Coagulator fluorescent bleaching recovery technology (Drop-FRAP)-based biomacromolecule interaction modulator screening platform

Abstract

The invention relates to a method for screening a macromolecular interaction regulator based on a fluorescent bleaching recovery technology. According to the method, a condensation body fluorescence bleaching recovery technology is utilized, and the change of a fluorescence signal is observed in real time, so that the regulation effect of a small molecule compound on biomacromolecules is tracked. The method disclosed by the invention has high sensitivity and repeatability, can be applied to a platform for researching and screening potential candidate drugs of biomacromolecule interaction, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the fields of biomolecular condensates and drug R & D. Specifically, the present invention relates to a screening platform for modulators of biomacromolecular interactions based on the condensate fluorescence recovery after photobleaching technique (Drop-FRAP). Background Art

[0002] Proteins are one of the most fundamental molecules in living organisms and play important roles within cells, such as catalyzing chemical reactions, transmitting signals, maintaining cell structure, etc. Protein-protein interaction is a very important research area in biology.

[0003] The interaction between proteins is an important part of the complex biochemical reaction network within cells. The interaction between proteins has important effects on organisms. For example, it maintains life processes, maintains the normal functions of tissues and organs, regulates metabolism, affects gene expression, and maintains structural integrity.

[0004] In addition, the interaction between proteins can also be used in drug development. For example, by studying protein interactions, new drug targets can be discovered, providing a basis for the design and R & D of new drugs; it helps to design drug molecules with higher affinity and specificity; by regulating specific protein interactions, the therapeutic effect of drugs can be enhanced; understanding protein interactions can help predict and reduce the side effects of drugs and improve drug safety; research based on protein interactions helps to achieve personalized medicine and select the most suitable treatment method according to the biological characteristics of patients.

[0005] Generally speaking, in-depth understanding of protein interactions is of great value for revealing the mechanisms of life processes in organisms, the pathogenesis of diseases, and the R & D of new drugs.

[0006] However, there is still a lack of methods in the prior art for rapidly and real-time monitoring the regulatory effects of small molecule compounds on biomolecular interactions. Summary of the Invention

[0007]

[0007] One object of the present invention is to provide a method for screening modulators of biomacromolecular interactions (including inhibitors and promoters) based on the condensate fluorescence recovery after photobleaching technique (Drop-FRAP).

[0008] Another object of the present invention is to provide a method for rapidly screening direct protein-protein interaction (PPI) inhibitors.

[0009] In the first aspect of the present invention, there is provided a method for screening modulators of macromolecular interactions, including the steps of:

[0010] a) Provide an engineered cell containing a target condensate A with dynamic fluidity; the target condensate A contains a first fusion protein formed by the fusion of a first target molecule X, a phase separation scaffold protein Y, and a first fluorescent protein F1 element, and the cell also co-expresses a second fusion protein, which is a fusion protein formed by the fusion of a first target molecule B and a second fluorescent protein F2 element;

[0011] Wherein, the first target molecule X and the first target molecule B interact with each other, so that the first fusion protein and the second fusion protein form a condensate complex A-B through the interaction of the first target molecule X and the first target molecule B;

[0012] b. In the absence of a candidate compound, perform photobleaching treatment on the cell, and measure the fluorescence signals of the condensate complex A-B in the cell before and after the photobleaching treatment to obtain the first fluorescence signal measurement data;

[0013] c. In the presence of the candidate compound, perform photobleaching treatment on the cell, and measure the fluorescence signals of the condensate complex A-B in the cell before and after the photobleaching treatment to obtain the second fluorescence signal measurement data;

[0014] d. Compare the first fluorescence signal measurement data and the second fluorescence signal measurement data to evaluate the effect of the candidate compound on the interaction between the first target molecule X and the first target molecule B;

[0015] Wherein, when the candidate compound promotes the interaction, the candidate compound is an interaction promoter; when the candidate compound inhibits the interaction, the candidate compound is an interaction inhibitor,

[0016] And the steps of steps b and c can be interchanged or carried out simultaneously.

[0017] In another preferred example, the target condensate A emits a fluorescence signal of a first wavelength.

[0018] In another preferred example, the fluorescence signal of the first wavelength is the fluorescent protein F1.

[0019] In another preferred example, the fluorescent protein F1 forms part of the first fusion protein.

[0020] In another preferred example, the first fluorescent protein F1 element is located at the N-terminus, C-terminus, or a combination thereof of the first fusion protein.

[0021] In another preferred example, the second fusion protein emits a fluorescence signal of a second wavelength.

[0022] In another preferred example, the fluorescence signal of the second wavelength is the fluorescent protein F2.

[0023] In another preferred example, the fluorescent protein F2 forms part of the second fusion protein.

[0024] In another preferred example, the second fluorescent protein F2 element is located at the N-terminus, C-terminus or a combination thereof of the second fusion protein.

[0025] In another preferred example, the target condensate A contains the fluorescent protein F1; the first target molecule B contains the fluorescent protein F2.

[0026] In another preferred example, the fluorescent protein F2 is the first target molecule B.

[0027] In another preferred example, the first wavelength and the second wavelength are distinguishable.

[0028] In another preferred example, the fluorescent protein F1 and the fluorescent protein F2 are two fluorescent proteins with different colors.

[0029] In another preferred example, the engineered cell is obtained by transfecting (transient transfection) the DNA sequence construct 1 and the DNA sequence construct 2 into the cell, so that the cell co-expresses the target condensate A and the second fusion protein;

[0030] Wherein, the DNA sequence construct 1 includes a first target molecule X sequence, a phase separation scaffold protein Y sequence and a first fluorescent protein F1 element sequence;

[0031] The DNA sequence construct 2 includes a first target molecule B sequence and a second fluorescent protein F2 element sequence.

[0032] In another preferred example, in steps b and c, the laser wavelength used for the photobleaching treatment is the same; and the laser wavelength is 480 nm - 580 nm; preferably, 488 nm or 561 nm.

[0033] In another preferred example, in steps b and c, the photobleaching treatment refers to spot laser irradiation of the region where the condensate complex A - B is located in the cell.

[0034] In another preferred example, the time of the laser irradiation is 0.001 - 1 s; preferably 0.001 - 0.1 s.

[0035] In another preferred example, the method further includes the steps:

[0036] e. In the presence of a positive compound, perform a photobleaching treatment on the cells, and measure the fluorescence signals of the condensate complex A-B in the cells before and after the photobleaching treatment to obtain the third fluorescence signal measurement data; wherein, the positive compound is a known regulator of the first target molecule X and the first target molecule B;

[0037] f. Based on the second fluorescence signal measurement data and the third fluorescence signal measurement data, evaluate the effect of the candidate compound on the interaction between the first target molecule X and the first target molecule B relative to the positive compound.

[0038] In another preferred example, in step e, the wavelength and irradiation time of the laser used for photobleaching are the same as those in steps b and c.

[0039] In another preferred example, the third fluorescence signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 element in the second fusion protein, and the recovery intensities before and after bleaching are denoted as RI postive 。

[0040] In another preferred example, in step f, compare RI postive and RI test ratio (RI test and RI postive ), denoted as U;

[0041] Wherein, when the positive compound is an inhibitor of the interaction,

[0042] When U < 1, and the smaller the U value, the better the inhibitory effect of the candidate compound on the interaction than the positive compound;

[0043] When U > 1, and the larger the U value, the less effective the candidate compound is in inhibiting the interaction than the positive compound;

[0044] Or when the positive compound is a promoter of the interaction,

[0045] When U < 1, and the smaller the U value, the less effective the candidate compound is in promoting the interaction than the positive compound;

[0046] When U > 1, and the larger the U value, the better the promoting effect of the candidate compound on the interaction than the positive compound.

[0047] In another preferred example, the phase separation scaffold protein Y sequence has the sequence listing shown in SEQ ID No: 1 - SEQ ID No: 16.

[0048] In another preferred embodiment, the phase separation scaffold protein Y is a protein sequence with characteristics of a disordered structure.

[0049] In another preferred embodiment, the phase separation scaffold protein Y is a protein sequence predicted by Main Page-Phase to have characteristics of a disordered structure.

[0050] In another preferred embodiment, the first fluorescence signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 component in the second fusion protein, and the recovery intensities before and after bleaching are denoted as RI ref ; and

[0051] The second fluorescence signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 component in the second fusion protein, and the recovery intensities before and after bleaching are denoted as RI test .

[0052] In another preferred embodiment, in step (d), the ratio of RI test and RI ref is denoted as Z; test / RI ref )

[0053] wherein, when Z < 1, it indicates that the candidate compound inhibits the interaction, and the smaller the Z value, the greater the inhibitory effect;

[0054] while when Z > 1, it indicates that the candidate compound promotes the interaction, and the greater the Z value, the greater the promoting effect.

[0055] In another preferred embodiment, when RI test is 90% of RI ref , the candidate compound is an interaction inhibitor. Preferably, when RI test is 80%, 75%, 50%, 30%, 15%, 5% of RI ref , the candidate compound is an interaction inhibitor;

[0056] When RI test is 110% of RI ref , the candidate compound is an interaction inhibitor. Preferably, when RI test is 125%, 150%, 170%, 190%, 200% of RI ref , the candidate compound is an interaction promoter.

[0057] In another preferred embodiment, the blank control used for RI ref is DMSO.

[0058] In another preferred example, the recovery intensity (RI) is calculated by the following formula Q1:

[0059] RI = W1 - W0 (Q1)

[0060] In the formula,

[0061] W1 is the plateau value of the fluorescence intensity recovery curve of the second fluorescent protein F2 element after photobleaching treatment;

[0062] W0 is the minimum value of the fluorescence intensity of the second fluorescent protein F2 element after photobleaching treatment.

[0063] In another preferred example, the plateau value of the fluorescence intensity recovery curve is the relative fluorescence intensity of any point in the curve plateau or the average value of multiple data points.

[0064] In another preferred example, Y1 is the relative fluorescence intensity at the t-th second after bleaching treatment, and t is 30 - 1000.

[0065] In another preferred example, t is 50 - 500 s, preferably 90 - 300 s, more preferably 120 - 240 s.

[0066] In another preferred example, the bleaching depth (PD) is calculated by the following formula Q2:

[0067] PD = W 100％ -W0 (Q2)

[0068] In the formula,

[0069] W 100％ is the fluorescence intensity value of the second fluorescent protein F2 element before photobleaching treatment;

[0070] W0 is the minimum value of the fluorescence intensity of the second fluorescent protein F2 element after photobleaching treatment.

[0071] In another preferred example, RI ref and RI test are normalized to calculate the intensity recovery rate, and the calculation method is as follows:

[0072] Among them, the calculation formula for the recovery rate of RI ref is RI ref / PD ref , and its normalized recovery rate value is denoted as 100%;

[0073] RI test The calculation formula for the recovery rate of is RI test / PD test , and its normalized recovery rate value is calculated by the following formula Q3:

[0074] RItest * = (RI test / PD test ) / (RI ref / PD ref ) × 100% (Q3).

[0075] In another preferred example, when RI test * < 100%, it indicates that the candidate compound inhibits the interaction, and the smaller the RI test * value, the greater the inhibitory effect;

[0076] While when RI test * > 100%, it indicates that the candidate compound promotes the interaction, and the larger the RI test * value, the greater the promoting effect.

[0077] In another preferred example, when RI test * < 90%, the candidate compound is an interaction inhibitor. Preferably, when RI test < 80%, 75%, 50%, 30%, 15%, 5%, the candidate compound is an interaction inhibitor;

[0078] When RI test * > 110%, the candidate compound is an interaction promoter. Preferably, when RI test * > 125%, 150%, 170%, 190%, 200%, the candidate compound is an interaction promoter.

[0079] In another preferred example, in step (b), it further includes: in the absence of the candidate compound, performing photobleaching treatment on the cells, and measuring the first-wavelength fluorescence signal from the first fusion protein in the condensate complex A - B in the cells before and after the photobleaching treatment to obtain the fifth fluorescence signal measurement data;

[0080] And step (c) further includes: in the presence of the candidate compound, performing photobleaching treatment on the cells, and measuring the first-wavelength fluorescence signal from the first fusion protein in the condensate complex A - B in the cells before and after the photobleaching treatment to obtain the sixth fluorescence signal measurement data.

[0081] In another preferred example, in step (b), based on the fifth fluorescence signal measurement data, a selected region (ROI) is determined, and the first-wavelength fluorescence signal measurement data in the selected region is obtained.

[0082] In another preferred example, in step (c), based on the measured data of the sixth fluorescence signal, a selected region (ROI) is determined, and the measured data of the fluorescence signal of the first wavelength in the selected region is obtained.

[0083] In another preferred example, the size of the selected region (ROI) is 1 - 100 μm. 2 .

[0084] In another preferred example, the method is carried out under real-time fluorescence monitoring.

[0085] In another preferred example, the fluorescent proteins F1 and F2 are selected from the group consisting of GFP, EYFP, mCherry, mStrawberry, dTomato, EBFP and their mutants.

[0086] In a second aspect of the present invention, there is provided a device for determining whether a candidate substance is a macromolecular interaction regulator, comprising:

[0087] A1. A test module, which is configured to incubate engineered cells in the presence or absence of the candidate substance; the cells contain a target condensate A with dynamic fluidity; the target condensate A contains a first fusion protein formed by fusion of a first target molecule X, a phase separation scaffold protein Y and a first fluorescent protein F1 element, and the cells also co-express a second fusion protein, which is a fusion protein formed by fusion of a first target point molecule B and a second fluorescent protein F2 element;

[0088] Wherein, the first target molecule X and the first target point molecule B interact with each other, so that the first fusion protein and the second fusion protein form a condensate complex A - B through the interaction of the first target molecule X and the first target point molecule B;

[0089] A2. A laser bleaching module, which is configured to bleach the condensate complex A - B in the cells by laser irradiation;

[0090] A3. A data acquisition module, which is configured to:

[0091] - In the absence of the candidate compound, collect the first fluorescence signal measurement data of the fluorescence signals of the condensate complex A - B in the cells before and after photobleaching treatment; and

[0092] - In the presence of the candidate compound, collect the second fluorescence signal measurement data of the fluorescence signals of the condensate complex A - B in the cells before and after photobleaching treatment;

[0093] A4. Interaction regulator evaluation module, the interaction regulator evaluation module is configured to: compare the first fluorescence signal measurement data and the second fluorescence signal measurement data, so as to obtain an evaluation result of whether the candidate substance is a regulator of the interaction between the first target molecule X and the first target molecule B; and

[0094] A5. Output module, the output module is used to output the evaluation result.

[0095] In another preferred example, the data acquisition module is further configured to:

[0096] - In the presence of a positive compound, collect third fluorescence signal measurement data of the fluorescence signals of the condensate complex A-B in the cell before and after photobleaching treatment; wherein, the positive compound is a known regulator of the first target molecule X and the first target molecule B.

[0097] In another preferred example, the interaction regulator evaluation module is configured to:

[0098] Compare the second fluorescence signal measurement data and the third fluorescence signal measurement data, so as to evaluate the influence of the candidate compound on the interaction between the first target molecule X and the first target molecule B relative to the positive compound.

[0099] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Brief Description of the Drawings

[0100] Figure 1 This is a schematic diagram of the technical route, based on fluorescence recovery after photobleaching of CoPIC phase separation macromolecular interactions. Perform photobleaching operation (photo bleach) on the target fluorescence in the selected region (region of interest), and then track the recovery of the target signal in real time. For example, the fluorescence recovery curve shown in the schematic diagram of the right curve in the figure. When a small molecule that inhibits biomacromolecular interaction (or PPI inhibitor) is added, as Figure 1 shown below, after the same bleaching operation, the inhibited biomacromolecular interaction cannot achieve the fluorescence signal recovery phenomenon, that is, the red curve cannot return to its original state. Among them, client represents the client protein. During the process of protein-protein interaction, K_on (association rate constant) and K_off (dissociation rate constant) are two important parameters describing the kinetics of protein interaction.

[0101] Figure 2The one labeled with DMSO is the control group, and the other four small molecules are all treatment groups with STAT3 dimerization inhibitors. The green signal represents the STAT3 protein fused with the NUP98 phase separation scaffold, and the red signal represents the client STAT3 protein. This system is used to detect STAT3 dimerization inhibitors. The area within the white box in the figure is the region where fluorescence recovery after photobleaching occurs.

[0102] Figure 3 For the Figure 2 experimental data statistics, the green curve represents the recovery kinetic curve of the phase separation scaffold protein, and the red curve represents the client recovery kinetic curve; the recovery efficiency of the red curve represents whether the small molecule has the ability to inhibit STAT3 dimerization.

[0103] Figure 4 For another example of protein-protein interaction fact, it is the detection of MDM2-P53 interaction inhibitor. Respectively, it is the DMSO control, and RG7388 is the experimental group. The green signal is the MDM2 droplet fused with the phase separation scaffold protein, and the red signal is the P53 client. The area within the white square in the figure is the region where fluorescence recovery after photobleaching is carried out.

[0104] Figure 5 For the Figure 4 experimental data statistics, the red curve represents the real-time fluorescence recovery of the client P53, and the green curve represents the fluorescence recovery of the phase separation scaffold protein. The proportion of the red curve recovery indicates the strength of the effect of the small molecule inhibiting the interaction. Detailed implementation mode

[0105] After extensive and in-depth research, the inventors have provided for the first time a brand-new method for real-time tracking and identifying inhibitors of biological macromolecules. Specifically, the present invention utilizes the condensate fluorescence recovery after photobleaching technology to observe the changes in fluorescence signals in real time, so as to track the regulatory effects of small molecule compounds on biological macromolecules. The method of the present invention has the characteristics of high sensitivity, short detection time, and low transfection requirements. Based on this, the inventors have completed the present invention.

[0106] Terms

[0107] As used herein, the term "condensate" is a stable protein structure formed by the fusion expression of a phase separation scaffold protein and a target molecule; it has dynamic fluidity; the target molecule can move and flow inside and outside the condensate.

[0108] As used herein, the term "interaction" or "interactome" refers to enzyme-substrate interactions, receptor-ligand interactions, and other intracellular protein-protein interactions with affinity. Inhibitors / promoters can participate in various post-translational modifications, such as phosphorylation, acetylation, dephosphorylation, acetylation, and so on; or they can directly act on the structural interface of non-enzyme type interacting macromolecule-macromolecule, such as blocking receptor-ligand binding.

[0109] The term "K_on (association rate constant)" represents the rate constant of protein interaction, that is, the speed of protein binding. The higher the K_on value, the faster the protein binding speed and the faster the formation of the complex;

[0110] The term "K_off (dissociation rate constant)" represents the rate constant of protein interaction, that is, the speed of protein dissociation. The higher the K_off value, the faster the protein dissociation speed and the lower the stability of the complex.

[0111]

[0112] These two rate constants are used to define the kinetic process of interaction, thereby revealing the speed of binding and dissociation between proteins. Understanding K_on and K_off is crucial for understanding the strength, stability, and kinetic process of protein interactions. Changes in these parameters can affect the formation and dissociation of protein complexes, and thus regulate biological processes such as cell signal transduction and metabolic regulation. In the present invention, K_on and K_off represent the dissociation and binding speeds between interacting protein pairs, and reflect the stability of the condensate complex A-B.

[0113] As used herein, the term "phase separation scaffold protein" refers to a protein that can spontaneously form phase separation droplets in cells, does not have direct physical interaction with target molecules B and X, and has intrinsic disorder in its primary sequence, which is conducive to the formation of condensates in cells.

[0114] As used herein, the term "fluorescence bleaching" refers to quenching fluorescence by laser irradiation. Fluorescence bleaching only bleaches the fluorescent group and does not affect the interaction between proteins. Usually, the wavelength for bleaching fluorescence is between 480 - 580 nm, for example, 488 nm or 561 nm.

[0115] As used herein, the terms "target molecule" and "target point molecule" refer to a group of interacting biological macromolecules, such as proteins and enzymes. The biological macromolecules that can be used as "target molecules" and "target point molecules" in the present application can be signal pathway proteins. For example, apoptosis pathway proteins: Bcl-2 family: including Bcl-2, Bcl-xl, etc., which regulate cell survival and apoptosis. Caspases: such as caspase-3, caspase-8, which are key proteins for executing apoptosis. MDM2-p53 signal pathway. Cell proliferation and survival signal pathway proteins: PI3K-AKT-mTOR pathway: including PI3 kinase, Akt, and mTOR, which are involved in the regulation of cell growth, survival, and metabolism. MAPK pathway: including ERK, JNK, and p38, which are involved in cell proliferation, differentiation, and stress response. Cell surface receptors: receptor tyrosine kinases (RTKs): such as EGFR, Insulin receptor, which regulate growth factor signals. G protein-coupled receptors (GPCRs): such as β-adrenergic receptor, which regulate cell signal transduction. Wnt / β-catenin signal pathway: β-catenin: is degraded under normal conditions but accumulates when Wnt signal is activated and interacts with proteins such as BCL9 and TCF4. JAK-STAT3 signal pathway.

[0116] As used herein, "modulator" or "interaction modulator" refers to a small molecule compound that modulates the interaction between target molecules and target point molecules, including inhibitors and promoters. The presence of the interaction modulator affects the bleaching recovery rate and recovery ratio of the fluorescent label in the condensate.

[0117] The target point molecules that can be used in the present application can be signal pathway protein inhibitors known in the art. For example, common interaction pairs and their corresponding inhibitors.

[0118]

[0119] As used herein, the term "fluorescent protein" refers to a class of luminescent proteins with stable chemical properties, strong penetrability, and colors that are easy to observe under a microscope, including but not limited to GFP, EYFP, mCherry, mStrawberry, dTomato, EBFP, and their respective mutants. As used herein, fluorescent protein F1 and fluorescent protein F2 are respectively labeled with target molecules connected to the phase separation scaffold protein and target point molecules co-expressed in the cell, and the fluorescent colors emitted by the two are different.

[0120] As used herein, the difference between direct PPI inhibitors and indirect inhibitors is as follows: Suppose there are three proteins, 1, 2, and 3, in a system. 1 and 2 interact directly, and 3 is responsible for phosphorylating 1. Only phosphorylated 1 can interact directly with 2. Then, a small molecule that disrupts the 1-2 interaction can target either the phosphorylation of 3-1 (indirectly) or directly target the 1-2 interaction interface (directly). From the result, the 1-2 interaction is disrupted, but it is impossible to distinguish whether it is direct or indirect.

[0121] The FRAP experiment of the present application can identify whether a small molecule compound is a direct inhibitor in a very short time, which cannot be achieved by traditional methods.

[0122] Fluorescence Recovery After Photobleaching (FRAP)

[0123] Fluorescence Recovery After Photobleaching (FRAP) is a high-resolution microscopy technique widely used in the biological field to study molecular movement and molecular interactions within cells. The basic principle of FRAP involves selectively quenching fluorescent tags within a cell or a specific region within a cell, and then observing the dynamics of their re-diffusion and recovery of fluorescence in that region over a certain period of time. This technique provides important information about the movement, diffusion, and interactions of biomolecules by quantitatively analyzing the rate and pattern of recovery.

[0124] In recent years, the application of FRAP technology in biological research has been continuously expanding, especially in the study of condensates within cells. Condensates are specific subcellular structures formed by the aggregation of biomacromolecules (such as proteins, nucleic acids, etc.), which have unique functions and biological importance. FRAP technology can be used to study the dynamic behavior and interactions of molecules within condensates.

[0125] The application of protein interactions in condensates is an important area of FRAP technology. By fluorescently labeling different proteins and performing FRAP experiments, the relative concentration, stability of proteins in condensates, and their interactions with other molecules can be revealed. This is crucial for understanding the regulatory mechanisms of cell signaling, gene expression, and cell structure. More importantly, FRAP can also be used to screen and evaluate potential protein interaction inhibitors, providing a powerful tool for drug development.

[0126] The application of FRAP technology in biological research has gone beyond traditional molecular motion research and become an indispensable tool for in-depth exploration of the structure of intracellular condensates and the interaction of biological macromolecules. FRAP is a very effective technical means for studying molecular dynamics changes and has played an important role in phase separation research. This technology provides rich data and insights for the biological field and is expected to promote more innovative research, especially in the fields of protein interaction and drug development.

[0127] Screening platform for regulators of biological macromolecule interactions based on condensate fluorescence recovery after photobleaching (Drop-FRAP)

[0128] The present invention discloses a method for screening regulators of biological macromolecule interactions based on fluorescence recovery after photobleaching. Mainly through methods such as plasmid transfection, cells are fused to express phase separation scaffold proteins, target molecules, and fluorescent proteins to form stable condensates; at the same time, target molecules that interact with the target molecules are also co-expressed in the cells. Due to the dynamic fluidity of the condensates, the target molecules can move and flow inside and outside the condensates. After fluorescence bleaching, candidate compounds are added, and by real-time monitoring of the fluorescence signals before and after fluorescence bleaching, the regulatory effects of the candidate compounds on biological macromolecules can be dynamically traced.

[0129] Since fluorescence bleaching only bleaches the fluorescent proteins and does not affect the interaction between the target molecules and the target molecules. In the absence of candidate compounds, the fluorescence signal will gradually recover to a fluorescence signal level equivalent to that before bleaching. The addition of candidate compounds will affect the bleaching recovery rate and recovery ratio of the fluorescent proteins in the condensates, thereby showing the regulatory effects of the candidate compounds in real time.

[0130] In addition, the method of the present invention is also applicable to cells with heterogeneous transfection. In cells with heterogeneous transfection, a small number of condensate lipid droplets formed by phase separation scaffold proteins are found, and the method of the present invention can be used for tracking, and the detection speed can reach the millisecond level.

[0131] The method of the present invention highlights the characteristics of dynamic and continuous traceability, making it an efficient tool for showing inhibitory effects in real time.

[0132] The specific working principle is as follows:

[0133] First, by fusing the expression of the phase separation scaffold protein and the target molecule, stable target condensates A are formed. These condensates have dynamic fluidity, that is, the target molecules can move and flow inside and outside the condensates, and this characteristic is one of the key features of this technology.

[0134] At the same time, the target molecule B that interacts with the condensate is also co-expressed. This target molecule B can interact with the target molecule in the condensate to form a condensate complex A-B. SeeFigure 1 , by fluorescence recovery after photobleaching (FRAP), the fluorescent protein conjugated to the target molecule B within the condensate is photobleached. Due to the dynamic nature of the condensate, the target molecule B will exhibit mobility over time while remaining traceable.

[0135] Subsequently, by introducing an interaction inhibitor or promoter, the inhibitory / promoting effect can be monitored and quantitatively determined in real time. The presence of the interaction inhibitor / promoter will affect the bleaching recovery rate and recovery ratio of the fluorescent label in the condensate, thereby enabling the real-time demonstration of the inhibitory / promoting effect of the interaction. This method highlights the dynamic and continuously traceable characteristics, making it an efficient tool for real-time demonstration of the inhibitory effect.

[0136] Therefore, the present invention describes a method for fusing a phase separation scaffold protein, a target molecule, and a target molecule, combined with the fluorescence recovery after photobleaching method, to achieve dynamic real-time tracking of interaction inhibitors. The characteristic of this technology is its ability to demonstrate the inhibitory effect in real time, providing an innovative tool for the study of biomacromolecular interactions and drug development.

[0137] Specifically, the phase separation scaffold protein in the present invention has an amino acid sequence selected from SEQ ID NO: 1 - SEQ ID NO: 15, wherein LCD represents low complexity domain, a low complexity sequence. SEQ ID NO: 16 is the nucleotide sequence of SEQ ID NO: 1.

[0138]

[0139]

[0140] Compared with the prior art, the main advantages of the present invention include:

[0141] 1. Real-time tracking and observation of biomacromolecular interactions: Through continuous image acquisition, the method of the present invention can demonstrate the dynamic movement and interaction of molecules within the target condensate in real time. This real-time tracking characteristic enables researchers to obtain detailed information about biomacromolecular interactions, such as kinetics, rates, and patterns, rather than just static images. This not only helps to understand intracellular processes more deeply but also provides more comprehensive data, contributing to biological research and drug development.

[0142] 2. Rapid verification of whether a small molecule is a direct (protein-protein interaction) PPI inhibitor: By introducing a potential inhibitor into condensates and immediately performing a FRAP experiment, it is possible to quickly verify whether a small molecule has direct PPI inhibitory activity. This has significant advantages for the field of drug development, especially for cell-based PPI inhibitor screening methods. Traditional intracellular PPI inhibitor screening methods are usually time-consuming and expensive, while condensate FRAP technology can provide a faster and more reliable method for screening direct PPI inhibitors, promising to accelerate the process of new drug discovery.

[0143] The following will further elaborate on the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0144] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0145] The materials, reagents, instruments, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources. In the following quantitative tests, three repeated experiments are set up, and the results are averaged. In the following embodiments, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0146] Material:

[0147] Fetal bovine serum: Purchased from GIBCO, ThermoFisher Scientific

[0148] Charcoal-treated fetal bovine serum: Purchased from VivaCell

[0149] Pen-Strep: Purchased from GIBCO, ThermoFisher Scientific

[0150] RG-7388 small molecule: Purchased from MCE

[0151] Ruxolitinib small molecule: Purchased from MCE

[0152] MI-773 small molecule: purchased from MCE

[0153] RG-7388 small molecule: purchased from MCE

[0154] NPT-10344 small molecule: purchased from Bioduro

[0155] NPT-10345 small molecule: purchased from Bioduro

[0156] NPT-10346 small molecule: purchased from Bioduro

[0157] DMEM: purchased from GIBCO, ThermoFisher Scientific

[0158] 96-well flat-bottom imaging plate (black bottom with clear lid): purchased from Perkin Elmer

[0159] The human kidney epithelial cell line HEK293T was purchased from the National Resource Bank of Model and Characteristic Experimental Cells (https: / / www.cellbank.org.cn / ) and maintained in DMEM containing 10% fetal bovine serum and 100 Units / ml Pen-Strep at 37 °C and 5% CO2.

[0160] Instrument: Nikon A1 LFOV

[0161] Example 1: Detection of STAT3 dimerization inhibitor

[0162] 1. Preparation of recombinant vectors

[0163] Synthesize and construct DNA molecules of GFP-NUP98N-STAT3 and mCherry-STAT3 artificially (shown as SEQ ID NO: 1-2 in Table 1 below). Insert the obtained DNA molecules into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (including the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites, to obtain recombinant eukaryotic expression vectors pcDNA3.1-GFP-NUP98N-STAT3 and pcDNA3.1-mCherry-STAT3 respectively. According to the manufacturer's instructions, extract the plasmids using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) and store them at -20 °C.

[0164] Table 1: Constructs used in Example 1

[0165]

[0166] Among them, the NUP98N scaffold protein has the amino acid sequence shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:16.

[0167] 2. Expression of Recombinant DNA

[0168] Using the liposome transfection method commonly known in the art, the plasmid DNA prepared in Example 1 was transiently expressed in cells as described below.

[0169] (1) Liposome transfection:

[0170] Prepare cell culture medium and inoculate HEK293T cells into a culture dish to reach an appropriate cell density. According to the instructions of the liposome transfection reagent, mix the purified DNA prepared in Example 1 with the transfection reagent at a specified ratio to form a transfection complex. Add the transfection complex to the cell culture medium, and then place it in a constant temperature incubator to culture the cells for a certain period of time under appropriate conditions (such as 37°C, 5% CO2).

[0171] (2) Expression detection:

[0172] According to the target protein to be expressed, select an appropriate method for detection. For example, a fluorescence microscope can be used to observe the expression of red fluorescent protein (mCherry) or / and green fluorescent protein (GFP) in cells.

[0173] 3. Verification of STAT3 Dimerization Inhibitor

[0174] After culturing the transfected cell line obtained in step 2 for 12 - 18 hours, perform a condensate FRAP experiment using a laser confocal scanning microscope, as Figure 1 shown.

[0175] Add the small molecule to be tested or DMSO (as the control group) to the confocal well of cell culture, gently mix up and down three times, and immediately start looking for double-positive condensates under the confocal microscope for the FRAP experiment. Select about 3 droplets for simultaneous bleaching each time, and select unbleached droplets and unbleached non-droplet regions as controls for final data processing. The confocal results are as Figure 2 shown.

[0176] Among them, the small molecule inhibitors are Ruxolitinib, NPT-10344, NPT-10345, and NPT-10346 respectively.

[0177] Table 2

[0178]

[0179] *Note: The RI / PD value of the blank control is taken as 100%; the lower the normalized recovery rate, the stronger the transient direct inhibitory effect of the test compound on protein-protein interaction.

[0180] 4. FRAP Data Analysis

[0181] Based on the data analysis of FRAP, the fluorescence recovery of different treatment groups was statistically analyzed, and the calculation formula is as follows:

[0182] Relative recovery signal (expressed as a percentage and excluding the side effect of bleaching) = [(I_t - I_min) / (I_max - I_min)] * [(I_unbleached_max - I_min) / (I_unbleached_t - I_min)] * 100%

[0183] I_t: Fluorescence signal intensity at time point t.

[0184] I_min: The minimum value of the signal intensity in the bleached area, usually obtained at the initial time point after bleaching.

[0185] I_max: The maximum value of the signal intensity in the unbleached area, usually obtained in the reference image.

[0186] I_unbleached_max: The maximum value of the signal intensity in the unbleached area, usually obtained in the reference image.

[0187] I_unbleached_t: The signal intensity of the unbleached area at time point t.

[0188] For each time point, calculate the percentage of the relative recovery signal and plot the recovery curve, where the time point is along the horizontal axis and the percentage of the relative recovery signal is along the vertical axis. Use data analysis software such as MATLAB, GraphPad Prism or a plugin in ImageJ to complete. The FRAP recovery curve and the percentage contribution of the recovery components are as Figure 3 shown, so as to help explain the dynamic behavior of the fluorescent marker in the cell.

[0189] Example 2: Verification of P53 / MDM2 Interaction Inhibitor

[0190] 1. Preparation of Recombinant Vectors

[0191] Synthesize artificial DNA molecules encoding mCherry-P53 and encoding GFP-NUP98N-MDM2 (shown as SEQ ID NO: 3-4 in Table 3 below). Insert the obtained DNA molecules into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (including the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites, to obtain recombinant eukaryotic expression vectors pcDNA3.1-mCherry-P53 and pcDNA3.1-GFP-NUP98N-MDM2 respectively. According to the manufacturer's instructions, use an endotoxin-free plasmid extraction kit (purchased from Nanjing Cowin Biotech Co., Ltd.) to extract the plasmids and store them at -20°C.

[0192] Table 3: Constructs used in Example 2

[0193]

[0194] 2. Expression of recombinant DNA

[0195] Use the liposome transfection method commonly known in the art to transiently express the cells with the purified DNA prepared in Example 2 as described below.

[0196] (1) Liposome transfection:

[0197] Prepare cell culture medium and inoculate HEK293T cells into a culture dish to reach an appropriate cell density. According to the instructions of the liposome transfection reagent, mix the purified DNA prepared in Example 2 with the transfection reagent in a specified ratio to form a transfection complex. Add the transfection complex to the cell culture medium and gently shake the culture dish to evenly distribute the complex on the cells. Place the culture dish in a constant temperature incubator and culture the cells under appropriate conditions (such as 37°C, 5% CO2) for a certain period of time.

[0198] (2) Expression detection:

[0199] Select an appropriate method for detection according to the target protein expressed. For example, a fluorescence microscope can be used to observe the expression of red fluorescent protein (mCherry) or / and green fluorescent protein (GFP) in cells.

[0200] 3. Verification of P53 / MDM2 interaction inhibitor

[0201] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A method for screening macromolecular interaction regulators, characterized in that, Including steps: a) providing an engineered cell, wherein the cell contains a target aggregate A having dynamic fluidity; the target aggregate A contains a first fusion protein formed by the fusion of a first target molecule X, a phase separation scaffold protein Y, and a first fluorescent protein F1 element, and the cell further co-expresses a second fusion protein formed by the fusion of a first target molecule B and a second fluorescent protein F2 element; Wherein, the first target molecule X and the first target molecule B interact with each other, so that the first fusion protein and the second fusion protein form an aggregate complex AB through the interaction between the first target molecule X and the first target molecule B; b. performing photobleaching treatment on the cell in the absence of the candidate compound, and measuring the fluorescence signal of the condensate complex AB in the cell before and after the photobleaching treatment to obtain first fluorescence signal measurement data; c. performing photobleaching treatment on the cell in the presence of the candidate compound, and measuring the fluorescence signal of the condensate complex AB in the cell before and after the photobleaching treatment to obtain second fluorescence signal measurement data; d. comparing the first fluorescence signal measurement data and the second fluorescence signal measurement data to evaluate the effect of the candidate compound on the interaction between the first target molecule X and the first target molecule B; Wherein, when the candidate compound promotes the interaction, the candidate compound is an interaction enhancer; when the candidate compound inhibits the interaction, the candidate compound is an interaction inhibitor. Furthermore, steps b and c can be interchanged or performed simultaneously.

2. The method according to claim 1, wherein The engineered cells are prepared by transfecting (transiently transfecting) DNA sequence construct 1 and DNA sequence construct 2 into cells, so that the cells co-express target aggregate A and the second fusion protein; Wherein, the DNA sequence construct 1 includes a first target molecule X sequence, a phase separation backbone protein Y sequence and a first fluorescent protein F1 element sequence; The DNA sequence construct 2 includes a first target molecule B sequence and a second fluorescent protein F2 element sequence.

3. The method according to claim 1, characterized in that, In step b and step c, the laser wavelength used in the photobleaching treatment is the same; and the laser wavelength is 480nm-580nm; preferably, 488nm or 561nm.

4. The method according to claim 1, wherein In step b and step c, the photobleaching treatment refers to targeted laser irradiation of the area where the condensate complex AB in the cell is located.

5. The method according to claim 1, characterized in that, The method further comprises the steps of: e. performing photobleaching treatment on the cell in the presence of a positive compound, and measuring the fluorescence signal of the condensate complex AB in the cell before and after the photobleaching treatment to obtain third fluorescence signal measurement data; wherein the positive compound is a known modulator of the first target molecule X and the first target molecule B; f. Evaluate the effect of the candidate compound on the interaction between the first target molecule X and the first target molecule B relative to the positive compound based on the second fluorescence signal measurement data and the third fluorescence signal measurement data.

6. The method according to claim 1, wherein The described phase separation scaffold protein Y sequence has a sequence listing as shown in SEQ ID No: 1 - SEQ ID No:

16.

7. The method according to claim 1, wherein The first fluorescence signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 element in the second fusion protein, and the recovery intensities before and after bleaching are denoted as RI ref ; and The second fluorescence signal described is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 element in the second fusion protein, and the recovery intensities before and after bleaching are denoted as RI test .

8. The method according to claim 7, wherein In step (d), compare RI test with RI ref and record the ratio (RI test / RI ref ) as Z; Among them, when Z < 1, it indicates that the candidate compound inhibits the interaction, and the smaller the Z value, the greater the inhibitory effect; while when Z > 1, it indicates that the candidate compound promotes the interaction, and the greater the Z value, the greater the promoting effect.

9. The method according to claim 1, characterized in that The described method is carried out under real-time fluorescence monitoring.

10. An apparatus for determining whether a candidate substance is a macromolecular interaction modulator, characterized in that, It includes: A1. A test module configured to incubate engineered cells in the presence or absence of the candidate substance; The cell contains a target condensate A with dynamic fluidity; the target condensate A contains a first fusion protein formed by fusion of a first target molecule X, a phase separation scaffold protein Y, and a first fluorescent protein F1 element, and the cell also co-expresses a second fusion protein, which is a fusion protein formed by fusion of a first target point molecule B and a second fluorescent protein F2 element; Among them, the first target molecule X and the first target point molecule B interact with each other, so that the first fusion protein and the second fusion protein form a condensate complex A - B through the interaction of the first target molecule X and the first target point molecule B; A2. A laser bleaching module configured to bleach the condensate complex A - B in the cell by laser irradiation; A3. A data acquisition module configured to: - In the absence of the candidate compound, collect first fluorescence signal measurement data of the fluorescence signals of the condensate complex A - B in the cell before and after photobleaching treatment; and - In the presence of the candidate compound, collect second fluorescence signal measurement data of the fluorescence signals of the condensate complex A - B in the cell before and after photobleaching treatment; A4. An interaction regulator evaluation module configured to: compare the first fluorescence signal measurement data and the second fluorescence signal measurement data to obtain an evaluation result on whether the candidate substance is a regulator of the interaction between the first target molecule X and the first target point molecule B; and A5. An output module for outputting the evaluation result.

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