WO2025162029A1 - Biomacromolecular interaction regulator screening platform based on condensate drop-fluorescence recovery after photobleaching (drop-frap) - Google Patents

Abstract

A macromolecular interaction regulator screening method, comprising the steps of: providing an engineered cell, the cell containing a target condensate A having dynamic fluidity, the target condensate A containing a first fusion protein formed by a target molecule X, a phase separation scaffold protein Y and a first fluorescent protein F1 module, the cell further co-expressing a second fusion protein, and the second fusion protein being a fusion protein formed by a target molecule B and a second fluorescent protein F2 module; by means of interaction of the target molecule X and the target molecule B, forming a condensate complex A-B; in the presence or absence of a candidate compound, separately performing photobleaching treatment on the cell, and measuring fluorescence signals obtained before and after the photobleaching treatment of the condensate complex A-B in the cell; and performing comparison for the change of the fluorescence signals, and evaluating the impact of the candidate compound on the interaction of the target molecule X and the target molecule B.

Description

A platform for screening biomacromolecule interaction modulators based on Drop-FRAP (fluorescence recovery after photobleaching) Technical Field

The present invention belongs to the field of bio-aggregates and drug research and development, and in particular, relates to a bio-macromolecule interaction regulator screening platform based on condensate fluorescence recovery after photobleaching (Drop-FRAP) technology.

Background Art

Proteins are among the most fundamental molecules in living organisms, playing crucial roles within cells, such as catalyzing chemical reactions, transmitting signals, and maintaining cell structure. Protein-protein interactions are a crucial research area in biology.

Protein-protein interactions are essential components of the complex biochemical networks within cells. These interactions have crucial impacts on organisms, such as maintaining life processes, supporting the normal function of tissues and organs, regulating metabolism, influencing gene expression, and maintaining structural integrity.

In addition, protein-protein interactions 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 development of new drugs; it helps to design drug molecules with higher affinity and specificity; and it enhances the therapeutic effects of drugs by regulating specific protein interactions. Understanding protein interactions can help predict and reduce drug side effects and improve drug safety. Research based on protein interactions can help achieve personalized medicine and select the most appropriate treatment method based on the patient's biological characteristics.

Overall, a deep understanding of protein interactions is of great value in revealing the mechanisms of life processes in organisms, the pathogenesis of diseases, and the development of new drugs.

However, the existing technology still lacks methods for rapid and real-time monitoring of the regulatory effects of small molecule compounds on biomolecular interactions.

Summary of the Invention

One object of the present invention is to provide a method for screening biomacromolecule interaction regulators (including inhibitors and promoters) based on drop-FRAP (Drop-FRAP) technology.

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

In a first aspect of the present invention, a method for screening macromolecular interaction regulators is provided, comprising the steps of:

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 a coacervate complex A-B through the interaction between the first target molecule X and the first target molecule B;

b. performing a photobleaching treatment on the cell in the absence of the candidate compound, and measuring the fluorescence signals of the condensate complex A-B in the cell before and after the photobleaching treatment to obtain first fluorescence signal measurement data;

c. performing a photobleaching treatment on the cell in the presence of the candidate compound, and measuring the fluorescence signals of the condensate complex A-B 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.

In another preferred embodiment, the target aggregate A emits a fluorescence signal of a first wavelength.

In another preferred embodiment, the fluorescent signal of the first wavelength is fluorescent protein F1.

In another preferred embodiment, the fluorescent protein F1 constitutes a part of the first fusion protein.

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

In another preferred embodiment, the second fusion protein emits a fluorescent signal of a second wavelength.

In another preferred embodiment, the fluorescent signal of the second wavelength is fluorescent protein F2.

In another preferred embodiment, the fluorescent protein F2 constitutes a part of the second fusion protein.

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

In another preferred embodiment, the target aggregate A contains fluorescent protein F1; the first target molecule B contains fluorescent protein F2.

In another preferred embodiment, the fluorescent protein F2 is the first target molecule B.

In another preferred embodiment, the first wavelength and the second wavelength are distinguishable.

In another preferred embodiment, the fluorescent protein F1 and the fluorescent protein F2 are two fluorescent proteins with different colors.

In another preferred embodiment, the engineered cells are produced 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.

In another preferred embodiment, 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.

In another preferred embodiment, in step b and step c, the photobleaching treatment refers to targeted laser irradiation of the area where the condensate complex A-B in the cell is located.

In another preferred embodiment, the laser irradiation time is 0.001-1s, preferably 0.001-0.1s.

In another preferred embodiment, the method further comprises the steps of:

e. performing a photobleaching treatment on the cell in the presence of a positive compound, and measuring the fluorescence signals of the condensate complex A-B 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.

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

In another preferred embodiment, the third fluorescent signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 component in the second fusion protein, and the recovery intensity before and after photobleaching is recorded as RI _postive .

In another preferred embodiment, in step f, the ratio of RI _postive to RI _test (RI _test and RI _postive ) is compared, denoted as U;

Wherein, when the positive compound is the inhibitor of the interaction,

When U is less than 1, and the smaller the U value is, it means that the candidate compound has a better inhibitory effect on the interaction than the positive compound;

When U>1, and the larger the U value, the greater the inhibitory effect of the candidate compound on the interaction is, the less effective the positive compound is.

Or when the positive compound is a promoter of the interaction,

When U<1, and the smaller the U value, it means that the candidate compound has a weaker promoting effect on the interaction than the positive compound;

When U>1, and the larger the U value, it means that the candidate compound has a better promoting effect on the interaction than the positive compound.

In another preferred example, the phase separation skeleton protein Y sequence has a sequence list as shown in SEQ ID No: 1-SEQ ID No: 16.

In another preferred embodiment, the phase separation scaffold protein Y is a protein sequence having disordered structural characteristics.

In another preferred embodiment, the phase separation skeleton protein Y is a protein sequence with disordered structural characteristics predicted by Main Page-Phase.

In another preferred embodiment, the first fluorescent signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 element in the second fusion protein, and the recovery intensity before and after bleaching is recorded as RI _ref ; and

The second fluorescent signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 component in the second fusion protein, and the recovery intensity before and after photobleaching, which is recorded as RI _test .

In another preferred embodiment, in step (d), the ratio of RI _test and RI _ref (RI _test /RI _ref ) is compared and recorded as Z;

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

When Z>1, it means that the candidate compound promotes the interaction, and the larger the Z value, the greater the promotion effect.

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%, or 5% of RI _ref , the candidate compound is an interaction inhibitor.

When RI _test is 110% of RI _ref , the candidate compound is an interaction inhibitor. Preferably, when RI _test is 125%, 150%, 170%, 190%, or 200% of RI _ref , the candidate compound is an interaction enhancer.

In another preferred embodiment, the blank control used in RI _ref is DMSO.

In another preferred embodiment, the recovery strength (RI) is calculated by the following formula Q1:
RI＝W1－W0 (Q1)

Where,

W1 is the platform value of the fluorescence intensity recovery curve of the second fluorescent protein F2 element after photobleaching treatment;

W0 is the minimum fluorescence intensity of the second fluorescent protein F2 element after photobleaching.

In another preferred embodiment, the platform value of the fluorescence intensity recovery curve is the relative fluorescence intensity of any point in the curve platform or the average value of multiple data points.

In another preferred embodiment, Y1 is the relative fluorescence intensity at t seconds after bleaching, where t is 30-1000.

In another preferred embodiment, t is 50-500s, preferably 90-300s, more preferably 120-240s.

In another preferred embodiment, the bleaching depth (PD) is calculated by the following formula Q2:
PD＝W _100％ -W0 (Q2)

Where,

W _100% is the fluorescence intensity value of the second fluorescent protein F2 element before photobleaching treatment;

W0 is the minimum fluorescence intensity of the second fluorescent protein F2 element after photobleaching.

In another preferred embodiment, RI _ref and RI _test are normalized to calculate the strength recovery rate, and the calculation method is as follows:

The recovery rate of RI _ref is calculated as RI _ref /PD _ref , and the normalized recovery rate value is recorded as 100%;

The recovery rate of RI _test is calculated as RI _test /PD _test , and the normalized recovery rate value is calculated using the following formula Q3:
RI _test *=(RI _test /PD _test )/(RI _ref /PD _ref )×100% (Q3).

In another preferred embodiment, when RI _test * is less than 100%, it indicates that the candidate compound inhibits the interaction, and the smaller the RI _test * value, the greater the inhibitory effect;

When RI _test *>100%, it means that the candidate compound promotes the interaction, and the larger the RI _test * value, the greater the promotion effect.

In another preferred embodiment, when RI _test * is less than 90%, the candidate compound is an interaction inhibitor. Preferably, when RI _test is less than 80%, 75%, 50%, 30%, 15%, or 5%, the candidate compound is an interaction inhibitor.

When RI _test *>110%, the candidate compound is an interaction inhibitor. Preferably, when RI _test *>125%, 150%, 170%, 190%, 200%, the candidate compound is an interaction enhancer.

In another preferred embodiment, step (b) further comprises: performing a photobleaching treatment on the cell in the absence of the candidate compound, and measuring the first wavelength fluorescence signal from the first fusion protein of the condensate complex A-B in the cell before and after the photobleaching treatment to obtain fifth fluorescence signal measurement data;

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

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

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

In another preferred embodiment, the size of the selected region of interest (ROI) is 1-100 μm ² .

In another preferred embodiment, the method is performed under real-time fluorescence monitoring.

In another preferred embodiment, the fluorescent proteins F1 and F2 are selected from the group consisting of GFP, EYFP, mCherry, mStrawberry, dTomato, EBFP and mutants thereof.

In a second aspect of the present invention, there is provided an apparatus for determining whether a candidate substance is a macromolecular interaction modulator, comprising:

A1. A test module configured to incubate engineered cells in the presence or absence of a candidate substance; the cells contain a target aggregate A having dynamic fluidity; the target aggregate A comprises a first fusion protein formed by a fusion of a first target molecule X, a phase-separating scaffold protein Y, and a first fluorescent protein F1 element; and the cells further co-express a second fusion protein formed by a 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 A-B through the interaction between the first target molecule X and the first target molecule B;

A2. A laser bleaching module, wherein the laser bleaching module is configured to bleach the condensate complex A-B in the cell by laser irradiation;

A3. Data acquisition module, the data acquisition module is configured to:

- collecting first fluorescence signal measurement data from the fluorescence signal of the condensate complex A-B in the cell before and after photobleaching treatment in the absence of the candidate compound; and

- collecting second fluorescence signal measurement data from the fluorescence signal of the condensate complex A-B in the cell before and after photobleaching treatment in the presence of the candidate compound;

A4. An interaction modulator evaluation module, wherein the interaction modulator evaluation module is configured to: compare the first fluorescence signal measurement data and the second fluorescence signal measurement data to obtain an evaluation result of whether the candidate substance is a modulator of the interaction between the first target molecule X and the first target molecule B; and

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

In another preferred embodiment, the data acquisition module is further configured to:

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

In another preferred embodiment, the interaction regulator evaluation module is configured as follows:

The second fluorescence signal measurement data and the third fluorescence signal measurement data are compared to 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.

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 described in detail 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 listed here one by one.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of this technology route, which is based on fluorescence photobleaching recovery of CoPIC phase-separated macromolecular interactions. The target fluorescence is photobleached in a selected region (region of interest), and then the recovery of the target signal is tracked in real time, such as the fluorescence recovery curve shown in the curve diagram on the right side of the figure. When a small molecule that inhibits the interaction of biomacromolecules (or PPI inhibitors) is added, as shown below in Figure 1, after the same photobleaching operation, the inhibited biomacromolecule interaction cannot achieve fluorescence signal recovery, that is, the red curve cannot be restored to its original state. Among them, client represents the client protein. In the process of protein-protein interaction, K_on (association rate constant) and K_off (dissociation rate constant) are two important parameters that describe the dynamics of protein interaction.

Figure 2 shows the DMSO control group, while the other four small molecules are treated with STAT3 dimerization inhibitors. The green signal represents STAT3 protein fused to the NUP98 phase-separated backbone, and the red signal represents the client STAT3 protein. This system is used to detect STAT3 dimerization inhibitors. The white box in the figure indicates the region where fluorescence recovery after photobleaching occurs.

Figure 3 shows the data statistics for the experiment in Figure 2. The green curve represents the recovery kinetics curve of the phase-separated scaffold protein, and the red curve represents the client recovery kinetics curve. The recovery efficiency of the red curve represents whether the small molecule has the ability to inhibit STAT3 dimerization.

Figure 4 shows another example of protein-protein interaction, detecting an inhibitor of the MDM2-P53 interaction. The images are shown as a DMSO control and an experimental group with RG7388. The green signal represents MDM2 droplets fused to a phase-separating scaffold protein, and the red signal represents the P53 client. The white box in the image indicates the region where fluorescence recovery after photobleaching occurs.

Figure 5 shows the statistical data from the experiment in Figure 4. The red curve represents the real-time fluorescence recovery of client P53, and the green curve represents the fluorescence recovery of the phase-separated scaffold protein. The recovery ratio of the red curve indicates the effectiveness of the small molecule inhibitory interaction.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventors have developed a novel method for real-time tracking and identification of biomacromolecule inhibitors. Specifically, the present invention utilizes condensate fluorescence recovery after photobleaching (FDR) technology to observe changes in fluorescence signals in real time, thereby tracking the regulatory effects of small molecule compounds on biomacromolecules. This method boasts high sensitivity, short detection times, and minimal transfection requirements. Based on this, the inventors completed the present invention.

the term

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

As used herein, the term "interaction" or "interaction" 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, and acetylation; they can also act directly at the structural interface of non-enzymatic macromolecules, such as blocking receptor-ligand binding.

The term "K_on (association rate constant)" represents the rate constant of protein interaction, that is, the speed at which proteins bind. A higher K_on value indicates faster protein binding and faster complex formation.

The term "K_off (dissociation rate constant)" represents the rate constant of protein interaction, that is, the rate at which the protein dissociates. A higher K_off value indicates a faster protein dissociation rate and a lower stability of the complex.

These two rate constants are used to define the kinetics of the interaction, thereby revealing the speed of association and dissociation between proteins. Understanding K_on and K_off is crucial for understanding the strength, stability, and kinetics of protein interactions. Changes in these parameters can affect the formation and dissociation of protein complexes, thereby regulating biological processes such as cell signaling and metabolic regulation. In the present invention, K_on and K_off represent the dissociation and association rates between interacting protein pairs, and express the stability of the condensate complex A-B.

As used herein, the term "phase-separating scaffold protein" refers to a segment of protein that can spontaneously form phase-separated droplets within cells, does not have direct physical interactions with target molecules B and X, and has an intrinsically disordered primary sequence that is conducive to the formation of aggregates within cells.

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

As used herein, the terms "target molecule" and "target molecule" refer to a group of biomacromolecules with interactions, such as proteins and enzymes. Biomacromolecules that can be used as "target molecules" and "target molecules" in this application can be signaling 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, are key proteins for executing apoptosis. MDM2-p53 signaling pathway. Cell proliferation and survival signaling pathway proteins: PI3K-AKT-mTOR pathway: including PI3 kinase, Akt and mTOR, involved in cell growth, survival and metabolic regulation. MAPK pathway: including ERK, JNK and p38, involved in cell proliferation, differentiation and stress response. Cell surface receptors: receptor tyrosine kinase (RTK): such as EGFR, Insulin receptor, regulate growth factor signals. G protein-coupled receptor (GPCR): such as β-adrenergic receptor, regulates cell signal transduction. Wnt/β-catenin signaling pathway: β-catenin is degraded under normal conditions but accumulates when Wnt signaling is activated and interacts with proteins such as BCL9 and TCF4. JAK-STAT3 signaling pathway.

As used herein, "modulator" or "interaction modulator" refers to a small molecule compound that modulates the interaction between a target molecule and a target site molecule, including inhibitors and promoters. The presence of an interaction modulator affects the photobleaching recovery rate and recovery ratio of the fluorescent marker in the aggregate.

The target 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.

As used herein, the term "fluorescent protein" refers to a class of luminescent proteins with stable chemical properties, strong penetrability, and colors that are easily observed 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, respectively, label target molecules linked to phase-separating scaffold proteins and target molecules co-expressed in cells, and the two emit different fluorescence colors.

As used herein, the distinction between direct and indirect PPI inhibitors is as follows: Assume there are three proteins in a system: proteins 1, 2, and 3. Proteins 1 and 2 interact directly, and protein 3 is responsible for phosphorylating protein 1, which then allows it to interact directly with protein 2. Therefore, a small molecule that disrupts the 1-2 interaction can target either the phosphorylation of protein 3-1 (indirect) or the 1-2 interaction interface (direct). The results indicate that both disrupt the 1-2 interaction, but it is impossible to distinguish whether this is direct or indirect.

The FRAP experiment of the present application can identify whether a small molecule compound is a direct inhibitor in a very short time, which is impossible with traditional methods.

Photobleaching-recovery assay (FRAP)

Fluorescence Recovery After Photobleaching (FRAP) is a high-resolution microscopy technique widely used in biology to study intracellular molecular motion and interactions. The basic principle of FRAP involves selectively quenching fluorescent tags within a cell or a specific region within a cell, and then observing the dynamic characteristics of their re-diffusion and recovery of fluorescence within that region over a specific time period. This technique provides valuable information about the motion, diffusion, and interactions of biomolecules by quantitatively analyzing the speed and pattern of recovery.

In recent years, the application of FRAP technology in biological research has continued to expand, particularly in the study of intracellular condensates. Condensates are specialized subcellular structures formed by the aggregation of biomacromolecules (such as proteins and nucleic acids), which possess unique functions and biological importance. FRAP technology can be used to study the dynamic behavior and interactions of molecules within condensates.

The application of FRAP technology to protein interactions within aggregates is a key area of application. By fluorescently labeling different proteins and performing FRAP experiments, it is possible to reveal the relative concentration, stability, and interactions of proteins with other molecules within aggregates. This is crucial for understanding the regulatory mechanisms of cell signaling, gene expression, and cell structure. Importantly, FRAP can also be used to screen and evaluate potential inhibitors of protein interactions, providing a powerful tool for drug discovery.

The application of FRAP technology in biological research has transcended traditional studies of molecular motion, becoming an indispensable tool for in-depth exploration of the structure of intracellular aggregates and the interactions of biomacromolecules. FRAP is a highly effective technique for studying molecular dynamics and has played a significant role in phase separation research. This technology provides a wealth of data and insights for the biological field, and is expected to drive further innovative research, particularly in the areas of protein interactions and drug discovery.

A screening platform for biomacromolecule interaction modulators based on Drop-FRAP (fluorescence recovery after photobleaching)

The present invention discloses a method for screening biomacromolecule interaction regulators based on fluorescence recovery after photobleaching technology. The method primarily involves using methods such as plasmid transfection to enable cell fusion and expression of a phase-separated scaffold protein, a target molecule, and a fluorescent protein to form stable aggregates. Simultaneously, the cells also co-express target molecules that interact with the target molecules. Because the aggregates have dynamic fluidity, the target molecules can move and flow within and outside the aggregates. After fluorescence photobleaching, candidate compounds are added. By real-time monitoring of the fluorescence signals before and after fluorescence photobleaching, the regulatory effects of the candidate compounds on the biomacromolecules can be dynamically tracked.

Because fluorescence bleaching only bleachs the fluorescent protein and does not affect the interaction between the target molecule and the target molecule, the fluorescence signal will gradually recover to the level equivalent to the fluorescence signal before bleaching in the absence of the candidate compound. However, the addition of the candidate compound will affect the bleaching recovery rate and recovery ratio of the fluorescent protein in the aggregate, thus demonstrating the regulatory effect of the candidate compound in real time.

In addition, the method of the present invention is also applicable to cells with heterogeneous transfection. If a small number of lipid droplets formed by phase-separated skeletal proteins are found in the heterogeneously transfected cells, they can be tracked using the method of the present invention, and the detection speed can reach milliseconds.

The method of the present invention highlights the characteristics of dynamic and continuous traceability, making it a tool for efficiently demonstrating the inhibitory effect in real time.

The specific working principle is as follows:

First, by fusing the phase-separation backbone protein with the target molecule, a stable target aggregate A is formed. These aggregates have dynamic fluidity, that is, the target molecules can move and flow in and out of the aggregates, which is one of the key features of this technology.

At the same time, target molecule B, which interacts with the condensate, is also co-expressed. This target molecule B can interact with the target molecule within the condensate, forming a condensate complex A-B. As shown in Figure 1, fluorescence recovery after photobleaching is used to bleach the fluorescent protein attached to target molecule B within the condensate. Due to the dynamic nature of the condensate, target molecule B exhibits temporal mobility while remaining traceable.

Subsequent introduction of interaction inhibitors or promoters allows for real-time monitoring and quantitative determination of inhibitory/promoting effects. The presence of interaction inhibitors/promoters influences the photobleaching recovery rate and recovery ratio of the fluorescent marker within the aggregates, thereby demonstrating the inhibitory/promoting effects in real time. This method, with its dynamic and continuous traceability, is a highly effective tool for demonstrating inhibitory effects in real time.

Therefore, this paper describes a method for dynamic, real-time tracking of interaction inhibitors by integrating a phase-separating scaffold protein, a target molecule, and a target site molecule, combined with fluorescence recovery after photobleaching. This technology is unique in that it can demonstrate the inhibitory effect in real time, providing an innovative tool for studying biomacromolecular interactions and drug development.

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

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

1. Real-time tracking and observation of biomacromolecular interactions: Through continuous image acquisition, the method of this invention can visualize the dynamic motion and interactions of molecules within the target condensate in real time. This real-time tracking capability enables researchers to obtain detailed information about biomacromolecular interactions, such as kinetics, rates, and patterns, rather than just static images. This not only facilitates a deeper understanding of intracellular processes but also provides more comprehensive data, aiding biological research and drug development.

2. Rapidly verify whether a small molecule is a direct (protein interaction) PPI inhibitor: By introducing a potential inhibitor into the aggregate and immediately performing a FRAP experiment, it is possible to quickly verify whether the small molecule has a direct PPI inhibitory effect. This has significant advantages in the field of drug development, especially in cell-based PPI inhibitor screening methods. Traditional intracellular PPI inhibitor screening methods are usually time-consuming and expensive, while aggregate FRAP technology can provide a faster and more reliable direct PPI inhibitor screening method, which is expected to accelerate the process of new drug discovery.

The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. Quantitative experiments in the following examples were performed in triplicate, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing refers to the 5′-terminal nucleotide of the corresponding DNA/RNA, and the last position refers to the 3′-terminal nucleotide of the corresponding DNA/RNA.

Material:

Fetal bovine serum: purchased from GIBCO, ThermoFisher Scientific

Activated carbon-treated fetal bovine serum: purchased from VivaCell

Pen-Strep: purchased from GIBCO, ThermoFisher Scientific

RG-7388 small molecule: purchased from MCE

Ruxolitinib small molecule: purchased from MCE

MI-773 small molecule: purchased from MCE

RG-7388 small molecule: purchased from MCE

NPT-10344 small molecule: purchased from Bioduro

NPT-10345 small molecule: purchased from Bioduro

NPT-10346 small molecule: purchased from Bioduro

DMEM: purchased from GIBCO, ThermoFisher Scientific

96-well flat-bottom imaging plate (black bottom with transparent cover): purchased from Perkin Elmer

The human renal epithelial cell line HEK293T was purchased from the National Model and Specialty Experimental Cell Resource Bank (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 .

Instrument: Nikon A1 LFOV

Example 1: Detection of STAT3 dimerization inhibitors

1. Preparation of recombinant vector

Artificially synthesized and constructed DNA molecules for GFP-NUP98N-STAT3 and mCherry-STAT3 (shown in SEQ ID NOs. 1-2 in Table 1 below). The resulting DNA molecules were inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (containing the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites to generate the recombinant eukaryotic expression vectors pcDNA3.1-GFP-NUP98N-STAT3 and pcDNA3.1-mCherry-STAT3, respectively. Plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the manufacturer's instructions and stored at -20°C.

Table 1: Constructs used in Example 1

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

2. Expression of recombinant DNA

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

(1) Liposome transfection:

Prepare cell culture medium and seed HEK293T cells into a culture dish to achieve an appropriate cell density. According to the instructions for the liposome transfection reagent, mix the purified DNA prepared in Example 1 with the transfection reagent in the specified ratio to form a transfection complex. Add the transfection complex to the cell culture medium and then place the dish in a constant temperature incubator and culture the cells under appropriate conditions (e.g., 37°C, 5% _CO2 ) for a specified period of time.

(2) Expression detection:

Depending on the target protein being expressed, select an appropriate detection method. For example, you can use a fluorescence microscope to observe the expression of red fluorescent protein (mCherry) and/or green fluorescent protein (GFP) in cells.

3. Validation of STAT3 dimerization inhibitors

After culturing the transfected cell line obtained in step 2 for 12-18 hours, a FRAP experiment of aggregates was performed using a laser confocal scanning microscope, as shown in FIG1 .

The small molecule to be tested or DMSO (as a control group) was added to the cell culture confocal well and gently mixed up and down three times. Then, the double positive aggregates were immediately searched for under the confocal microscope for FRAP experiment. About three droplets were selected for simultaneous bleaching each time. Unbleached droplets and unbleached non-droplet areas were selected as controls for final data processing. The confocal results are shown in Figure 2.

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

Table 2

*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 interaction.

4. FRAP Data Analysis

According to the FRAP data analysis, the fluorescence recovery of different treatment groups was statistically analyzed, and the calculation formula is as follows:

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

I_t: Fluorescence signal intensity at time point t.

I_min: Minimum signal intensity of the bleached area, usually obtained at the initial time point after bleaching.

I_max: The maximum value of the signal intensity in the unbleached area, usually obtained in the baseline image.

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

I_unbleached_t: Signal intensity of the unbleached area at time point t.

For each time point, calculate the percentage of relative recovered signal and plot a recovery curve with time along the horizontal axis and the percentage of relative recovered signal along the vertical axis. This can be accomplished using data analysis software such as MATLAB, GraphPad Prism, or a plugin in ImageJ. The FRAP recovery curve, along with the percentage contribution of the recovered component, is shown in Figure 3, helping to interpret the dynamic behavior of the fluorescent marker within the cell.

Example 2: Validation of P53/MDM2 interaction inhibitors

1. Preparation of recombinant vector

Artificially synthesize DNA molecules encoding mCherry-P53 and GFP-NUP98N-MDM2 (shown in SEQ ID NOs. 3-4 in Table 3 below). The resulting DNA molecules were inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (containing the restriction enzyme recognition sequence) between the HindIII and EcoRI restriction enzyme recognition sites to generate the recombinant eukaryotic expression vectors pcDNA3.1-mCherry-P53 and pcDNA3.1-GFP-NUP98N-MDM2, respectively. Plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the manufacturer's instructions and stored at -20°C.

Table 3: Constructs used in Example 2

2. Expression of recombinant DNA

Using the liposome transfection method generally known in the art, the purified DNA prepared in Example 2 was transiently expressed in cells as described below.

(1) Liposome transfection:

Prepare cell culture medium and seed HEK293T cells into a culture dish to achieve an appropriate cell density. According to the instructions for the liposome transfection reagent, mix the purified DNA prepared in Example 2 with the transfection reagent in the 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 incubate the cells under appropriate conditions (e.g., 37°C, 5% _CO2 ) for a specified period of time.

(2) Expression detection:

Depending on the target protein being expressed, select an appropriate detection method. For example, you can use a fluorescence microscope to observe the expression of red fluorescent protein (mCherry) and/or green fluorescent protein (GFP) in cells.

3. Validation of P53/MDM2 interaction inhibitors

All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims (10)

A method for screening macromolecular interaction regulators, characterized by comprising the steps of: 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 a coacervate complex A-B through the interaction between the first target molecule X and the first target molecule B; b. performing a photobleaching treatment on the cell in the absence of the candidate compound, and measuring the fluorescence signals of the condensate complex A-B in the cell before and after the photobleaching treatment to obtain first fluorescence signal measurement data; c. performing a photobleaching treatment on the cell in the presence of the candidate compound, and measuring the fluorescence signals of the condensate complex A-B 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. The method according to claim 1, wherein the engineered cells are produced by transfecting (transiently transfecting) DNA sequence construct 1 and DNA sequence construct 2 into cells, so that the cells co-express the 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. The method of claim 1, wherein in step b and step c, the laser wavelength used for the photobleaching treatment is the same; and the laser wavelength is 480 nm to 580 nm; preferably, 488 nm or 561 nm. The method according to claim 1, characterized in that in step b and step c, the photobleaching treatment refers to targeted laser irradiation of the area where the condensate complex A-B is located in the cell. The method according to claim 1, further comprising the steps of: e. performing a photobleaching treatment on the cell in the presence of a positive compound, and measuring the fluorescence signals of the condensate complex A-B 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. The method as claimed in claim 1 is characterized in that the phase separation skeleton protein Y sequence has a sequence table as shown in SEQ ID No: 1-SEQ ID No: 16. The method of claim 1, wherein the first fluorescent signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 element in the second fusion protein, and the recovery intensity before and after photobleaching is recorded as RI _ref ; and The second fluorescent signal is the fluorescence of the second wavelength emitted by the second fluorescent protein F2 component in the second fusion protein, and the recovery intensity before and after photobleaching, which is recorded as RI _test . The method of claim 7, wherein in step (d), the ratio of RI _test to RI _ref (RI _test /RI _ref ) is compared and recorded as Z; Wherein, when Z<1, it indicates that the candidate compound inhibits the interaction, and the smaller the Z value, the greater the inhibitory effect; When Z>1, it means that the candidate compound promotes the interaction, and the larger the Z value, the greater the promotion effect. The method according to claim 1, characterized in that the method is carried out under real-time fluorescence monitoring. A device for determining whether a candidate substance is a macromolecular interaction modulator, characterized by comprising: A1. A test module configured to incubate engineered cells in the presence or absence of a candidate substance; the cells contain a target aggregate A having dynamic fluidity; the target aggregate A comprises a first fusion protein formed by a fusion of a first target molecule X, a phase-separating scaffold protein Y, and a first fluorescent protein F1 element; and the cells further co-express a second fusion protein formed by a 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 A-B through the interaction between the first target molecule X and the first target molecule B; A2. A laser bleaching module, wherein the laser bleaching module is configured to bleach the condensate complex A-B in the cell by laser irradiation; A3. Data acquisition module, the data acquisition module is configured to: - collecting first fluorescence signal measurement data from the fluorescence signal of the condensate complex A-B in the cell before and after photobleaching treatment in the absence of the candidate compound; and - collecting second fluorescence signal measurement data from the fluorescence signal of the condensate complex A-B in the cell before and after photobleaching treatment in the presence of the candidate compound; A4. An interaction modulator evaluation module, wherein the interaction modulator evaluation module is configured to: compare the first fluorescence signal measurement data and the second fluorescence signal measurement data to obtain an evaluation result of whether the candidate substance is a modulator of the interaction between the first target molecule X and the first target molecule B; and A5. Output module, the output module is used to output the evaluation result.