A cell screening model for label-free g-protein coupled receptor and application thereof
A BLT1 receptor screening model was constructed using label-free cell integrated pharmacology technology. By utilizing the RWG biosensor, the technical problems existing in the prior art were solved, achieving high efficiency and simplified operation, and enabling efficient screening of drugs for BLT1 receptor-related diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2026-06-26
AI Technical Summary
Existing GPCR detection methods mainly target a single signaling pathway and cannot simultaneously detect the activation of multiple signaling pathways. They also require fluorescent labels or additional indicators, are cumbersome to operate, have an impact on cells, and cannot effectively screen drugs for BLT1 receptor-related diseases.
Using label-free cell-integrated pharmacology, a label-free high-throughput screening model for the BLT1 receptor was constructed by converting the dynamic mass redistribution of intracellular components induced by drugs into wavelength shift response signals using a resonant waveguide grating (RWG) biosensor. Efficient screening was then achieved using the Epic optical biosensor.
It achieves non-invasive, high spatiotemporal resolution, high sensitivity, and high throughput screening, simplifies the operation, and can screen out highly active ligands of the BLT1 receptor, broadening the clinical application range of compounds and providing a model system for drug screening of BLT1-related diseases.
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Figure CN122278772A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to cell screening models, specifically to a label-free G protein-coupled receptor cell screening model and its applications. Background Technology
[0002] G protein-coupled receptors (GPCRs) are among the most important membrane receptors in cell signaling and are also one of the most promising drug targets in small molecule drug development, with over 30% of drugs targeting GPCRs [ASHauser, M. M. Attwood, M. Rask-Andersen, et al. Nat. Rev. Drug Discov. 2017, 16, 829]. Leukotriene B4 is well-known as one of the most potent neutrophil chemotactic agents, playing a crucial role in host infection defense through interaction with the high-affinity receptor BLT1. Besides neutrophils, BLT1 is expressed in many other cell types, including almost all immune cell types and some non-immune cells such as epithelial cells, fibroblasts, endothelial cells, smooth cells, and muscle cells [R. He, Y. Chen, Q. Cai. Pharmacol Res. 2020, 158, 104857]. Therefore, BLT1 is involved in the pathogenesis of various inflammatory and immune diseases, including asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis [T. Yokomizzo, T. Shimizu. Immunol Rev. 2023, 317, 30]. In addition to acting on immune cells, the leukotriene B4-BLT1 axis can also affect the biological functions of tissue structural cells, thereby initiating and / or spreading pathological inflammation in affected tissues [R. He, Y. Chen, Q. Cai. Pharmacol Res. 2020, 158, 104857]. Therefore, the search for and development of highly active BLT1 ligands is essential. Blocking the leukotriene B4-BLT1 axis with antagonists to inhibit pathological inflammation may slow disease progression or reduce disease severity.
[0003] Currently, high-throughput screening methods mainly include traditional radioligand-receptor binding assays, GTPγS binding assays, cyclic adenosine monophosphate (cAMP) assays, calcium flux assays, reporter gene assays, receptor endocytosis assays, and β-arrestin recruitment assays. However, these methods all have certain limitations. Most GPCR assays target only one signaling pathway and cannot simultaneously detect the activation of multiple signaling pathways. Furthermore, they require the addition of fluorescent labels or additional indicators, making the operation cumbersome, and the indicators can also have some impact on cells [W. Thomsen, J. Frazer, D. Unett. Curr Opin Biotechnol. 2005, 16, 655; L.M. Mayr, D. Bojanic. Curr Opin Pharmacol. 2009, 9, 580]. Novel label-free integrative pharmacology technology utilizes label-free resonant waveguide grating (RWG) biosensors to transform the dynamic mass redistribution of intracellular components induced by drugs into a holistic, dynamic wavelength shift response signal, known as dynamic mass reset (DMR) signal. This technology is characterized by being non-invasive, having high spatiotemporal resolution, high sensitivity, high throughput, suitability for target-pathway integration studies, ease of operation, and short experimental cycles. The detection process requires no labeling or additional indicators, providing a more realistic reflection of drug effects at the whole-cell level [M. Morse, H. Sun, E. Tran, et al. BMCPharmacol Toxicol. 2013, 14, 17]. Currently, there is no method for constructing a screening model for the membrane receptor BLT1 using this technology. Therefore, this invention employs label-free integrative pharmacology technology to construct a label-free high-throughput screening model for the BLT1 receptor, which can significantly improve the efficiency of BLT1 ligand screening. This is of great significance for elucidating the pharmacological and physiological functions of BLT1 and provides a model system for drug screening in diseases closely related to the BLT1 receptor. Summary of the Invention
[0004] To address the problems mentioned in the background art, the purpose of this invention is to provide a cell screening model for label-free G protein-coupled receptors and its applications.
[0005] To achieve the above objectives, the technical solution of this invention, utilizing novel label-free cell integration pharmacology technology, is as follows:
[0006] A label-free cell screening model for G protein-coupled receptors (GCPs), specifically a BLT1 receptor cell screening model, using the Flp-In cell line stably expressing BLT1. TM -CHO-BLT1.
[0007] The cell line Flp-In TM-CHO-BLT1 is used to transfer the BLT1 plasmid into Flp-In TM -CHO cells were used to obtain the Flp-In cell line, which highly expresses the BLT1 receptor. TM -CHO-BLT1.
[0008] Application of the label-free G protein-coupled receptor cell screening model, wherein the label-free G protein-coupled receptor cell screening model is used in high-throughput screening of compounds with BLT1 receptor for the prevention and / or treatment of related diseases.
[0009] The compound is used in the preparation of medicaments for the prevention and / or treatment of BLT1 receptor-related diseases.
[0010] The disease is one or more of the following: asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, immune complex-mediated glomerulonephritis, and immune diseases.
[0011] A method for screening drugs for diseases closely related to G protein-coupled receptors, based on label-free cell integrative pharmacology, utilizing the aforementioned stable BLT1-expressing cell line Flp-In TM -CHO-BLT1, using known agonists and antagonists, establishes a cell screening model for the BLT1 receptor. Based on the similarity and specificity of the DMR signal spectrum of the test sample with the characteristic DMR signal spectra of known agonists and antagonists, the agonist or antagonist activity of the test sample is determined, and drugs closely related to diseases associated with G protein-coupled receptors are screened.
[0012] The known agonist is leukotriene B4, and the known antagonist is BIIL-260.
[0013] The label-free cell integrated pharmacology technology utilizes a resonant waveguide grating (RWG) biosensor to convert the dynamic redistribution of intracellular components caused by drugs into a holistic, dynamic wavelength shift response signal. This signal is the response value (pm) of wavelength change, which is realized through an Epic optical biosensor 384 microplate.
[0014] The BLT1 receptor cell screening model involves seeding Flp-In into cell-compatible 384-well microplates with optical biosensing capabilities. TM -CHO-BLT1 cells were seeded at a density of 1.5 × 10⁻⁶ cells. 4 Cells per well, with a cell culture medium volume of 40 μL per well, and a cell culture time of 18–24 h after seeding.
[0015] The screening protocol for agonists is as follows:
[0016] (1) The sample dissolved in HBSS buffer and the BLT1 receptor agonist leukotriene B4 (concentrations of 200 nM, 66.6667 nM, 22.2222 nM, 7.4074 nM, 2.4691 nM, 0.823 nM, 0.2743 nM, 0.0914 nM, 0.0305 nM, 0.0102 nM, 0.0034 nM, 0.0011 nM, 0.0004 nM, and 0.0001 nM) were added to the inoculated Flp-In. TM -CHO-BLT1 cells were placed in 384-well microplates and monitored in real time on an Epic instrument for 60 minutes. If the DMR characteristic spectrum of the sample and the BLT1 receptor agonist leukotriene B4 had similar contours, the DMR characteristic spectrum of the sample and the BLT1 receptor agonist leukotriene B4 was considered to be similar.
[0017] (2) The BLT1 receptor antagonist BIIL-260 dissolved in HBSS buffer (concentrations of 10000 nM, 3333.333 nM, 1111.111 nM, 370.37 nM, 123.457 nM, 41.152 nM, 13.717 nM, 4.572 nM, 1.524 nM, 0.508 nM, 0.169 nM, 0.056 nM, 0.019 nM, 0.006 nM) was added to the inoculated Flp-In. TM -CHO-BLT1 cells were pretreated in 384-well microplates for 60 min, and then the sample (sample concentration: the concentration at which the sample in step (1) caused 80%-100% DMR characteristic signal intensity) was added to the wells of the cell plate containing the antagonist BIIL-260. The DMR characteristic signal was detected. If the intensity of this DMR characteristic signal was lower than that in step (1), the sample was determined to be an agonist of the BLT1 receptor.
[0018] The screening protocol for antagonists is as follows:
[0019] (1) The sample dissolved in HBSS buffer and the BLT1 receptor agonist leukotriene B4 (concentrations of 200 nM, 66.6667 nM, 22.2222 nM, 7.4074 nM, 2.4691 nM, 0.823 nM, 0.2743 nM, 0.0914 nM, 0.0305 nM, 0.0102 nM, 0.0034 nM, 0.0011 nM, 0.0004 nM, and 0.0001 nM) were added to the inoculated Flp-In. TM -CHO-BLT1 cells were placed in 384-well microplates and monitored in real time on an Epic instrument for 60 min to obtain DMR characteristic signal spectra.
[0020] (2) If the sample in step (1) does not induce a DMR signal spectrum, add agonist leukotriene B4 (concentration of agonist leukotriene B4: the concentration at which leukotriene B4 induces 80%-100% DMR characteristic signal intensity in step (1)) to the cell plate in which the sample was added in step (1), and detect the DMR characteristic signal spectrum; if this DMR characteristic signal is weaker than the signal of leukotriene B4 in step (1), it can be determined that the sample is an antagonist of BLT1 receptor.
[0021] The advantages and beneficial effects of this invention are as follows:
[0022] The label-free BLT1 cell model established in this invention enables high-throughput screening of commercially available synthetic and natural small molecule libraries or natural product extracts / mixtures to obtain highly active ligands for the BLT1 receptor. The BLT1 receptor is closely related to the occurrence and development of inflammatory and immune diseases such as asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis. Based on the correlation between the target and the disease, the clinical application scope of the screened compounds can be broadened. Attached Figure Description
[0023] Figure 1 Different concentrations of the BLT1 receptor agonist leukotriene B4 in Flp-In TM -CHO-BLT1 concentration-response dependence curve on cells and pretreatment with Flp-In TM Concentration-response dependent curves of DMR signal spectra of fixed concentration of leukotriene B4 in -CHO-BLT1 cells after 60 min.
[0024] Figure 2 Pretreatment of Flp-In with different concentrations of the BLT1 receptor antagonist BIIL-260 TM Concentration-response dependent curves of DMR signal spectra of fixed concentration of leukotriene B4 in -CHO-BLT1 cells after 60 min. Detailed Implementation
[0025] The present invention will now be further illustrated with examples. These examples are merely illustrative and not intended to limit the scope of the invention.
[0026] The BLT1 cell screening model constructed in this invention does not require fluorescent labeling and the detection process does not require the addition of additional indicators. It features target pathway integration response, no cell damage, reliable detection results, high sensitivity, high screening throughput, short cycle time, and simple operation. It can be used to search for highly active ligands of the BLT1 receptor from natural product libraries, metabolite libraries, and combinatorial chemistry libraries, providing a model system for drug screening of diseases closely related to the BLT1 receptor. Current research indicates that the BLT1 receptor is involved in the pathogenesis of inflammatory and immune diseases such as asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis. Therefore, the BLT1 cell screening model established in this invention can be used to obtain lead compounds for related diseases.
[0027] The novel label-free cell-integrated pharmacology technology used in this model is based on label-free resonant waveguide grating (RWG) biosensors. It transforms the dynamic mass redistribution of intracellular components caused by drugs into a holistic, dynamic wavelength shift response signal, called dynamic mass reset (DMR) signal. It features non-invasiveness, high spatiotemporal resolution, high sensitivity, high throughput, target-pathway integration research, simple operation, and short experimental cycle. The detection process does not require labeling or the addition of additional indicators, and more realistically reflects the effect of drugs at the whole level of living cells.
[0028] Chinese hamster ovarian cells Flp-In TM -CHO cells (catalog number: R75807) were obtained from Gibco. An inverted microscope was purchased from OLYMPUS. Leukotriene B4 (catalog number: 20110) was purchased from Beijing Ruierxinde Technology Co., Ltd., and the receptor antagonist BIIL-260 (catalog number: HY-114641A) was purchased from Guangzhou Weijia Technology Co., Ltd. Culture medium F12K (catalog number: C11765500BT) was purchased from Gibco. Lipofectamine TM 3000 transfection reagent (catalog number: L3000015) was purchased from Gibco. Hygromycin B (catalog number: H8080-1g) was purchased from Tianjin Bai'ao Hengkang Biotechnology Co., Ltd. Balanced salt solutions HBSS (catalog number: 14065-056) and HEPES (catalog number: 15630-080) were purchased from Gibco. Epic optical biosensor 384-well microplates were purchased from Corning. The detection platform was Corning's third-generation... The imaging system, purchased from Corning, detects the wavelength shift caused by dynamic mass resetting (DMR) of cells.
[0029] Example 1: Flp-In TM Construction of stable CHO-BLT1 cell lines
[0030] Specific transfection methods:
[0031] 1) Transform the BLT1 plasmid into Flp-In TM -CHO cells were used to obtain the Flp-In cell line, which highly expresses the BLT1 receptor. TM -CHO-BLT1, lipo3000: P3000: plasmid (pOG44: pcDNA) TM 5 / FRT-BLT1=9:1)=21.7μL:40μL:20μg.
[0032] 2) Eight hours after transfection, replace with 10 mL of fresh F12 complete culture medium (10% FBS).
[0033] 3) 24 h after transfection, replace with 12 mL of fresh F12 complete medium containing 400 μg / μL hygromycin B.
[0034] 4) After screening the culture for 7-10 days, replace the medium with 12 mL of fresh F12 complete medium containing 400 μg / μL hygromycin B every 2-3 days to obtain a stable cell line Flp-In that highly expresses the BLT1 receptor. TM -CHO-BLT1.
[0035] Example 2: BLT1 receptor agonist leukotriene B4 in Flp-In TM Characterization on -CHO-BLT1 cells
[0036] The above embodiments obtained Flp-In in the logarithmic growth phase. TM -CHO-BLT1 cells were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶ cells / well. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. Equilibrate on the imaging system for 120 min; rescan the baseline for 2 min, then apply different concentrations of the BLT1 receptor agonist leukotriene B4 (200 nM, 66.6667 nM, 22.2222 nM, 7.4074 nM, 2.4691 nM, 0.823 nM, 0.2743 nM, 0.0914 nM, 0.0305 nM, 0.0102 nM, 0.0034 nM, 0.0011 nM, 0.0004 nM, 0. Leukotriene B4 (0.0001 nM) was added to microplates at a concentration of 10 μL per well, with three replicates. The plates were placed on an Epic instrument for real-time DMR signal monitoring for 60 min. The baseline was then rescanned for 2 min. Leukotriene B4 at a concentration that elicits 80%-100% of the characteristic DMR signal intensity (equivalent to 1 nM leukotriene B4 according to the curve) was added to the microplates at a concentration of 10 μL per well, with three replicates. The plates were then placed on an Epic instrument for real-time DMR signal monitoring for 60 min. Experimental results showed that leukotriene B4 activated the BLT1 receptor to produce a concentration-dependent DMR signal response. The concentration-response curves were monophasic "S"-shaped and all reached saturation. The highest DMR response value reached approximately 300 pm. Its EC50... 50 The value was 0.09±0.01 nM; leukotriene B4 desensitized BLT1 receptors in a concentration-dependent manner, with the concentration response curves exhibiting a monophasic "S" shape and all reaching saturation responses, and its IC50 value was 0.09±0.01 nM. 50 The value was 0.39 ± 0.05 nM. These results indicate that the BLT1 receptor is functionally expressed and can be used for agonist screening and characterization.
[0037] Example 3: BLT1 receptor antagonist BIIL-260 in Flp-In TM Characterization on -CHO-BLT1 cells
[0038] The above embodiments obtained Flp-In in the logarithmic growth phase. TM -CHO-BLT1 cells were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶ cells / well. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. Equilibrate on the imaging system for 120 min. Different concentrations of the BLT1 receptor antagonist BIIL-260 (10000 nM, 3333.333 nM, 1111.111 nM, 370.37 nM, 123.457 nM, 41.152 nM, 13.717 nM, 4.572 nM, 1.524 nM, 0.508 nM, 0.169 nM, 0.056 nM, 0.019 nM, 0.0 0.6 nM leukotriene B4 was added to microplates for cell pretreatment for 60 min, with 10 μL added to each well (3 replicates). The baseline was rescanned for 2 min. Then, leukotriene B4 at a concentration that elicits 80%-100% of the characteristic DMR signal intensity (2 nM leukotriene B4 according to the curve) was added to the microplates, with 10 μL added to each well (3 replicates). The microplates were then placed on an Epic instrument for real-time DMR signal monitoring for 60 min. Experimental results showed that BIIL-260 antagonized the BLT1 receptor in a concentration-dependent manner, with a monophasic "S"-shaped concentration response curve that reached saturation. Its IC50 value was [missing information]. 50 The value was 37.26 ± 5.12 nM. These results indicate that the model can be used for the screening and characterization of antagonists.
[0039] This invention establishes a label-free BLT1 screening model based on label-free cell integrative pharmacology technology. This model has the advantages of not requiring fluorescent labeling and not requiring the addition of additional indicators in the detection process. It can efficiently and reliably screen commercially available synthetic and natural small molecule libraries or natural product extracts / mixtures to obtain highly active ligands for the BLT1 receptor, providing lead compounds for targeted therapy of BLT1-related diseases.
[0040] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A cell screening model for label-free G protein-coupled receptors, characterized in that: The cell screening model for label-free G protein-coupled receptor is the cell screening model for BLT1 receptor, and the specific model is Flp-In cell line stably expressing BLT1 TM - CHO-BLT1.
2. The cell screening model for label-free G protein-coupled receptors according to claim 1, characterized in that: The cell line Flp-In TM -CHO-BLT1 is used to transfer the BLT1 plasmid into Flp-In TM -CHO cells were used to obtain the Flp-In cell line, which highly expresses the BLT1 receptor. TM -CHO-BLT1.
3. The application of the cell screening model of the label-free G protein-coupled receptor as described in claim 1, characterized in that: The label-free G protein-coupled receptor cell screening model is used in high-throughput screening of compounds with BLT1 receptor for the prevention and / or treatment of related diseases.
4. The application of the cell screening model of label-free G protein-coupled receptors according to claim 3, characterized in that: The compound is used in the preparation of medicaments for the prevention and / or treatment of BLT1 receptor-related diseases.
5. The application of the cell screening model of label-free G protein-coupled receptors according to claim 4, characterized in that: The disease is one or more of the following: asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, immune complex-mediated glomerulonephritis, and immune diseases.
6. A method for screening drugs for diseases closely related to G protein-coupled receptors, characterized in that: Based on label-free cell integrative pharmacology, using the Flp-In cell line stably expressing BLT1 as described in claim 1, TM -CHO-BLT1, using known agonists and antagonists, establishes a cell screening model for the BLT1 receptor; based on the similarity and specificity of the DMR signal spectrum of the test sample with the characteristic DMR signal spectrum of known agonists and antagonists, the agonist or antagonist activity of the test sample is determined, and drugs closely related to diseases of G protein-coupled receptors are screened.
7. The method for screening drugs for diseases closely related to G protein-coupled receptors according to claim 6, characterized in that: The known agonist is leukotriene B4, and the known antagonist is BIIL-260.