to-background ratio threshold is 4. First, the signal-to-background ratio (S / B) is measured under different light intensities (3%, 6%, 12%, 25%, 50%, and 100%, respectively). MCF-7 cell samples transfected with C4Y are selected, and their signal and background values ​​are measured at an exposure time of 300ms. The average signal-to-background ratio of the 20 brightest cells in the same field of view is taken as the signal-to-background ratio for that field of view (signal-to-background ratio refers to the original grayscale value of the cell image (I0.05)). raw ) and the background gray value (I) of the area around the cell where there is no obvious fluorescent region bgThe ratio of the signal-to-background ratio is calculated, and then the average of the signal-to-background ratios from three different fields of view at the same aperture is taken as the final signal-to-background ratio for the current light intensity and the current channel; the result is as follows. Figure 2B As shown in the left figure, the final signal-to-background ratio of the DD channel did not exceed 4 at 3% light intensity, while at 6% and above light intensity, the final signal-to-background ratio of all three channels exceeded 5.

[0135] In this embodiment, by manually finding a suitable field of view and focusing, six fields of view are captured for each hole. After multiple measurements, the imaging time for a single hole does not exceed 3 minutes.

[0136] Regarding the degree of photobleaching, this embodiment measures the photobleaching degree at 6% and 12% light intensities, setting the bleaching threshold to 5%. At 6% light intensity, donor excitation light is continuously applied for 10 minutes, with three-channel imaging performed every minute. Each channel has an exposure time of 300ms. The average photobleaching degree of at least four transfected CFP cells and transfected YFP cells is calculated. Within a 3-minute single-well imaging time, the photobleaching degree x is calculated from the AA channel of transfected YFP cells. A The degree of photobleaching was calculated using the DD channel of CFP-transfected cells. D All less than 5% (e.g.) Figure 2B (Middle right image); while at 12% light intensity, x A and x D All exceed 5% (e.g.) Figure 3 (As shown).

[0137] Based on the above results, an excitation intensity of 6% was selected. At this point, the signal-to-background ratio met the requirements, and the degree of photobleaching was relatively low.

[0138] S1-4. Perform Gaussian fitting on the fluorescence calibration slide image obtained by three-channel imaging. For each channel, create multiple masks and evaluate the uniformity of the fluorescence calibration slide image under different masks. Select the mask that makes the uniformity of the fluorescence calibration slide image obtained by three-channel imaging greater than the set uniformity threshold and perform a logical AND operation. The intersection region is used as the field of view analysis range.

[0139] Furthermore, the method for acquiring the fluorescence calibration slide image is as follows:

[0140] Locate the focal plane of the cells transfected with the FRET standard plasmid in the cell sample; without adjusting the focal length, replace the cell sample with a fluorescence calibration slide;

[0141] Under the excitation light intensity determined in the preliminary experiment, the exposure time was reduced to prevent image overexposure. At least three consecutive images of the three channels were taken, and the average gray value of each channel image was calculated pixel by pixel as the intensity distribution of fluorescence emitted by the fluorescence calibration slide of each channel.

[0142] Remove the fluorescence calibration slide and turn off the light source. Take three-channel images at least three times consecutively, and calculate the average gray value of each channel image pixel by pixel as the average thermal noise distribution of each channel.

[0143] The fluorescence calibration slide image for each channel is obtained by subtracting the camera's average thermal noise for each channel from the intensity distribution of fluorescence emitted by the fluorescence calibration slide pixel by pixel.

[0144] Furthermore, the mask manufacturing method is as follows:

[0145] Gaussian fitting was performed on the fluorescence calibration slide image of each channel. Regions with gray values ​​greater than the peak value within a certain range after fitting were marked as 1 pixel by pixel, and the remaining regions were marked as 0. The corresponding binarized image was obtained as the mask for each channel.

[0146] Furthermore, the formula for calculating the uniformity of the fluorescence calibration slide image is as follows:

[0147]

[0148] Where σ is the standard deviation of the gray values ​​of all pixels on the line connecting the maximum and minimum gray values ​​of the fluorescence calibration slide image, and μ is the average gray value of all pixels on the line connecting the maximum and minimum gray values ​​of the fluorescence calibration slide image.

[0149] In this embodiment, the uniformity threshold is set to 90%. First, the focal plane of the cell sample transfected with fluorescent protein is found under 20x magnification and 6% light intensity. Then, the cell sample is replaced with a CHROMA autofluorescent plastic slide (a type of fluorescence calibration slide). At an exposure time of 100 milliseconds, images of the DD, DA, and AA channels are captured five times consecutively (without overexposure). The average gray value is calculated pixel by pixel to obtain the intensity distribution of fluorescence emitted by the autofluorescent plastic slide. Next, the autofluorescent plastic slide is removed, and the light source is turned off. The three-channel images are captured multiple times consecutively, and the average gray value of multiple images is calculated pixel by pixel to obtain the average thermal noise of the camera. The average thermal noise of the camera is subtracted pixel by pixel from the three-channel images of the autofluorescent plastic slide to obtain the final three-channel autofluorescent plastic slide image.

[0150] Then, Gaussian fitting was performed on the obtained three-channel autofluorescent plastic slide images; regions with gray values ​​greater than 85% were marked as 1 pixel by pixel, and the remaining regions were marked as 0, thus obtaining three binarized images, which were added to the autofluorescent plastic slide images (e.g., Figure 2C Similarly, add 80% and 90% masks to the images of the autofluorescent plastic slides, respectively (e.g., ...). Figure 4The final images of the autofluorescent plastic slides are presented as heatmaps. The uniformity of the autofluorescent plastic slide images with 90%, 85%, and 80% masks added is calculated, and the results are shown in Table 1 below.

[0151] Table 1 Uniformity of images in each channel

[0152]

[0153] Based on the results in Table 1 above, the intersection area of ​​the 85% mask of the three-channel autofluorescent plastic slide image is taken as the field of view analysis range.

[0154] Through the above preprocessing, the experimental conditions of this embodiment were determined as follows: 4000 cells / well was used as the optimal cell seeding density; cell imaging was performed under 20x magnification and 6% excitation light intensity; and the region with a gray value peak of more than 85% of the autofluorescent plastic slide image after Gaussian fitting was selected for analysis.

[0155] S1-5. Then measure the system calibration coefficients a, b, c, d, G, k, and ε of the multimodal FRET fully automated imaging analyzer. A / ε D Where a, b, c, and d are spectral crosstalk coefficients, G is the sensitization quenching conversion factor, k is the ratio of donor to acceptor fluorescence intensity at equal concentrations in the absence of FRET, and ε A / ε D This is the ratio of the molar extinction coefficients of the acceptor and the donor. The measurement of the system calibration coefficient is existing technology and a conventional technique mastered by those skilled in the art; therefore, this application will not elaborate further.

[0156] After determining the experimental conditions in the preliminary experiments, live cell drug screening was carried out, such as... Figure 5 As shown, this embodiment uses the screening of Bcl-xL protein inhibitors as an example for illustration; firstly, it explains the source of plasmids and drugs in the live cell drug screening process:

[0157] The donor CFP-Bcl-xL plasmid was kindly provided by APGilmore (SUN B, CHEN H, WANG X, et al. Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export[J]. Cell Death Discovery, 2023, 9(1):37.); the receptor YFP-Bak plasmid was constructed as described by Sun (SUN B, CHEN H, WANG X, et al. Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export[J]. Cell Death Discovery, 2023, 9(1):37.). A1331852, ABT199, S63845, regorafenib (REGO), sorafenib (SOFA), and disulfiram (DSF) were all purchased from MCE (New Jersey, USA). Abivitinib (AC) was purchased from Glpbio (California, USA). Metformin (Met) was purchased from Solarbio (Beijing, China). Copper gluconate (II)(Cu) was purchased from Macklin (Shanghai, China).

[0158] Furthermore, live cells were seeded into multi-well plates containing culture medium at the cell seeding density determined in the preliminary experiment and cultured. Then, a mixed culture mode was used to form a transfection complex. The mixed culture mode refers to the co-transfection of cells with protein particles labeled with different concentrations of donor fluorescent protein and recipient fluorescent protein, mixed in one well at a transfection concentration ratio of a:b and b:a to form a transfection complex.

[0159] In this embodiment, MCF-7 cells were seeded at a density of 4000 cells / well into 96-well plates containing 10% FBSDMEM medium and cultured at 37°C in a 5% CO2 incubator for 12 hours. Then, each well was transiently transfected with 400 ng of plasmid, which was then mixed at a concentration ratio of 3:1 and 1:3 to form a transfection complex, ensuring that each well contained cells with significantly different donor and recipient expression levels. The cells were then treated with the test drugs (A1331852 and S63845) for 7 hours. A control group (DMOS treatment) and a drug group (positive control A1331852 and negative control S63845 treatment) were set up. Under the experimental conditions determined in the pre-experiment, including the imaging objective, excitation light intensity, and field of view analysis range, three-channel imaging (e.g., using a multimodal FRET fully automated imaging analyzer) was performed. Figure 7A (As shown).

[0160] Furthermore, when calculating the FRET three-channel imaging, cell data with a signal-to-background ratio (SBR) of any channel lower than a second SBR threshold are removed. In this embodiment, the second SBR threshold is 3. The donor-centered FRET efficiency E D FRET efficiency centered on the receptor can be measured using methods such as E-FERT; A It can be done through 3 3 -Measured using methods such as FRET; among which, the donor-centered FRET efficiency E D The calculation formula is as follows (see reference: ZAL T, GASCOIGNE NR J. Photobleaching-corrected FRET efficiency imaging of live cells[J]. Biophysical Journal, 2004, 86(6):3923-3939):

[0161]

[0162] FRET efficiency E centered on receptor A The calculation formula is (see reference: ERICKSON MG, ALSEIKHAN BA, PETERSON BZ, et al. Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells[J]. Neuron, 2001, 31(6): 973-985):

[0163]

[0164] donor-recipient concentration ratio R c The calculation formula is as follows (see reference: CHEN H, PUHL HL, KOUSHIK SV, et al. Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells[J]. Biophysical Journal, 2006, 91(5): L39-L41.):

[0165]

[0166] Among them, F c The fluorescence intensity at which the donor is sensitized to the acceptor is calculated using the following formula:

[0167] F c =I DA -a(I AA -cI DD )-d(I DD -bI AA ),

[0168] Among them, I DA This refers to the fluorescence intensity detected in the DA channel; I AA This refers to the fluorescence intensity detected in the AA channel; I DD The fluorescence intensity detected in the DD channel, where λ is the excitation wavelength and ε is the fluorescence intensity detected in the DD channel. A (λ) is the molar extinction coefficient of the receptor at the excitation wavelength λ, ε D (λ) is the molar extinction coefficient of the donor at the excitation wavelength λ.

[0169] Furthermore, in E D -R c With E A -(1 / R c In the linearly separated FRET two-hybrid assay (see reference: DU M, YANG F, MAI Z, et al. FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor[J]. Applied Physics Letters, 2018, 112(15): 153702.), the stoichiometric ratio N A / N D The maximum efficiency E can be achieved through donor-centered FRET. Dmax and the maximum efficiency of receptor-centered FRET EAmax The calculation formula is as follows:

[0170]

[0171] Among them, the maximum efficiency E of FRET centered on the receptor Amax The calculation method is as follows:

[0172] At the free donor concentration D free Much larger than the dissociation constant K d In cells, the efficiency of FRET centered on the receptor is increased. A The average value is used as the receptor-centered FRET maximum efficiency E Amax Or for E D -R c The graph is linearly fitted, and E is then... D With R c It is directly proportional, and its slope is the maximum efficiency E of FRET centered on the receptor. Amax ;;

[0173] donor-centered FRET maximum efficiency E Dmax The calculation method is as follows:

[0174] At free acceptor concentration A free Much larger than the dissociation constant K d In cells, the efficiency of FRET centered on the donor is increased. D The average value is used as the donor-centered FRET maximum efficiency E Dmax Or for E A -(1 / R c Linear fitting is performed on the graph, and E is obtained from this. A With 1 / R c It is directly proportional, and its slope is the maximum FRET efficiency E centered on the donor. Dmax .

[0175] Furthermore, regarding E D -R c Figure and E A -(1 / R c The graph is used for saturation analysis to calculate the saturation index sRatio, which refers to E. D -R c Figure and E A -(1 / R c The ratio of the area below the isotonic fitting line to the area above it in the figure, such as... Figure 6 As shown ( Figure 6 China-Israel E A -(1 / R c (Taking the diagram as an example), the calculation steps are as follows:

[0176] Calculate ED -R c Figure and E A -(1 / R c The isotonic fitting line of the graph;

[0177] To avoid interference from outlier data, E was adjusted based on actual protein binding and the direction of the isotonic fitting line. D -R c Figure and E A -(1 / R c The isotonic fitting data in the figure is initially screened to extract a set of isotonic fitting data points used to determine the area calculation region; in this embodiment, the range of the isotonic fitting data after the initial screening is 0 < 1 / R. c <5、0 <E A <1 or 0 <R c <5、0 <E D <1.

[0178] Determine E D -R c Figure and E A -(1 / R c The area for area calculation in the diagram is denoted by E. D -R c Figure and E A -(1 / R c In the graph, the x-axis and y-axis represent the lower and left boundaries, respectively. From the selected set of data points, find the midpoint of the multiple points with the largest x-coordinates. Use the x-coordinate of this midpoint as the right boundary and the y-coordinate as the upper boundary (e.g., the midpoint coordinates are (x...y)). m ,y m ), then x = x m As the right boundary, y = y m As the upper boundary, the upper boundary, lower boundary, left boundary, and right boundary constitute a closed area calculation region; at the same time, isotonic fitting data outside the area calculation region are removed to ensure accurate calculation of the trapezoidal integral in the subsequent process; wherein, if the upper boundary is not greater than the upper boundary threshold, the upper boundary is set as the upper boundary threshold; the upper boundary threshold is set based on the drug screening requirements, and in this embodiment, the upper boundary threshold is 5%, that is, if the ordinate of the midpoint coordinate y m If the value is no greater than 5%, then the upper boundary is y = 5%, where 5% refers to E. D or E A .

[0179] In the area calculation region, let A be the area below the isotonic fitting line and B be the area above it, and calculate E. D -R c Figure and E A -(1 / R c The saturation index sRatio corresponding to the graph is A / B.

[0180] In this embodiment, if at least one sRatio is less than 2, and E Dmax E Amax If all values ​​are greater than 5%, the data is considered unsaturated and a complete FRET two-hybrid analysis cannot be performed (i.e., accurate E values ​​cannot be obtained simultaneously). Dmax and E Amax If the data from this group are discarded, cell culture and drug screening should be performed again; if sRatio is greater than 2, or E Dmax E Amax If any one of the values ​​is less than 5%, then the data set is considered valid and retained.

[0181] Furthermore, this application uses a significant difference test to screen active and potential active substances, specifically:

[0182] Calculate the donor-centered FRET maximum efficiency E in multiple experiments for each drug group. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D The average value, and compared with the donor-centered maximum FRET efficiency E in multiple experiments of the control group. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D The average value was tested for significance.

[0183] If a certain drug group E Dmax The average value and E Amax The average value was simultaneously compared with the control group E Dmax The average value and E Amax If the average values ​​of the samples are significantly different, then the test drug in that drug group is an active substance.

[0184] If a certain drug group E Dmax The average value or E Amax Any one of the average values ​​compared to the control group E Dmax The average value or E Amax The average values ​​showed significant differences, and the stoichiometry N of the drug groups was also significantly different. A / N D The average value and the stoichiometry ratio of the control group N A / N D If the average values ​​of the drugs in a given drug group show a significant difference, then the drug being tested in that drug group is a potential active substance.

[0185] In this embodiment, as Figure 7BAs shown, 87 DMSO-treated cells (control group Ctrl group) were analyzed in 24 fields of view; the results were calculated according to the corresponding formula and E. A -(1 / R c The graph shows that E tends to a constant value. A (2.5<1 / R c <3.5) Taking the average to obtain E Amax =0.193; according to the corresponding calculation formula and E D -R c The graph shows that E tends to a constant value. D (0.8 <R c <3.5) Taking the average to obtain E Dmax =0.115. Similarly, as... Figure 7C As shown, in 24 fields of view, 91 cells treated with A1331852 (positive drug group) were analyzed, and the E values ​​tended to be constant. A (2<1 / R c <3.5) and E D (0.8 <R c The average of values ​​<3.5) is used to obtain E. Amax =0.014 and E Dmax =0.015. For example... Figure 7D As shown, 87 cells treated with S63845 (negative drug group) were analyzed in 24 fields of view, and the E values ​​tended to be constant. A (2.5<1 / R c <3.5) and E D (0.8 <R c The average of values ​​<3.5) is used to obtain E. Amax =0.192 and E Dmax =0.106. According to the formula for calculating the stoichiometry, the stoichiometry of the Bak-Bcl-xL complex (Bak-Bcl-xL) in the control group, positive drug group, and negative drug group were 0.597 (0.115 / 0.193), 1.075 (0.015 / 0.014), and 0.552 (0.106 / 0.192), respectively. The Ex ratio of Bcl-xL to Bak can be obtained using FRET two-hybrid analysis. Dmax and E Amax Values. These values ​​were lower than the control group after A1331852 treatment; therefore, A1331852 caused the release of the binding between Bcl-xL and Bak, thereby releasing Bak. In this case, stoichiometry is not applicable for analyzing the binding ratio of specific molecules (e.g., Figure 7E (Right figure). FRET two-hybrid analysis showed that S63845 does not affect the binding of Bcl-xL to Bak; after treatment with S63845, the Ek of Bcl-xL to Bak was reduced. Dmax and EAmax The values ​​and stoichiometric ratios were not significantly different from those of the control group (e.g. Figure 7E ).

[0186] This embodiment also uses the same method to screen six drugs (AC, DSF / Cu, Met, REGO, SOFA, and ABT199). Each compound was screened at a fixed concentration of 1 μM in each well, and at least three sets of experiments were performed for each drug group and control group to reduce the probability of false negatives or false positives. The treatment time for each compound was 7 hours; E0 was plotted for each experimental group. D -R c Figure and E A -(1 / R c (See the graph) and calculate the saturation index sRatio for each. If at least one sRatio is less than 2, and E Dmax E Amax If all values ​​are greater than 5%, discard the data for this group and repeat cell culture and drug screening; if all values ​​are greater than 2, or E Dmax E Amax If any one of the values ​​is less than 5%, the data is retained; the filtering results are as follows: Figure 8 As shown.

[0187] Because the dissociation of Bcl-xL from Bak leads to the self-homogenization of Bak in mitochondria, resulting in apoptosis, compounds that unbind Bcl-xL and Bak are considered the active substances, defined as ExL. Dmax The average value and E Amax The mean values ​​were significantly different from the corresponding mean values ​​of the control group (Ctrl group, DMSO treatment); compounds that altered the binding of Bcl-xL and Bak were considered potentially active substances, defined as E... Dmax The average value or E Amax One of the mean values ​​was significantly different from the corresponding mean value of the control group (Ctrl group), and the mean value of the stoichiometric ratio was also significantly different from that of the control group (Ctrl group). Based on this criterion, the E of DSF / Cu was found to be significantly different. Amax The average value and stoichiometric ratio (N) A / N D The mean value of DSF / Cu was significantly different from that of the control group (Ctrl group), resulting in a receptor-donor stoichiometry of 0.702, making DSF / Cu a potential active substance that could alter the binding of Bcl-xL to Bak. The remaining compounds were considered ineffective against Bcl-xL and Bak. Table 2 presents the detailed results of the drug treatment; all data are expressed as mean ± standard error of at least three independent experiments.

[0188] Table 2. Results of FRET two-hybrid analysis under eight drug treatments.

[0189]

[0190]

[0191] It should be noted that, for the sake of simplicity, the aforementioned method embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, because according to the present invention, some steps can be performed in other orders or simultaneously.

[0192] Based on the same idea as the FRET two-hybrid analysis-based live-cell drug screening method in the above embodiments, the present invention also provides a FRET two-hybrid analysis-based live-cell drug screening system, which can be used to perform the above-described FRET two-hybrid analysis-based live-cell drug screening method. For ease of explanation, the schematic diagram of an embodiment of a FRET two-hybrid analysis-based live-cell drug screening system only shows the parts relevant to the embodiments of the present invention. Those skilled in the art will understand that the illustrated structure does not constitute a limitation on the device, and it may include more or fewer components, combine certain components, or have different component arrangements.

[0193] like Figure 9 As shown, another embodiment of the present invention provides a live cell drug screening system based on FRET two-hybrid analysis, including a detection condition determination module and a drug screening module;

[0194] The detection condition determination module is used to determine experimental conditions in preliminary experiments using cell samples transfected with the FRET standard plasmid on a multi-well plate.

[0195] Determine the cell seeding density;

[0196] Determine the imaging objective lens;

[0197] Determine the excitation light intensity;

[0198] Determine the scope of the field of view analysis;

[0199] Calibration parameters a, b, c, d, G, k, ε for measuring the FRET system of a wide-field fluorescence microscope. A / ε D Where a, b, c, and d are spectral crosstalk coefficients, G is the sensitization quenching conversion factor, k is the ratio of donor to acceptor fluorescence intensity at equal concentrations in the absence of FRET, and ε A / ε D The ratio of the molar extinction coefficients of the acceptor and the donor;

[0200] The drug screening module performs live-cell drug screening based on FRET two-hybrid analysis, according to the experimental conditions determined by the detection conditions.

[0201] According to the cell seeding density determined in the preliminary experiment, cells transfected with gene-marked drug target protein particles were cultured in multi-well plates until the cells expressed donor and acceptor proteins. Drug groups and control groups were set up. Among them, the drug groups included cell groups treated with multiple drugs, and cell groups treated with the same drug had multiple sets of experiments. The control group also had multiple sets of experiments.

[0202] Under the experimental conditions of imaging objective, excitation light intensity, and field of view analysis range determined in the preliminary experiment, the drug group and the control group were placed under a wide-field fluorescence microscope for FRET three-channel imaging; the FRET three channels include the DD channel, AA channel, and DA channel.

[0203] For FRET three-channel imaging, cell data with a signal-to-background ratio below a second signal-to-background ratio threshold in any channel were removed, and then the donor-centered FRET efficiency E was calculated. D FRET efficiency centered on receptors A donor-recipient concentration ratio R c And draw E D -R c Figure and E A -(1 / R c )picture;

[0204] Use E D -R c With E A -(1 / R c The linearly segregated FRET two-hybrid analysis method yielded the donor-centered FRET maximum efficiency E. Dmax The maximum efficiency of receptor-centered FRET E Amax and stoichiometry N A / N D ;

[0205] Calculate E D -R c Figure and E A -(1 / R c The saturation index sRatio in the graph was used to filter the data from the drug group and the control group;

[0206] Based on the screening data, a significance test was performed on the drug group and the control group to obtain the active and potential active substances in the drug group.

[0207] It should be noted that the FRET two-hybrid analysis-based live cell drug screening system of the present invention corresponds one-to-one with the FRET two-hybrid analysis-based live cell drug screening method of the present invention. The technical features and beneficial effects described in the above-mentioned embodiment of the FRET two-hybrid analysis-based live cell drug screening method are applicable to the embodiment of the FRET two-hybrid analysis-based live cell drug screening system. For details, please refer to the description in the embodiment of the method of the present invention, which will not be repeated here.

[0208] Furthermore, in the above embodiment of a live cell drug screening system based on FRET two-hybrid analysis, the logical division of each program module is merely illustrative. In practical applications, the above functions can be assigned to different program modules as needed, for example, for the sake of corresponding hardware configuration requirements or software implementation convenience. That is, the internal structure of the live cell drug screening system based on FRET two-hybrid analysis can be divided into different program modules to complete all or part of the functions described above.

[0209] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0210] 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 drug screening in living cells based on FRET two-hybrid analysis, characterized in that, The method comprises the following steps: Step 1: Pre-experiment is performed on a cell sample transfected with a FRET standard plasmid on a multi-well plate to determine experimental conditions: Determine the cell inoculation density; Determine the imaging objective; Determine the excitation light intensity; Determine the field of view analysis range; Calibration parameters a, b, c, d, G, k, e of a FRET system of a wide-field fluorescence microscope A / e D ; wherein a, b, c, d are spectral crosstalk coefficients, G is a sensitized quenching conversion factor, k is the ratio of fluorescence intensities of equal concentrations of donor and acceptor in the absence of FRET, e A / e D is the ratio of molar extinction coefficients of acceptor and donor; Step 2: Perform live cell drug screening based on FRET double-hybrid analysis according to the experimental conditions determined in the pre-experiment: According to the cell inoculation density determined in the pre-experiment, the cells transfected with the gene-labeled drug target protein plasmid are cultured on a multi-well plate until the cells express the donor protein, and a drug group and a control group are set; the drug group comprises a plurality of drug-treated cell groups, and each drug-treated cell group has a plurality of experiments; the control group also has a plurality of experiments; Under the experimental conditions of the imaging objective and the excitation light intensity determined in the pre-experiment, the drug group and the control group are placed under a wide-field fluorescence microscope for FRET three-channel imaging; the FRET three-channel includes a DD channel, an AA channel and a DA channel; For FRET three-channel imaging, the data of cells with any channel signal background ratio lower than the second signal background ratio threshold were rejected, and then the donor-centered FRET efficiency E was calculated within the field of view analysis range determined in the pre-experiment D , the acceptor-centered FRET efficiency E A , and the donor concentration ratio R c , and the E D -R c figure and the E A -(1 / R c ) figure were drawn; E D -R c with E A -(1 / R c ) linear separation of FRET Dmax , donor-centered FRET efficiency E Amax and stoichiometry N A / N D ; E D -R c Figure and E A -(1 / R c ) Figure; the saturation index sRatio is defined as E D -R c Figure and E A -(1 / R c ) Figure; the ratio of the area under the isosbestic line to the area above the isosbestic line. Based on the data after screening, significant difference test is performed on the drug group and the control group to obtain active substances and potential active substances in the drug group.

2. A live cell drug screening method based on FRET two-hybrid assay as claimed in claim 1, wherein, The determination of the experimental conditions is as follows: Test a plurality of different cell inoculation densities and different proportions of the FRET standard plasmid on a multi-well plate, analyze the cell distribution and the transfection efficiency, exclude the cell inoculation densities that cause cell accumulation, little or no contact and low transfection efficiency, and select the cell inoculation density with sufficient contact between cells and high transfection efficiency; Evaluate the cell morphology and the number of cells in a single field of view under different magnification objectives, and select the imaging objective with the number of cells in a single field of view greater than a set cell threshold and clear cell morphology; For the cell sample transfected with FRET standard plasmid, FRET three-channel imaging was performed under different excitation light intensities to evaluate the three-channel signal background ratio and the degree of photobleaching, estimate the single-well imaging time, and select the excitation light intensity as the excitation light intensity, which is greater than the first signal background ratio threshold and the degree of acceptor photobleaching x A and the degree of donor photobleaching x D is less than the set bleaching threshold within the single-well imaging time. Perform Gaussian fitting on the fluorescence calibration slide images obtained by three-channel imaging, make a plurality of masks for each channel, evaluate the uniformity of the fluorescence calibration slide images under different masks, select the masks with the uniformity of the fluorescence calibration slide images greater than a set uniformity threshold for the three-channel imaging and perform logical AND operation, and take the intersection region as the field of view analysis range.

3. A live cell drug screening method based on FRET two-hybrid assay as claimed in claim 2, wherein, The signal-to-background ratio calculation method for each channel is as follows: Take the average signal-to-background ratio of a plurality of cells with the brightest signal in the same field of view as the signal-to-background ratio of the field of view, and take the average value of the signal-to-background ratios of a plurality of different fields of view in the same well as the signal-to-background ratio of the current light intensity and the current channel; The light bleaching degree calculation method is as follows: Continuously irradiate the donor excitation light on the cells transfected with the FRET donor plasmid and the FRET acceptor plasmid respectively and individually, and take pictures of the DD or AA channel images before and after light bleaching at the same interval; For the donor photobleaching degree x D , the calculation formula is: x D = (I DD -I DP ) / I DD , wherein I DD and I DP are the fluorescence intensities of the cells transfected with only the donor in the image of the donor detection channel before and after photobleaching, respectively; For the extent of photobleaching of the acceptor x A , the formula is: x A = (I AA -I AP ) / I AA , wherein I AA and I AP are the fluorescence intensities of the cells transfected with the acceptor only in the image of the acceptor detection channel before and after photobleaching, respectively; The single-well imaging time is determined by the number of fields of view taken per well and is affected by the operation skill of the operator, the transfection degree of the cells and the imaging speed of the instrument.

4. The live-cell drug screening method based on FRET two-hybrid analysis as described in claim 2, characterized in that, The acquisition method of the fluorescence calibration slide image is as follows: Find the focal plane of the transfected fluorescent protein cells in the cell sample transfected with the FRET standard plasmid; replace the cell sample with a fluorescence calibration slide; Under the excitation light intensity determined in the pre-experiment, take three-channel images at least three times in succession by reducing the exposure time, and calculate the average gray value of each channel image pixel by pixel as the intensity value distribution of the fluorescence emitted by the fluorescence calibration slide of each channel. Remove the fluorescence calibration slide and turn off the light source, take three-channel images at least 3 times in succession, and calculate the average gray value of each channel image pixel by pixel as the camera average thermal noise distribution of each channel; Subtract the camera average thermal noise of the corresponding channel from the intensity value distribution of each channel of the fluorescence calibration slide to obtain the fluorescence calibration slide image of each channel; The mask making method is: Each channel of the fluorescence calibration slide image is respectively Gaussian fitted, and the region with a fitting gray value greater than a certain range of the peak value is marked as 1 pixel by pixel, and the remaining region is marked as 0 to obtain the corresponding binary image as the mask of each channel. The calculation formula of the fluorescence calibration slide image uniformity is: ， Wherein, σ is the standard deviation of the gray value of all pixel points on the line connecting the maximum gray value point and the minimum gray value point in the fluorescence calibration slide image, and μ is the average gray value of all pixel points on the line connecting the maximum gray value point and the minimum gray value point in the fluorescence calibration slide image.

5. The live-cell drug screening method based on FRET two-hybrid analysis as described in claim 1, characterized in that, The cells transfected with the gene-labeled drug target protein plasmid are cultured on a multi-well plate, and the specific steps are as follows: The living cells are inoculated into the multi-well plate containing the culture medium according to the cell inoculation density determined by the pre-experiment for culture; Different concentrations of donor fluorescent protein-labeled and acceptor fluorescent protein-labeled protein plasmids are used to co-transfect cells, and then mixed in a well according to the transfection concentration ratio of a:b and b:a to form a transfection complex.

6. The live-cell drug screening method based on FRET two-hybrid analysis as described in claim 1, characterized in that, the donor-centered FRET efficiency E D measured by the E-FERT method; The FRET efficiency E centered on the acceptor A by 3 3 measured by the -FRET method.

7. A live cell drug screening method based on FRET two-hybrid assay as claimed in claim 1 wherein, The stoichiometric ratio N A / N D The calculation formula is: ​ ， where E Dmax is the maximum efficiency of FRET with the donor as the center, E Amax is the maximum efficiency of FRET with the acceptor as the center; The FRET maximum efficiency E Amax is calculated as follows: At free donor concentration D free much greater than dissociation constant K d The average of the acceptor-centered FRET efficiencies E A in the cells is taken as the maximum acceptor-centered FRET efficiency E Amax ; or a linear fit is performed on the plot of E D - R c , and the slope of this fit is taken as the maximum acceptor-centered FRET efficiency E Amax . The donor-centered FRET maximum efficiency E Dmax is calculated as: In cells with free acceptor concentration A free much greater than dissociation constant K d , the average of donor-centered FRET efficiency E D is taken as the donor-centered FRET maximum efficiency E Dmax ; or a linear fit is performed on the plot of E A vs. (1 / R c ) with the slope taken as the donor-centered FRET maximum efficiency E Dmax .

8. The live-cell drug screening method based on FRET two-hybrid analysis as described in claim 1, characterized in that, The saturation index sRatio calculation step is: Calculation E D - R c Figure and E A - (1 / R c ) isocline of the isosmotic fit line; According to the actual protein binding condition and the trend of the isosmotic fitting line, the E D -R c Figure and E A -(1 / R c ) figure isosmotic fitting data for the initial screening, screening for determining the area of isosmotic fitting data point set calculation area; determining E D -R c figure and E A -(1 / R c ) figure, the middle point of the multiple points with the largest abscissa from the screened isosbestic fitting data point set is found, the abscissa of the middle point is taken as the right boundary, and the ordinate of the middle point is taken as the upper boundary, and E D -R c figure and E A -(1 / R c ) figure, the x-axis and y-axis are the lower boundary and left boundary; the upper boundary, lower boundary, left boundary, and right boundary constitute a closed area calculation region; if the upper boundary is not greater than the upper boundary threshold, the upper boundary is set to the upper boundary threshold; In the area calculation region, the area under the isosbestic fitting line is recorded as A, and the area above is recorded as B, and E is calculated D -R c Figure and E A -(1 / R c ) The corresponding saturation index sRatio = A / B.

9. A live cell drug screening method based on FRET two-hybrid assay as claimed in claim 1 wherein, The significant difference test of the drug group and the control group is specifically: ​ The average of the donor-centered FRET maximum efficiency E Dmax , acceptor-centered FRET maximum efficiency E Amax , and stoichiometry ratio N A / N D of the multiple experiments of each drug group is compared with the average of the donor-centered FRET maximum efficiency E Dmax , acceptor-centered FRET maximum efficiency E Amax , and stoichiometry ratio N A / N D of the multiple experiments of the control group for significance difference test. If the average of E Dmax and the average of E Amax are significantly different from the average of E Dmax and the average of E Amax , respectively, the drug in the drug group is an active substance. If either the mean of E Dmax or the mean of E Amax is significantly different from the mean of E Dmax or the mean of E Amax and the mean of the stoichiometric ratio N A / N D of the drug group is significantly different from the mean of the stoichiometric ratio N A / N D of the control group, then the drug in the drug group is a potential active substance.

10. A live cell drug screening system based on FRET two-hybrid analysis, characterized in that, The system is applied to the live cell drug screening method based on FRET double-hybrid analysis of claim 1, and the system comprises a detection condition determination module and a drug screening module; The detection condition determination module is used to determine the experimental conditions by using the cells transfected with the FRET standard plasmid on the multi-well plate: Determine the cell inoculation density; Determine the imaging objective; Determine the excitation light intensity; Determine the field of view analysis range; Calibration parameters a, b, c, d, G, k, e of a FRET system of a wide-field fluorescence microscope A / e D ; wherein a, b, c, d are spectral crosstalk coefficients, G is a sensitized quenching conversion factor, k is the ratio of fluorescence intensities of equal concentrations of donor and acceptor in the absence of FRET, e A / e D is the ratio of molar extinction coefficients of acceptor and donor; The drug screening module performs live cell drug screening based on FRET double-hybrid analysis according to the experimental conditions determined by the detection condition determination module: According to the cell inoculation density determined by the pre-experiment, the cells transfected with the gene-labeled drug target protein plasmid are cultured on a multi-well plate until the cells express the donor and acceptor proteins, and the drug group and the control group are set; the drug group comprises a plurality of cell groups treated with different drugs, and each cell group treated with the same drug has a plurality of experiments; the control group also has a plurality of experiments; Under the experimental conditions of the imaging objective, the excitation light intensity and the field of view analysis range determined by the pre-experiment, the drug group and the control group are placed under a wide-field fluorescence microscope for FRET three-channel imaging; the FRET three-channel includes the DD channel, the AA channel and the DA channel; For FRET three-channel imaging, the data of cells with any channel signal background ratio lower than the second signal background ratio threshold were rejected, and then the donor-centered FRET efficiency E D , acceptor-centered FRET efficiency E A and donor concentration ratio R c were calculated, and the E D -R c figure and E A -(1 / R c ) figure were plotted. E D -R c with E A -(1 / R c ) linear separation of FRET Dmax , donor-centered FRET efficiency E Amax and stoichiometry ratio N A / N D ; E D -R c Figure and E A -(1 / R c ) Figure; the ratio of the area under the isosbestic line to the area above the isosbestic line in the E D -R c Figure and E A -(1 / R c ) Figure; the ratio of the area under the isosbestic line to the area above the isosbestic line in the E Based on the data after screening, the significant difference test of the drug group and the control group is performed to obtain the active substances and potential active substances in the drug group.

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