CN112391351A - Opioid receptor allosteric modulator screening model and application - Google Patents
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Abstract
The invention relates to a model for screening an opioid receptor allosteric modulator and application thereof. The invention utilizes the characteristics of HEK293-Delta stable cell strain of high expression Delta opioid receptor and a high-throughput real-time fluorescence detection analysis system (FLIPR) platform to construct a model for screening the Delta opioid receptor regulator. The Delta opioid receptor allosteric modulator constructed based on the high-throughput real-time fluorescence detection analysis system (FLIPR) platform has the characteristics of novelty, high sensitivity, reliability, simple and convenient operation, short test period, high-throughput screening and the like, and is used for discovering the Delta opioid receptor allosteric modulator from a natural product library, a metabolite library and a combinatorial chemistry library, central nervous system diseases closely related to the Delta opioid receptor, such as addiction, pain, depression, Parkinson's disease, learning, cognition and the like, and drug screening of diseases such as heart disease, ischemic stroke, cancer and the like.
Description
Technical Field
The invention relates to a screening model and application of an opioid receptor allosteric modulator, in particular to a Delta opioid receptor allosteric modulator screening model and application.
Background
G protein-coupled receptors (GPCRs) are seven transmembrane receptors, the number of the G protein-coupled receptors is up to 800, the G protein-coupled receptors are the most important membrane receptors in cell signal transduction, and the G protein-coupled receptors play an important role in regulation and control in the occurrence and development of diseases, so the G protein-coupled receptors are the most concerned drug targets in the development of small molecule drugs. About 34% of the drug targets currently approved by the FDA are GPCRs. The Delta opioid receptor is a G protein-coupled receptor with 7 transmembrane domains that, in addition to pain regulation and addiction, is also widely involved in a variety of physiological and pathological activities, such as the regulation of learning, memory, and endocrine, cardiovascular, respiratory, and immune systems. The Delta opioid receptor allosteric modulators bind to sites other than the natural ligand binding site, are capable of modulating the interaction between heterodimeric subunits or domains, and have high specificity and drug safety while maintaining temporal and spatial controllability of receptor signaling. Therefore, the allosteric modulators play an important role in the development of novel Delta receptor target drugs.
The current screening methods of allosteric modulators mainly include traditional radioligand receptor binding assays, GTP γ S binding assays, and β -arrestin recruitment assays. However, these methods have certain limitations, and the conventional radioligand receptor binding assay requires washing and filtration, and has the disadvantages of long assay period and low throughput.
FLIPR Tetra is a high-throughput CCD imaging plate reader, is suitable for life science research and high-throughput drug screening, and can quantitatively detect intracellular calcium flow in the research of molecular biology, botany, genetics, zoology, microbiology, pathology, pathophysiology and the like, particularly in the calcium flow detection of GPCRs in drug screening. Calcium flux detection is an important detection index in biology, and internationally, a calcium flux quantitative detection technology for GPCRs is a compound screening technology which is more and more widely applied. FLIPR can also be used for more accurate quantitative and qualitative detection of fluorescence, using CCD direct imaging, more suitable for increasing GPCR cytology studies. Therefore, a screening model of the Delta receptor allosteric modulator is established based on the FLIPR platform, and the method has the characteristics of reliable detection result, high sensitivity, high screening flux, short period, simplicity and convenience in operation and the like, can greatly improve the discovery efficiency of the Delta opioid receptor allosteric modulator, has important significance for explaining the pharmacology and physiological functions of the Delta allosteric modulator, and provides guidance for drug screening of central nervous system diseases closely related to the Delta opioid receptor, such as addiction, pain, depression, Parkinson's disease, learning, cognition and the like, and diseases such as heart disease, ischemic stroke, cancer and the like.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a cell screening model of Delta receptor allosteric modulators by means of a high-throughput real-time fluorescence detection and analysis (FLIPR) platform so as to realize the screening of the Delta receptor allosteric modulators based on the change of intracellular calcium flow signals.
In order to achieve the aim, a Delta opioid receptor allosteric modulator cell screening model is provided by means of a high-throughput real-time fluorescence detection analysis system (FLIPR), and is used for high-throughput screening of Delta opioid receptor allosteric modulators and drug screening of central nervous system diseases closely related to Delta opioid receptors, such as addiction, pain, depression, Parkinson's disease, learning, cognition and the like, and diseases such as heart disease, ischemic stroke, cancer and the like.
The technical scheme of the invention is as follows:
based on a high-throughput real-time fluorescence detection analysis system (FLIPR), a cell screening model of the Delta opioid receptor allosteric modulator is established by using a cell line HEK293-Delta which stably expresses the Delta opioid receptor and by means of a known Delta opioid receptor agonist. And judging the positive regulation effect of the sample to be detected on the Delta opioid receptor agonist according to the improvement of the activity of the sample to be detected on the Delta opioid receptor agonist.
Wherein, the signal detected by the high-throughput real-time fluorescence detection and analysis system (FLIPR) is a fluorescence signal value (RFU) and is realized by a high-throughput real-time fluorescence detection and analysis system (FLIPR) Calcium-6 detection kit.
Wherein the Delta opioid receptor allosteric modulator cell screening model is characterized in that HEK293-Delta cells are inoculated in a 96-microplate, and the density of the inoculated cells is 6.0-8.0 multiplied by 104One cell per well, the volume of the cell culture solution is 100 mu L per well, and the cell culture time after inoculation is 18-24 h.
The application of a cell screening model of Delta opioid receptor allosteric modulators includes the following steps of screening a sample to be tested for positive modulator activity:
(1) adding a sample to be detected into a 96 micro-porous plate inoculated with HEK293-Delta cells, wherein the concentration of each group of the sample to be detected is 10 mu M, 2.5 mu M, 0.625 mu M, 0.156 mu M and 0 mu M, and detecting the fluorescence signal value (RFU) of the sample to be detected;
(2) if no or weak fluorescence signal is detected in the step (1), adding Delta opioid receptor agonist enkephalin to each sample group to be detected in the microplate of the HEK293-Delta cells in the step (1), wherein the concentration is 0.06-1000 nM, and detecting the fluorescence signal value (RFU) of the enkephalin;
(3) if the enkephalin in step (2) obtains EC according to fluorescence signal value (RFU)50The value decreases as the concentration of the test sample increases, and the test sample can be judged to be a positive modulator of the Delta opioid receptor agonist.
The Delta receptor allosteric modulator screening model is used for screening a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain the Delta receptor allosteric modulator.
The invention can carry out high-throughput screening on a commercial small molecule library, an autonomously prepared natural product extract, a component or compound library and a chemical modifier by utilizing the established Delta opioid receptor allosteric modulator cell screening model to obtain the Delta opioid receptor modulator. In addition, according to the correlation between the target and the disease, the Delta opioid receptor is found to play an important role in central nervous system diseases, such as addiction, pain, depression, Parkinson's disease, learning, cognition and the like, and diseases, such as heart disease, ischemic stroke, cancer and the like, and the drug screening of the related diseases can also be carried out.
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FIG. 1: fluorescence signal values (RFU) on HEK293-Delta cells of BMS986187 at different concentrations;
FIG. 2: fluorescence signal value (RFU) dose-response curves of enkephalins on HEK293-Delta cells after 5min pretreatment of HEK293-Delta cells with BMS986187 at different concentrations.
Detailed Description
The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
Example 1: fluorescence signal values of Delta opioid receptor agonist modulator BMS986187 on HEK293-Delta cells
Human embryonic kidney cells HEK293-Delta cells were obtained from the institute of chemical and physical, university of Chinese academy of sciences, and purchased from OLYMPUS under an inverted microscope. Enkephalins (cat # 1889) and BMS986187 (cat # 5983) were purchased from Tocris. DMEM (Cat. No. 01-052-1ACS) was purchased from Biological Industries; fetal bovine serum (FBS, cat # 10099141) was purchased from Gibco; balanced salt solutions HBSS (cat # 14065-. The kit of fluorescent dye Calcum-6 (cat # 3221567) was purchased from molecular devices, and the masking dye Amaranth (cat # A1016-50G) was purchased from Sigma; the cell culture plate is a 96-well plate (cat # 655090) dedicated to the high throughput real-time fluorescence detection and analysis system (FLIPR) and purchased from Greiner. Detection platform high throughput real-time fluorescence detection system, available from Molecular Devices, inc.
HEK293-Delta cells in logarithmic growth phase are inoculated into a 96 micro-porous plate special for a cell compatible high-flux real-time fluorescence detection and analysis system (FLIPR), the used culture medium is DMEM (containing 10% FBS), the inoculation volume of each hole is 100 mu L, and the inoculation density is 7.0 multiplied by 104And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%.Removing the culture medium from the cultured cells, adding 100 μ L of fluorescent dye solution (Calcium-6) into each well, and incubating at 37 deg.C for 1 h; remove the dye solution and add 100. mu.L Amararth (0.5mg/mL) per well and incubate for 5min at room temperature; BMS986187 is added into a microplate inoculated with HEK293-Delta cells, the concentration of each group of BMS986187 is 10 mu M, 2.5 mu M, 0.625 mu M, 0.156 mu M and 0 mu M, each group has 16 multiple wells, each well is added with 50 mu L, and calcium flow fluorescence signal detection is carried out at the wavelength of 520nm and 488 nm. Results as shown in fig. 1, the calcium flux fluorescence signal values of different concentrations of BMS986187 were consistent with the control group, indicating that BMS986187 was not able to cause changes in the calcium flux signal.
Example 2: fluorescence signal value of Delta opioid receptor agonist enkephalin on HEK293-Delta cells
HEK293-Delta cells in logarithmic growth phase are inoculated into a 96 micro-porous plate special for a cell compatible high-flux real-time fluorescence detection and analysis system (FLIPR), the used culture medium is DMEM (containing 10% FBS), the inoculation volume of each hole is 100 mu L, and the inoculation density is 7.0 multiplied by 104And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%. Removing the culture medium from the cultured cells, adding 100 μ L of fluorescent dye solution (Calcium-6) into each well, and incubating at 37 deg.C for 1 h; remove the dye solution and add 100. mu.L Amararth (0.5mg/mL) per well and incubate for 5min at room temperature; BMS986187 is added into a microplate inoculated with HEK293-Delta cells, the concentration of BMS986187 is 10 MuM, 2.5 MuM, 0.625 MuM, 0.156 MuM, 0 MuM is placed in a drug plate A, each concentration is 16 multiple wells; diluting enkephalin 4 times from 1000nM, and placing 8 concentrations of 1000nM, 250nM, 62.5nM, 15.63nM, 3.91nM, 0.98nM, 0.24nM and 0.06 nM in drug plate B, each concentration being 2 duplicate wells; the drug plates A and B were loaded automatically using a FLIPR instrument, 50. mu.L per well, and calcium flux fluorescence signal (RFU) detection was performed at 520nm and 488nm wavelengths. As shown in FIG. 2 and Table 1, BMS986187 can improve the activity of enkephalin on Delta opioid receptors, the activity of the enkephalin is increased along with the increase of the concentration of BMS986187, and the EC of the enkephalin50The value decreased with increasing concentration of BMS986187, indicating that BMS986187 is Delta positive allosteric modulator of the opioid receptor agonist enkephalin.
TABLE 1 EC of enkephalin obtained by calculating RFU value of enkephalin of different concentrations on HEK293-Delta cell after HEK293-Delta cell is treated by BMS986187 of different concentrations for 5min50Value of
The invention establishes a Delta opioid receptor modulator cell screening model based on a high-throughput real-time fluorescence detection system, and the model can efficiently and reliably screen a commercial small molecule library, an autonomously prepared natural product extract, a component or compound library and a chemical modifier so as to obtain the Delta opioid receptor modulator and central nervous system diseases closely related to the Delta opioid receptor, such as addiction, pain, depression, Parkinson's disease, learning, cognition and the like, and medicines for diseases such as heart disease, ischemic stroke, cancer and the like.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A model for screening allosteric modulators of Delta opioid receptors, characterized by: the method is characterized in that a model for screening the Delta opioid receptor regulator is constructed by utilizing the characteristics of HEK293-Delta stable cell strain of the high-expression Delta opioid receptor and utilizing a high-throughput real-time fluorescence detection analysis system platform.
2. The model for screening allosteric modulators of Delta opioid receptors according to claim 1, characterized by: HEK293-Delta cells are inoculated in a 96 micro-porous plate special for a FLIPR platform, and the density of the inoculated cells is 6.0-8.0 multiplied by 104One cell per well, the volume of the cell culture solution is 100 mu L per well, and the cell culture time after inoculation is 18-24 h.
3. The application of a Delta opioid receptor allosteric modulator screening model is characterized in that: adding a sample to be detected and enkephalin into a FLIPR special 96-hole cell culture plate inoculated with HEK293-Delta cells, wherein if the activity of the enkephalin is improved along with the increase of the concentration of the sample to be detected, the fact that the sample to be detected can positively regulate the activity of the enkephalin is shown, and the sample to be detected can be judged to be a positive regulator of Delta opioid receptors.
4. The use of a model for screening allosteric modulators of Delta opioid receptors according to claim 3, wherein: the method specifically comprises the following steps:
[1] adding an allosteric positive molecule BMS986187 of Delta receptor agonist enkephalin dissolved in HBSS buffer salt, fixing the concentration, wherein the concentration range is 0-10 mu M, into a 96-hole cell culture plate inoculated with HEK293-Delta cells, and detecting a calcium flow signal;
[2] continuously adding Delta receptor stimulant enkephalin into the 96-hole cell culture plate added with the Delta opioid receptor allosteric positive molecule BMS986187 in the step (1), wherein the concentration of the Delta receptor stimulant enkephalin is 0.06 nM-1000 nM, and detecting a calcium flow signal;
[3] the activity of the enkephalin obtained in the step [2] is increased along with the increase of the concentration of BMS986187, the EC50 value of the enkephalin is reduced along with the increase of the concentration of BMS986187, and the sample to be detected can be judged to be the positive regulator of the Delta opioid receptor agonist.
5. The use of a model for screening allosteric modulators of Delta opioid receptors according to claim 3, wherein: the Delta receptor allosteric modulator screening model is used for screening a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain the Delta receptor allosteric modulator.
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Citations (3)
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| US20170056377A1 (en) * | 2015-08-31 | 2017-03-02 | National Health Research Institutes | Opioid receptor modulators and use thereof |
| US20170370929A1 (en) * | 2014-12-17 | 2017-12-28 | Bristol-Myers Squibb Company | Positive allosteric modulators of the delta-opioid receptor |
| CN111349610A (en) * | 2018-12-21 | 2020-06-30 | 泰州医药城国科化物生物医药科技有限公司 | Cell screening model of unmarked Delta receptor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170370929A1 (en) * | 2014-12-17 | 2017-12-28 | Bristol-Myers Squibb Company | Positive allosteric modulators of the delta-opioid receptor |
| US20170056377A1 (en) * | 2015-08-31 | 2017-03-02 | National Health Research Institutes | Opioid receptor modulators and use thereof |
| CN111349610A (en) * | 2018-12-21 | 2020-06-30 | 泰州医药城国科化物生物医药科技有限公司 | Cell screening model of unmarked Delta receptor |
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| AJAY S. YEKKIRALA 等: ""Clinically employed opioid analgesics produce antinociception via μ-δ opioid receptor heteromers in rhesus monkeys"", ACS CHEMICAL NEUROSCIENCE, vol. 3, no. 9 * |
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