A method for screening drugs targeting membrane protein receptors

Abstract

This invention provides a method for screening drugs targeting membrane protein receptors, comprising: attaching a fluorescent donor to the active domain of a live cell membrane protein receptor for fluorescent labeling; performing fluorescence imaging to detect the intrinsic fluorescence information of the fluorescent donor; constructing a two-dimensional fluorescence quenching interface on the live cell membrane, re-detecting the fluorescence information of the fluorescent donor and calculating its ratio with the intrinsic fluorescence information to obtain initial relative fluorescence information; stimulating live cells with the drug to be screened, detecting the fluorescence information of the fluorescent donor at different time points and calculating its ratio with the intrinsic fluorescence information to obtain relative fluorescence information at different time points, thereby obtaining a curve of relative fluorescence information changing over time, which is the characteristic curve of the drug to be screened; comparing the characteristic curve of the drug to be screened with the characteristic curves of different types of known agonists to determine the type of the drug to be screened. The method of this invention can accurately and comprehensively determine the type of the drug to be screened.

Description

Technical Field

[0001] This invention belongs to the field of drug screening and in situ detection technology of live cells. Specifically, this invention relates to a method for screening drugs targeting membrane protein receptors. More specifically, this invention relates to a method for screening drugs targeting membrane protein receptors by directly detecting the activation dynamics of membrane protein receptors on live cells. Background Technology

[0002] Membrane proteins are the main carriers of membrane system functions, responsible for signal transduction, transmembrane transport, energy exchange, and even cell migration. Membrane proteins are the most important drug targets, accounting for over 60% of currently known drug targets. For example, G protein-coupled receptors (GPCRs) are a class of 7-transmembrane proteins that participate in mediating various important physiological processes. Their family of proteins accounts for 40% of currently known drug targets, making them one of the most prevalent targets for best-selling drugs. Biochemical studies have shown that the main activation pathways of GPCR family proteins include G protein-binding pathways and G protein-independent pathways. The latter is mainly manifested in G protein-coupled receptor kinase (GRK)-dependent phosphorylation and β-arrestin binding-mediated negative feedback regulation. The key region for GPCR protein signaling pathway selection is the flexible tail structure (C-Tail) at the C-terminus of its intracellular domain. As the largest disordered domain on a GPCR, C-Tail exhibits structural diversity, presenting different conformations when binding to different ligand proteins. It is also a key region for post-expression modification and a binding target for β-arrestin. Therefore, understanding the structural dynamics of C-Tail, analyzing the protein signaling pathway responses corresponding to specific conformations, and screening for related membrane protein-targeting drugs are of great significance.

[0003] The screening of GPCR-related ligands has been in the research and development stage, and existing drug screening technologies still limit the development of GPCR-targeted drugs to varying degrees. For example, commonly used technologies such as cAMP detection, fluorometric imaging plate reader (FLIPR) based on calcium flow, Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) based on resonance energy transfer, and β-arrestin recruitment detection—a G protein-independent detection pathway—are all based on detecting the product after a certain pathway is completed, rather than relying on the molecular mechanism of receptor structural changes under the action of ligands. This means that any of these detection technologies can only detect one downstream protein signaling pathway, making it impossible to comprehensively evaluate the drug to be detected, thus significantly limiting the drug screening capabilities of these technologies.

[0004] Currently, one of the most commonly used techniques for detecting GPCR membrane protein receptors is the cAMP assay. Cyclic adenosine monophosphate (cAMP) is an important substance in cells involved in regulating metabolism and biological functions, acting as a "second messenger" for life information transmission, and is also a crucial link in the GPCR drug development pathway. In detection, cAMP levels are typically measured using a competitive method. Taking the most commonly used LANCE cAMP assay as an example, it is an immunoassay based on homogeneous time-resolved fluorescence energy transfer (TR-FRET). The principle of this assay is as follows... Figure 1 As shown, in the kit, cAMP molecules linked to rare earth elements (fluorescent donor molecules) are connected via cAMP antibodies and dye molecules (fluorescent acceptor molecules), with no other cAMP molecules present in the system at this stage. The fluorescent rare earth donor molecules are activated using 470 nm excitation light, and FRET (Fluorescent Electron Transduction) occurs between the rare earth and dye molecules, ultimately generating fluorescence at 670 nm. When ligand molecules are added to the cells, the concentration of cAMP molecules in the sample increases during cell signal transduction. A large number of unlabeled cAMP molecules compete with the rare earth-linked cAMP molecules in the kit. As the concentration of unlabeled cAMP molecules in the cells increases, the 670 nm fluorescence signal generated by FRET weakens, while the fluorescence signal of the rare earth element itself gradually increases in the 640 nm band. This detection method can be used for high-throughput drug discovery and has high sensitivity.

[0005] Current research indicates that the typical signaling pathway of GPCRs involves activating receptors located on the cell surface to transmit extracellular chemical signals into the cell. The G protein pathway and the β-arrestin pathway are the two main pathways. Traditional GPCR agonists activate the G protein signaling pathway upon binding to the receptor, while β-arrestin-preferred ligands primarily activate the β-arrestin pathway. Within the Gα subunit family, the Gαs and Gαi / o protein-coupled signaling pathways act on adenylate cyclase (AC), catalyzing the conversion of ATP to cyclic adenosine monophosphate (cAMP). However, receptors coupled to Gαs proteins directly activate this process, promoting cAMP production; while receptors coupled to Gαi / o proteins inhibit cAMP production. Therefore, while this downstream signaling product detection method has high sensitivity, it is limited by the need for prior knowledge of the drug coupling mechanism; furthermore, this method cannot screen for antagonists or Gαi / o coupled receptors.

[0006] Another commonly used detection method is BRET (Bioluminescence Resonance Energy Transfer) technology, which is based on bioluminescence. BRET is an enzymatic reaction in which a fluorescent donor enzyme spontaneously produces fluorescence in the presence of its corresponding substrate. The fluorescent acceptor in this case is another fluorescent protein whose emission spectrum overlaps with the BRET's, such as the mutant green fluorescent protein eYFP. Figure 2 As shown, luciferase (Rlu) is fused to a membrane protein receptor GPCR, and green fluorescent protein (GFP) is fused to β-Arrestin protein. When the agonist activates the β-Arrestin pathway, the GPCR binds to β-Arrestin, and the photons generated by luciferase are absorbed by GFP, producing fluorescence. Spectroscopic detection can clearly identify whether a ligand can activate the β-Arrestin pathway. This method is effective in identifying the bias effect of GPCRs.

[0007] The typical signaling pathways of GPCRs include the G protein pathway and the β-arrestin pathway. While the BRET technology can effectively identify the bias effect of GPCRs, it can only label one coupled protein at a time. This means that the results can only identify whether one pathway is activated, and it cannot make a comprehensive and accurate judgment on the type of unknown ligands. Summary of the Invention

[0008] Therefore, the purpose of this invention is to address the shortcomings of existing technologies by providing a method for drug screening targeting membrane protein receptors.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention provides a method for screening drugs targeting membrane protein receptors, comprising the following steps:

[0011] Step 1: Fluorescently label the membrane protein receptor by attaching a fluorescent donor to the active domain of the membrane protein receptor on the surface of living cells;

[0012] Step 2: Perform fluorescence imaging to detect the intrinsic fluorescence information of the fluorescence donor;

[0013] Step 3: Incubate the fluorescent acceptor with the live cells to construct a two-dimensional fluorescence quenching interface on the cell membrane of the live cells. Then, detect the fluorescence information of the fluorescent donor again and calculate its ratio with the intrinsic fluorescence information to obtain the initial relative fluorescence information.

[0014] Step 4: Stimulate the live cells with the drug to be screened, detect the fluorescence information of the fluorescent donor at different time points and calculate the ratio of its fluorescence information to the intrinsic fluorescence information to obtain the relative fluorescence information at different time points, thereby obtaining the curve of the relative fluorescence information changing with time, which is the characteristic curve of the drug to be screened.

[0015] Step 5: Compare the characteristic curve of the drug to be screened with the characteristic curves of different types of known agonists to determine the type of the drug to be screened;

[0016] All of the above steps are performed outside the body.

[0017] According to some embodiments of the present invention, the characteristic curve of the known agonist is obtained by the following method:

[0018] The live cells obtained in step 3 (i.e., live cells with a two-dimensional fluorescence quenching interface constructed on the cell membrane) are stimulated with a known agonist. The fluorescence information of the fluorescent donor at different time points is detected and the ratio of the fluorescence information to the intrinsic fluorescence information is calculated to obtain the relative fluorescence information at different time points. Thus, the curve of the relative fluorescence information changing with time is obtained, which is the characteristic curve of the known agonist.

[0019] According to some embodiments of the present invention, the screening method further includes the following steps:

[0020] Step 6: Determine the spatial conformation of the drug to be screened relative to the surface of the living cells;

[0021] Preferably, step 6 further includes the following steps:

[0022] Step 6-1: Using DNA molecules of different lengths as standards, construct a standard curve representing the relationship between relative fluorescence information and distance, wherein one end of the DNA molecules of different lengths is connected to a fluorescent donor and preferably the other end is connected to cholesterol, wherein the distance is the distance between the fluorescent donor and the fluorescent acceptor;

[0023] Step 6-2: Determine the spatial conformation of the known agonist relative to the surface of the living cells based on the standard curve and the characteristic curve of the known agonist;

[0024] Step 6-3: Determine the spatial conformation of the drug to be screened relative to the surface of the living cells, based on the type of the drug to be screened.

[0025] According to some embodiments of the present invention, the fluorescent donor is selected from one or more of fluorescent proteins, small molecule fluorescent dyes, fluorescent nanomaterials, and fluorescent microspheres.

[0026] Preferably, the fluorescent protein is selected from one or more of the following: mNeonGreen, Clover series green fluorescent proteins, GFP, BFP, CFP, YFP, EGFP, Venus, mOrange, Topaz, DsRed, mTFP1, mRFP1, mScarlet, mCherry, TagRFP, TurBoGFP, and their functional fragments or bioactive variants.

[0027] Preferably, the small molecule fluorescent dye is selected from one or more of the following: cyanines, coumarins, fluoresceins, rhodamines, porphyrins, fluoroborons, and diimides.

[0028] According to some embodiments of the present invention, the inherent fluorescence information is fluorescence intensity and / or fluorescence lifetime.

[0029] Preferably, the fluorescence imaging is fluorescence spectroscopy imaging and / or fluorescence spectrophotometer imaging.

[0030] Preferably, the fluorescence imaging is performed by measuring ensemble-level fluorescence lifetime using fluorescence lifetime imaging (FLIM) and / or by measuring single-molecule-level fluorescence intensity using total internal reflection fluorescence microscopy (TIRFM).

[0031] According to some embodiments of the present invention, the fluorescence acceptor is a lipophilic dye with low background fluorescence value; preferably, the lipophilic dye with low background fluorescence value is selected from one or more of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride, QSY fluorescence quencher series dyes and black hole fluorescence quencher series dyes.

[0032] According to some embodiments of the present invention, in step 3, the live cells are incubated with a lipophilic dye with a low background fluorescence value of 1-500 μM for 20-50 minutes so that the adsorption amount of the lipophilic dye with a low background fluorescence value on the cell membrane of the live cells reaches 6000-80000 molecules / square micrometer. Then, the cells are rinsed with buffer to remove excess lipophilic dye with a low background fluorescence value, thereby constructing a two-dimensional fluorescence quenching interface in the cell membrane of the live cells.

[0033] According to some embodiments of the present invention, the membrane protein receptor is a G protein-coupled receptor.

[0034] Preferably, the G protein-coupled receptor is the adrenaline receptor protein β2AR.

[0035] Preferably, the active domain is the carboxyl terminus and / or amino terminus of the G protein-coupled receptor, more preferably the carboxyl terminus of the G protein-coupled receptor.

[0036] According to some embodiments of the present invention, the drug to be screened is an agonist of a membrane protein receptor, preferably an agonist of a G protein-coupled receptor.

[0037] According to some embodiments of the present invention, the different types of known agonists include inverse agonists, G protein-biased agonists, β-Arrestin protein-biased agonists, and complete agonists.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The method of this invention belongs to the field of live cell in situ detection technology. Compared with traditional cAMP and BRET detection technologies, the method of this invention avoids crosstalk caused by indirect measurement of downstream product content in cAMP detection and is not limited by whether the drug coupling mechanism needs to be known beforehand. At the same time, the method of this invention also avoids the limitation of BRET detection, which can only identify whether one pathway is activated and cannot make an accurate and comprehensive judgment on the type of drug being screened.

[0040] This invention utilizes the conformational change of the active domain of a membrane protein receptor when it senses a specific ligand. It constructs a live-cell surface fluorescence decay measurement system, converting the conformational change of the active domain of the membrane protein receptor into a change in fluorescence signal. By detecting the change in fluorescence signal, the system can detect the temporal change in the structure of the active domain of the membrane protein receptor under drug stimulation. This technique can effectively distinguish between different types of membrane protein receptor drugs (e.g., agonists). Furthermore, if single-molecule fluorescence imaging is used to present the results, the molecular mechanisms of different coupling modes of membrane protein receptors under drug (e.g., agonist) stimulation can be further understood. The method of this invention has a broad detection range, is not limited to the type of drug, and has high practical value. Attached Figure Description

[0041] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0042] Figure 1 This is a schematic diagram illustrating the screening of drugs targeting GPCRs based on a competitive method of measuring cAMP levels.

[0043] Figure 2 This is a schematic diagram of fluorescence resonance energy transfer (BRET) technology based on bioluminescence.

[0044] Figure 3 This is a schematic diagram illustrating the technical principle of the method of the present invention.

[0045] Figure 4 This is a schematic diagram of the two-dimensional fluorescence quenching interface constructed in this invention.

[0046] Figure 5 These are characteristic curves obtained in Example 1 of the present invention targeting the adrenaline receptor protein β2AR under stimulation by different types of known agonists. A, B, C, and D correspond to the inverse agonist Carazolol, the G protein-biased agonist ICL 3-9, the β-Arrestin protein-biased agonist ICL 1-9, and the complete agonist isoproterenol (ISO), respectively.

[0047] Figure 6 The results of the fluorescence lifetime experiment of the living cell surface membrane protein receptor after adding carvedilol to the cell system in Example 1 of the present invention are shown.

[0048] Figure 7 The standard curve calibrated using DNA molecules of known length in Example 1 of this invention is shown.

[0049] Figure 8This invention illustrates the change in fluorescence lifetime of GFP fluorescent protein labeled with the carboxyl terminus of a cell surface membrane protein receptor after the addition of fully activated isoproterenol (ISO) to the cell system in Example 2 of this invention. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0051] This invention is based on fluorescence resonance energy transfer (FRET) technology and can be used to monitor sub-nanometer structural changes in biomolecules.

[0052] Generally, the fluorescence resonance energy transfer efficiency E between the donor and acceptor points in FRET is... T The formula for calculating (r) is as follows:

[0053] (1)

[0054] in:

[0055] E T (r) represents the fluorescence resonance energy transfer efficiency;

[0056] r represents the distance between the two fluorescent probes;

[0057] R0 represents the characteristic quenching distance, which is the distance at which the donor fluorescence intensity is quenched by half, i.e., E T The distance between the two fluorescent probes when (r) is 50%.

[0058] As can be seen from the above formula (1), the distance r between the two fluorescent probes (i.e., between the fluorescent donor and the fluorescent acceptor) is related to the fluorescence resonance energy transfer efficiency E. T The relationship between (r) is an inverse sixth power. The characteristic quenching distance R0 can be calculated from known intrinsic parameters of the fluorescent group, such as the spectral overlap between the fluorescent acceptor and the fluorescent donor, and the quantum efficiency of the fluorescent donor.

[0059] Here, R0 is calculated using the dipole approximation, and the specific calculation formula is as follows:

[0060] (2)

[0061] in:

[0062] κ represents the dipole orientation factor between two fluorescent molecules;

[0063] Q D Indicates the donor quantum yield;

[0064] εA Indicates the maximum extinction coefficient of the receptor;

[0065] N A Represents Avogadro's constant;

[0066] n represents the refractive index of the medium.

[0067] J(λ) represents the normalized donor emission spectrum F D With receptor absorption spectrum E A The spectral coincidence coefficient, with wavelength λ as the integral variable, is calculated using the following formula for J(λ):

[0068] (3)

[0069] Therefore, the characteristic quenching distance R0 between a donor molecule and an acceptor molecule undergoing FRET can be calculated. Based on R0, the following formula (4) can be used:

[0070] (4)

[0071] The energy transfer rate k can be obtained when the distance between a donor molecule and an acceptor molecule is r. T (r). Where, τ D It is the original fluorescence lifetime of the donor molecule, and its emission mode, i.e., the emission of photons at a transfer rate k. R The autoradiative transition back to the ground state occurs at a transfer rate k. NR Other non-radiative transitions occur. τ D It can be written as:

[0072] (5)

[0073] When a donor molecule and an acceptor molecule at a distance r undergo FRET, the energy transfer efficiency E T (r) is related to all the energy transfer mechanisms that occur within it, and can be expressed as:

[0074] (6)

[0075] Where, k T (r) can be calculated using formula (4), k R + k NR The value is τ D -1 , τ D This is the initial lifetime of the fluorescent acceptor molecule, which is a constant for a specific fluorescent molecule. Therefore, equation (6) can also be written as:

[0076] (7)

[0077] Building upon the aforementioned calculations of a FRET between a donor molecule and an acceptor molecule (i.e., a point-to-point FRET), this invention extends the point-to-point FRET to a point-to-multipoint FRET. For example... Figure 3 As shown, a quencher (such as a lipophilic black hole dye quencher) is embedded within the phospholipid bilayer of the cell membrane as a non-luminescent or weakly luminescent fluorescent acceptor at low luminescence intensity in the measured wavelength range, forming a fluorescent acceptor surface. All fluorescent acceptor molecules and one fluorescent donor molecule within the surface interact, and the result can be achieved through linear superposition. The energy transfer rate expression can be written as:

[0078] (8)

[0079] The integrated fluorescence resonance energy transfer efficiency E T (r) can be written as:

[0080] (9)

[0081] The actual measured light intensity (F) or lifetime (τ) of the fluorescent donor molecule in the experiment is related to the fluorescence resonance energy transfer efficiency E. T (r) has the following relation:

[0082] (10)

[0083] Where F0 and τ0 are the original fluorescence intensity or original fluorescence lifetime of the fluorescent acceptor molecule before FRET occurs.

[0084] Based on the above principles, this invention provides a method for drug screening targeting membrane protein receptors. This method is based on live-cell in situ fluorescence detection and directly screens for active drugs targeting membrane protein receptors by analyzing the structural change dynamics of disordered domains. The method includes the following steps:

[0085] Step 1: Fluorescently label the membrane protein receptor by attaching a fluorescent donor to the active domain of the membrane protein receptor on the surface of living cells.

[0086] The method of the present invention is applicable to various types of membrane protein receptors. Preferably, the membrane protein receptor is a G protein-coupled receptor. More preferably, the G protein-coupled receptor is the adrenaline receptor protein β2AR.

[0087] The active domain of a membrane protein receptor can be determined based on the specific membrane protein receptor. When the membrane protein receptor is a G protein-coupled receptor, the active domain is preferably the carboxyl terminus and / or amino terminus of the G protein-coupled receptor, and more preferably the carboxyl terminus of the G protein-coupled receptor.

[0088] In this invention, the fluorescent donor can be a fluorescent protein, a small molecule fluorescent dye, a fluorescent nanomaterial, or a fluorescent microsphere. The fluorescent protein can be selected from mNeonGreen, Clover series green fluorescent proteins, GFP, BFP, CFP, YFP, EGFP, Venus, mOrange, Topaz, DsRed, mTFP1, mRFP1, mScarlet, mCherry, TagRFP, TurBoGFP, or any functional fragment or bioactive variant thereof. The small molecule fluorescent dye can be selected from cyanines, coumarins, luciferins, rhodamines, porphyrins, fluoroborons, or diimides.

[0089] In a preferred embodiment, the fluorescent donor is a suitable fluorescent protein, such as green fluorescent protein (GFP), capable of generating fluorescence resonance energy transfer with the aforementioned fluorescent receptor surface, so as to ensure that after the fluorescently labeled membrane protein receptor is assembled onto the cell membrane surface of a living cell, the cell can perform normal functions, that is, the membrane protein receptor will not significantly interfere with the normal physiological processes of the cell.

[0090] Step 2: Perform fluorescence imaging to detect the intrinsic fluorescence information of the fluorescence donor.

[0091] In this invention, fluorescence imaging means include all techniques for detecting fluorescence intensity and / or fluorescence lifetime magnitude, including fluorescence spectrophotometric imaging and fluorescence photometer imaging, including measurements of fluorescence intensity and / or fluorescence lifetime at the ensemble level and at the single-molecule level.

[0092] In a preferred embodiment, the fluorescence imaging method is based on ensemble-level fluorescence lifetime measurement, preferably fluorescence lifetime imaging microscopy (FLIM). Fluorescent donor molecules are imaged using FLIM, and the average lifetime of the fluorescent donor molecules at the ensemble level is recorded in the FLIM experiment, denoted as τ0.

[0093] In another preferred embodiment, fluorescence imaging is performed based on single-molecule level fluorescence intensity measurements, preferably total internal reflection fluorescence microscopy (TIRFM). Fluorescent donor molecules are imaged using TIRFM, and the fluorescence intensity of a single fluorescent molecule is recorded and denoted as F0 in the TIRFM experiment. It is important to note that when using TIRFM for single-molecule detection, if the target protein is overexpressed in the cell, observable single-molecule imaging at the cell basal surface will not be possible. In this case, a strong laser can be used to illuminate the cell basal field of view to bleach the fluorescence of the target protein within the field of view, and then the observation of single-molecule fluorescence signals from the target protein diffusing from neighboring regions can be performed.

[0094] It should be noted that when fluorescence lifetime microscopy is used for fluorescence imaging, the fluorescence information referred to in this invention refers to the fluorescence lifetime of the fluorescent donor molecule. When total internal reflection fluorescence microscopy is used for fluorescence imaging, the fluorescence information referred to in this invention refers to the fluorescence intensity of the fluorescent donor molecule. The inherent fluorescence information of the fluorescent donor molecule is the aforementioned τ0 and / or F0.

[0095] Step 3: Incubate the fluorescent acceptor with the live cells to construct a two-dimensional fluorescence quenching interface on the cell membrane of the live cells. Then, detect the fluorescence information of the fluorescent donor again and calculate its ratio with the intrinsic fluorescence information to obtain the initial relative fluorescence information.

[0096] Constructing a two-dimensional fluorescence quenching interface for a living cell system is one of the most important and critical steps in the method of this invention.

[0097] First, a lipophilic dye with low background fluorescence value needs to be selected as the fluorescence acceptor to construct a two-dimensional fluorescence quenching interface in the cell membrane. The lipophilic dye with low background fluorescence value is a dye molecule that is lipophilic, can accumulate within the cell membrane system (i.e., the phospholipid bilayer), and emits weak or no light in the signal acquisition wavelength range.

[0098] In this invention, the lipophilic dye with low background fluorescence value can be selected from Dabsyl (4-(dimethylamino)azobenzene-4'-sulfonyl chloride), QSY fluorescence quencher series dyes, and BHQ (Black Hole Quencher) series dyes. Preferably, the lipophilic dye with low background fluorescence value is a black hole fluorescence quencher (Black Hole Quencher).

[0099] Next, incubating the live cell system with a 1-500 μM lipophilic dye for 20-50 min allows the adsorption capacity of the lipophilic dye on the cell membrane to reach approximately 6000-80000 molecules / square micrometer. Then, a buffer solution is added to wash away any excess lipophilic dye molecules. At this point, the two-dimensional fluorescence quenching interface of the live cell system is complete.

[0100] Taking the black hole fluorescence quencher BHQ-1 as an example, a 10 μM concentration of BHQ-1 was incubated with a live cell system for 30 min, so that the quencher adsorption amount on the cell membrane was about 7000 BHQ-1 molecules / square micrometer. Then, buffer solution was added to wash away the excess quencher molecules, thereby constructing a two-dimensional fluorescence quenching interface for the live cell system.

[0101] The constructed two-dimensional fluorescence quenching interface is as follows Figure 4 The double-layer membrane structure is shown in the figure.

[0102] Step 4: Obtain the characteristic curve of the drug to be screened.

[0103] Live cells with a two-dimensional fluorescence quenching interface were constructed by stimulating the cell membrane obtained in step 3 with the drug to be screened. Then, the fluorescence information (i.e., F or τ) of the fluorescent donor at different time points was detected, and the ratio of its fluorescence information to the intrinsic fluorescence information, i.e., F / F0 or τ / τ0, was calculated to obtain the relative fluorescence information at different time points. The curve of relative fluorescence information changing with time is the characteristic curve of the drug to be screened.

[0104] Step 5: Compare the characteristic curve of the drug to be screened with the characteristic curves of different types of known agonists to determine the type of the drug to be screened.

[0105] The method for obtaining the characteristic curve of a known agonist is similar to that for obtaining the characteristic curve of the drug to be screened. Following the method in step 4 above, the drug to be screened is replaced with a known agonist. Specifically, live cells with a two-dimensional fluorescence quenching interface are constructed by stimulating the cell membrane obtained in step 3 with the known agonist. Then, the fluorescence information of the fluorescent donor at different time points is detected, and its ratio to the intrinsic fluorescence information is calculated to obtain the relative fluorescence information at different time points. The curve showing the change of relative fluorescence information over time is the characteristic curve of the known agonist.

[0106] The method of the present invention is applicable to various types of drugs to be screened. Preferably, the drug to be screened is an agonist of a membrane protein receptor. More preferably, the agonist of a membrane protein receptor is an agonist of a G protein-coupled receptor.

[0107] The known types of agonists used in the method of this invention include inverse agonists, G protein pathway-biased agonists, Arrestin pathway-biased agonists, and full agonists. The addition of different agonists will have different effects on the activation pathway of membrane protein receptors, and the experimental results will show different characteristic curves.

[0108] Furthermore, as can be seen from the above principle, F / F0 or τ / τ0 is related to the fluorescence resonance energy transfer efficiency E. T There is a correspondence between (r) (see formula (10) for example), and E T (r) is also a function of the distance r between the fluorescent donor and the fluorescent acceptor (see, for example, Equation (1)). In this invention, the positional change of the site of the fluorescent donor-labeled membrane protein acceptor relative to the cell membrane can be converted into a change in fluorescence resonance energy transfer efficiency. The change in fluorescence resonance energy transfer efficiency can be reflected as a change in the relative decay magnitude of fluorescence lifetime (τ / τ0) or a change in the relative decay strength of fluorescence intensity (F / F0), that is, a change in relative fluorescence information. Therefore, the method of this invention can also study the spatial conformation of the drug to be screened relative to the surface of living cells by the change in relative fluorescence information. Therefore, the method of this invention further includes: Step 6: Determine the spatial conformation of the drug to be screened relative to the surface of the living cells.

[0109] Specifically, determining the spatial conformation of the drug to be screened relative to the surface of the living cells includes the following steps:

[0110] Step 6-1: Using DNA molecules of different lengths as standards, construct a standard curve of relative fluorescence information (i.e., F / F0 or τ / τ0) and the distance between the fluorescent donor and the fluorescent acceptor (i.e., the distance between the fluorescent donor and the two-dimensional fluorescence quenching interface).

[0111] Step 6-2: Using the standard curve and the characteristic curve of the known agonist, determine the change of the spatial conformation (distance) of the known agonist relative to the surface of the living cell over time.

[0112] Step 6-3: Based on the type of drug to be screened determined in Step 5 above, determine its spatial conformation relative to the surface of the living cells.

[0113] In step 6-1, DNA molecules of varying lengths, with one end attached to a fluorescent donor, are first inserted into the membrane of a living cell, and the initial fluorescence lifetime τ'0 or initial fluorescence intensity F'0 of the fluorescent donor is detected. To immobilize the DNA molecule on the cell membrane, cholesterol is attached to the other end.

[0114] Then, the fluorescent acceptor is adsorbed onto the cell membrane to construct a two-dimensional fluorescence quenching interface, which is the same as step 3 above.

[0115] Next, the fluorescence lifetime τ' or fluorescence intensity F' of DNA molecules of different lengths in the cell membrane system is measured, and F' / F'0 or τ' / τ'0 is calculated to obtain data on the distance of the fluorescent donor connected to the DNA molecule from different positions on the cell membrane.

[0116] Subsequently, a random number generator is used to generate the coordinates of fluorescent acceptor molecules within a cubic space with dimensions of 100 or 150 nm and a height of 4-5 nm. The number of these coordinates corresponds to the areal density of the fluorescent acceptor (quencher) distribution on the cell membrane. This cubic space represents the cytoplasmic space within the phospholipid bilayer of the cell membrane. The coordinates of the fluorescent donor molecule are placed at a distance *r* (i.e., the distance between the fluorescent donor and the two-dimensional fluorescence quenching interface) within the cubic space. Monte Carlo simulations are then used to obtain the distance-FRET efficiency curve between the fluorescent donor and the two-dimensional fluorescence quenching interface on the DNA molecule. The Monte Carlo simulation method can refer to relevant existing techniques. As an example, the following calculation procedure can be used to obtain a simulation curve that matches the aforementioned data, which is the standard curve of relative fluorescence information versus distance.

[0117] rc=-4:0.1:10;

[0118] c=3.1;

[0119] R0=5.2;

[0120] Total = zeros(length(rc), 1);

[0121] for i=1:length(rc)

[0122] for m=0:5000

[0123] Total(i)=Total(i)+fret_membrane_doubleLayer(R0,c,rc(i));

[0124] %TT(i,:)=1-Total / 1000;

[0125] end

[0126] end

[0127] T_T0 = 1 - Total. / 5000;

[0128] figure

[0129] plot(rc,T_T0);

[0130] xlabel('D(nm)')

[0131] ylabel('τ / τ0')

[0132] %err=norm(T_T0-y);

[0133] hold on

[0134] %{

[0135] grid on

[0136] xlabel('D(nm)')

[0137] ylabel('τ / τ0')

[0138] axis([ -inf inf 0.5 1])

[0139] b=(1-Total / 1000)';

[0140] %}

[0141] function [E]=fret_membrane_doubleLayer(R0,c,rc)

[0142] a=100;

[0143] N = round(a * a * c / 100);

[0144] % Assign random coordinates to each particle

[0145] X = zeros(1, N);

[0146] Y = zeros(1, N);

[0147] Z = zeros(1, N);

[0148] XX = zeros(1, N);

[0149] YY=zeros(1,N);

[0150] ZZ=zeros(1,N);

[0151] for i=1:N

[0152] X(i) = a * rand - a / 2;

[0153] Y(i) = a * rand - a / 2;

[0154] end

[0155] for i=1:N

[0156] XX(i) = a*rand - a / 2;

[0157] YY(i) = a * rand - a / 2;

[0158] ZZ(i) = -4;

[0159] end

[0160] d=zeros(1,N);

[0161] kt=0;

[0162] % Initialize Kt

[0163] for j=1:N

[0164] d(j)=sqrt(X(j)^2+Y(j)^2+rc^2);

[0165] kt = kt + (R0 / d(j))^6;

[0166] end

[0167] for k=1:N

[0168] d(k)=sqrt(XX(k)^2+YY(k)^2+(rc-ZZ(k))^2);

[0169] kt = kt + (R0 / d(k))^6;

[0170] end

[0171] %Calculate the overall reaction rate constant Kt

[0172] E=kt / (1+kt)

[0173] By implementing the above scheme, crosstalk caused by indirectly measuring the content of downstream products in cAMP detection can be effectively avoided, and the drug coupling mechanism does not need to be known in advance. At the same time, the method of the present invention can make an accurate and comprehensive judgment on the type of drug being screened, and also overcomes the screening limitation problem caused by the aforementioned BRET detection, which can only identify whether one pathway is activated.

[0174] Example 1

[0175] The method of this invention can be applied to any type of membrane protein receptor, such as G protein-coupled receptors (GPCRs). In this embodiment, a specific GPCR, namely the adrenaline receptor protein β2AR, is used as an example. However, it should be understood that this is only exemplary.

[0176] The specific process of the method provided in this embodiment is as follows:

[0177] Step 1: The carboxyl terminus of the adrenaline receptor protein β2AR was fused with green fluorescent protein mNeonGreen on HeLa cells (derived from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College).

[0178] Step 2: Using FLIM assay, the initial fluorescence lifetime τ0 of mNeonGreen for the adrenaline receptor protein β2AR on living cells was measured to be 2.97 ns.

[0179] Step 3: Using the black hole fluorescence quencher BHQ-1 as a lipophilic dye, a 10 μM solution of BHQ-1 was co-incubated with the live cell system for 30 min, allowing the quencher adsorption on the cell membrane to reach approximately 7000 BHQ-1 molecules per square micrometer. Excess quencher molecules were then washed away with buffer solution, thus constructing the two-dimensional fluorescence quenching interface for the live cell system. Without the addition of any other ligands, the mNeonGreen fluorescence lifetime was detected to be 2.2 ns, therefore τ / τ0 = 0.75.

[0180] Step 4: The cell systems constructed in Step 3 were stimulated by adding known inverse agonists Carazolol, G protein-biased agonists ICL 3-9, β-Arrestin protein-biased agonists ICL 1-9, and the complete agonist isoproterenol (ISO), respectively. The fluorescence lifetime of mNeonGreen fluorescent protein was measured over time, and τ / τ0 was calculated to obtain the results. Figure 5 The characteristic curves of different types of known agonists are shown.

[0181] Carvedilol, the drug to be screened, was added to the cell system constructed in step 3. The fluorescence lifetime of mNeonGreen fluorescent protein was detected over time, and τ / τ0 was calculated to obtain the results. Figure 6 The characteristic curve shown.

[0182] Step 5: By comparison Figure 5 and Figure 6 Carvedilol works by combining the effects of an inverse agonist with a biased agonist effect on the β-Arrestin pathway.

[0183] Step 6: Further study the spatial conformation of the relative cell membrane of the drug to be screened, carvedilol.

[0184] First, a standard curve was constructed. Double-stranded DNA molecules, labeled with rhodamine fluorescent molecules at one end and cholesterol molecules linked to the other, were used. Their double-strand lengths were 12 bp (4.1 nm), 16 bp (5.4 nm), 20 bp (6.8 nm), and 24 bp (8.2 nm), respectively. After insertion into the cell membrane, the angle between the DNA molecule and the cell membrane surface was approximately 60°. o Therefore, the distances between the DNA molecule and the quencher interface were 3.5 nm, 4.7 nm, 5.9 nm, and 7.1 nm, respectively. In the cell system constructed in step 3, FLIM technology was used to measure the τ0 of rhodamine as 2.83 ns. The corresponding fluorescence lifetimes of rhodamine fluorescent molecules on DNA molecules of different lengths on the cell membrane were measured to be 1.75 ns, 2.23 ns, 2.46 ns, and 2.82 ns, respectively. Their τ / τ0 values ​​are as follows: Figure 7 As shown by the solid circle in the middle.

[0185] Subsequently, a random number generator was used to generate coordinates of quencher molecules within a cubic space with dimensions of 100 or 150 nm and a height of 4-5 nm. These coordinates corresponded to the areal density of the quencher distributed on the cell membrane. This cubic space represented the cytoplasmic space within the phospholipid bilayer of the cell membrane. The coordinates of the fluorescent donor molecules were placed at a distance *r* (i.e., the distance between the fluorescent donor molecule and the quenching medium interface) within the cubic space. Monte Carlo simulations were then used to obtain the distance-FRET efficiency curve between the rhodamine fluorescent molecule on the DNA molecule and the two-dimensional fluorescence quenching interface. The density of quencher molecules within the space was adjusted to ensure a close match between the simulated and experimental curves, thus obtaining... Figure 7 The curve.

[0186] Combination Figure 5 and Figure 7 Analyze the spatial conformations of known types of agonists, among which Figure 5The red arrows in the diagram indicate the timing of agonist addition. (A) When the inverse agonist Carazolol is added to the system, the C-terminus of β2AR is in an inhibited state, and its spatial structure, far from the cell membrane surface—the G protein-binding state—is inhibited. Its average spatial position rapidly approaches the membrane surface with the addition of the inverse agonist, resulting in a rapid decrease in fluorescence intensity / fluorescence lifetime. (B) When the G protein-biased agonist ICL 3-9 is added to the system, the C-terminus of β2AR is in an activated state, bound to the G protein. Spatially, the C-terminus of β2AR is far from the cell membrane surface, resulting in a sharp increase in fluorescence intensity / fluorescence lifetime. As G protein in the cell is consumed and downstream negative feedback pathways are activated, the probability of the C-terminus of the receptor molecule approaching the membrane surface gradually increases after a few minutes. The G protein signaling pathway response ends, and the fluorescence intensity / fluorescence lifetime returns to its initial value. In other words, the G protein pathway response corresponds to the structural change of the C-terminus moving away from the cell membrane. The change in the average spatial position of the C-terminus observed after adding a G protein-biased agonist is a process of first rapidly moving away from the cell membrane surface, and then moving closer to the cell membrane surface. The change in fluorescence intensity / fluorescence lifetime is a process of rapid increase followed by decrease. (C) When the β-Arrestin protein-biased agonist ICL 1-9 is added to the system, the β-Arrestin pathway is selectively activated. The binding between β2AR and β-Arrestin protein causes the C-terminus of the receptor molecule on the membrane to approach the cell membrane surface. When a large number of β2AR molecules have their C-termini approaching the cell membrane surface, the average fluorescence intensity / fluorescence lifetime can be observed to decrease significantly under the action of the quenching interface. As time increases, the reaction gradually tends to equilibrium, and the fluorescence lifetime of the system tends to stabilize. (D) The complete agonist isoproterenol (ISO) can activate both the G protein pathway and the β-Arrestin pathway. Unlike the other two biased agonists, the mean fluorescence intensity / fluorescence lifetime of the system did not change significantly after the addition of the agonist ISO to the experimental system, even in the presence of the quenching interface. This result indicates that while the full agonist isoproterenol (ISO) increases G protein pathway signaling, causing the C-terminus to move further away from the cell membrane surface, it also increases β-Arrestin pathway signaling, causing the C-terminus to move closer to the cell membrane surface. The balance between these two factors results in the mean spatial position of the C-terminus relative to the cell membrane surface remaining constant for a period of time. Therefore, the mean fluorescence intensity / fluorescence lifetime of the system did not change significantly for a period after the addition of ISO. As time increases, the signal proportion of the β-Arrestin pathway gradually increases, causing the mean spatial position of the C-terminus to gradually move closer to the cell membrane surface, resulting in a gradual decrease in the mean fluorescence intensity / fluorescence lifetime.

[0187] Combination Figure 6 and Figure 7 Upon addition of carvedilol, due to its biased activation of the β-Arrestin pathway, the binding of β2AR to β-Arrestin protein causes the C-terminus of the receptor molecule to adhere closer to the cell membrane surface. Simultaneously, because of carvedilol's inhibitory effect on the G protein pathway, the conformation of β2AR with its C-terminus further away from the cell membrane decreases, resulting in a rapid decrease in the fluorescence lifetime of the fluorescent protein. After 5 minutes, the change in fluorescence lifetime reaches an inflection point, and its magnitude begins to slowly increase. At this point, the inhibited interaction between G protein and β2AR gradually recovers, and some of the C-terminus of β2AR, after binding to G protein, moves away from the cell membrane surface until equilibrium is reached. The effect of carvedilol is the result of the combined effect of an inverse agonist and biased activation of the β-Arrestin pathway. This is consistent with previous research findings on the interaction between carvedilol and β2AR.

[0188] Example 2

[0189] This embodiment uses an adrenaline receptor protein β2AR with GFP (green fluorescent protein) fused to its carboxyl terminus, and a lipophilic dye QSY7 as a quencher. In this embodiment, the fluorescence lifetime τ0 of the GFP fluorescent protein labeled with the carboxyl terminus of the adrenaline receptor protein β2AR on living cells is 2.70 ns, and the concentration of the lipophilic dye QSY7 is 330 μM.

[0190] The areal density of intracellular quencher QSY7 at 300 μM was determined using double-stranded DNA molecules labeled with cholesterol at one end and rhodamine at the other. The areal density was 50,000 QSY7 molecules / μm. The lengths of the double-stranded DNA molecules were 10 bp, 20 bp, and 30 bp, and their distances from the cell membrane were 3.4 nm, 5.9 nm, and 8.8 nm, respectively. The quenching results τ / τ0 of the rhodamine donor molecules on the three DNA molecules were 0.55, 0.89, and 0.99, respectively. Standard curves were constructed based on these data (not shown in the attached figure). Under these experimental conditions, the fluorescence lifetime of GFP fluorescent protein labeled with the C-terminus of β2AR decreased from an initial 2.70 ns to 2.44 ns after the addition of QSY7.

[0191] The addition of fully activated isoproterenol (ISO) yielded the following results: Figure 8 As shown, after adding ISO (isoproterenol) for 30 min, the fluorescence lifetime of the GFP fluorescent protein labeled with the β2AR active domain decreased from 2.44 ns to 2.36 ns.

[0192] Example 3

[0193] This embodiment uses adrenaline receptor protein β2AR with mScarlet red fluorescent protein fused to its carboxyl terminus, and lipophilic dye BHQ-1 as a quencher. In this embodiment, the fluorescence lifetime τ0 of the adrenaline receptor protein β2AR labeled with mScarlet fluorescent protein at its carboxyl terminus on living cells is 3.80 ns, and the concentration of lipophilic dye BHQ-1 is 10 μM.

[0194] The fluorescence lifetime of the β2AR C-terminal labeled mScarlet fluorescent protein decreased from an initial 3.80 ns to 2.89 ns after the addition of BHQ-1. Similar results to those in Example 1 were obtained with the addition of different types of agonists.

Claims

1. A method for screening drugs targeting membrane protein receptors, comprising the following steps: Step 1: Fluorescently label the membrane protein receptor by attaching a fluorescent donor to the active domain of the membrane protein receptor on the surface of living cells; Step 2: Perform fluorescence imaging to detect the intrinsic fluorescence information of the fluorescence donor; Step 3: Incubate the fluorescent acceptor with the live cells to construct a two-dimensional fluorescence quenching interface on the cell membrane of the live cells. Then, detect the fluorescence information of the fluorescent donor again and calculate its ratio with the intrinsic fluorescence information to obtain the initial relative fluorescence information. Step 4: Stimulate the live cells with the drug to be screened, detect the fluorescence information of the fluorescent donor at different time points and calculate the ratio of its fluorescence information to the intrinsic fluorescence information to obtain the relative fluorescence information at different time points, thereby obtaining the curve of the relative fluorescence information changing with time, which is the characteristic curve of the drug to be screened. Step 5: Compare the characteristic curve of the drug to be screened with the characteristic curves of different types of known agonists to determine the type of the drug to be screened; All of the above steps are performed outside the body.

2. The screening method according to claim 1, wherein, The characteristic curves of the known agonist were obtained by the following method: The live cells obtained in step 3 are stimulated with a known agonist. The fluorescence information of the fluorescent donor at different time points is detected and the ratio of the fluorescence information to the intrinsic fluorescence information is calculated to obtain the relative fluorescence information at different time points. The curve of the relative fluorescence information changing with time is obtained, which is the characteristic curve of the known agonist.

3. The screening method according to claim 1, wherein, The screening method further includes the following step: Step 6: Determine the spatial conformation of the drug to be screened relative to the surface of the living cells; Preferably, step 6 further includes the following steps: Step 6-1: Using DNA molecules of different lengths as standards, construct a standard curve representing the relationship between relative fluorescence information and distance, wherein one end of the DNA molecules of different lengths is connected to a fluorescent donor and preferably the other end is connected to cholesterol, wherein the distance is the distance between the fluorescent donor and the fluorescent acceptor; Step 6-2: Determine the spatial conformation of the known agonist relative to the surface of the living cells based on the standard curve and the characteristic curve of the known agonist; Step 6-3: Determine the spatial conformation of the drug to be screened relative to the surface of the living cells, based on the type of the drug to be screened.

4. The screening method according to claim 1, wherein, The fluorescent donor is selected from one or more of fluorescent proteins, small molecule fluorescent dyes, fluorescent nanomaterials, and fluorescent microspheres; Preferably, the fluorescent protein is selected from mNeonGreen, Clover series green fluorescent proteins, GFP, BFP, CFP, YFP, EGFP, Venus, mOrange, Topaz, DsRed, mTFP1, mRFP1, mScarlet, mCherry, TagRFP, TurBoGFP, and one or more of their functional fragments or bioactive variants; Preferably, the small molecule fluorescent dye is selected from one or more of the following: cyanines, coumarins, fluoresceins, rhodamines, porphyrins, fluoroborons, and diimides.

5. The screening method according to claim 1, wherein, The inherent fluorescence information is fluorescence intensity and / or fluorescence lifetime; Preferably, the fluorescence imaging is fluorescence spectroscopy imaging and / or fluorescence spectrophotometry imaging; Preferably, the fluorescence imaging is performed by measuring ensemble-level fluorescence lifetime using fluorescence lifetime imaging (FLIM) and / or by measuring single-molecule-level fluorescence intensity using total internal reflection fluorescence microscopy (TIRFM).

6. The screening method according to claim 1, wherein, The fluorescence acceptor is a lipophilic dye with low background fluorescence value; preferably, the lipophilic dye with low background fluorescence value is selected from one or more of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride, QSY fluorescence quencher series dyes and black hole fluorescence quencher series dyes.

7. The screening method according to claim 6, wherein, In step 3, the lipophilic dye with low background fluorescence value of 1-500 μM is incubated with the live cells for 20-50 minutes to make the adsorption amount of the lipophilic dye with low background fluorescence value on the cell membrane of the live cells reach 6000-80000 molecules / square micrometer. Then, the cells are washed with buffer to remove excess lipophilic dye with low background fluorescence value, thereby constructing a two-dimensional fluorescence quenching interface in the cell membrane of the live cells.

8. The screening method according to claim 1, wherein, The membrane protein receptor is a G protein-coupled receptor; Preferably, the G protein-coupled receptor is the adrenaline receptor protein β2AR; Preferably, the active domain is the carboxyl terminus and / or amino terminus of the G protein-coupled receptor, more preferably the carboxyl terminus of the G protein-coupled receptor.

9. The screening method according to claim 1, wherein, The drug to be screened is an agonist of a membrane protein receptor, preferably an agonist of a G protein-coupled receptor.

10. The screening method according to claim 1, wherein, The known types of agonists include inverse agonists, G protein-biased agonists, β-Arrestin protein-biased agonists, and full agonists.

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