CN111349610A - Cell screening model of unmarked Delta receptor - Google Patents
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Abstract
The invention provides a cell screening model of a non-labeled Delta receptor, and establishes a method for screening an agonist and an antagonist of the Delta receptor by utilizing a Delta stably expressed cell line based on a non-labeled cell integrative pharmacological technology. This method can also be used to study modulators that affect pathways downstream of the Delta receptor. The invention has the characteristics of no damage, high space-time resolution, high sensitivity, high flux, capability of target point-path integration research, simple operation, short experimental period and the like, does not need to add marks and additional indicators in the detection process, and more truly responds to the action of the medicament on the whole level of living cells; the method can greatly improve the discovery efficiency of Delta agonists, antagonists and pathway regulators, has great significance for describing the pharmacological and physiological functions of Delta, and simultaneously provides guidance for drug screening of relevant diseases such as analgesia and gastrointestinal motility, emotion, behavior, cardiovascular and the like which are participated by Delta receptors.
Description
Technical Field
The invention relates to the field of cell screening, in particular to a cell screening model of a marker-free Delta receptor.
Background
G-protein-coupled receptors (GPCRs) are the most important class of membrane receptors in cell signaling and are one of the most interesting Drug targets in the development of small molecule drugs, and about 34% of modern drugs directly target the receptor family [ Hauser, a.s., et al, Nature Reviews Drug Discovery 2017,16,829-842 ]. The Delta receptor is an opioid receptor and belongs to the family of G protein-coupled receptors. In 1977 Korsterlitz found an opioid receptor in the rat vas deferens, named Delta opioid receptor, and enkephalin was found to be a relatively selective endogenous ligand for the Delta opioid receptor; in 1992, the Delta receptor was successfully cloned, distributed in the cortex, olfactory bulb, hippocampus, amygdala, basal ganglia and hypothalamus; in addition, Delta opioid receptors are expressed on Chinese hamster ovary cells. Delta opioid receptors consist of 327 amino acids, have a relative molecular weight of approximately 40000, and have two glycosylation sites at the amino terminus. Delta receptors are involved in analgesia on the opioid spinal cord and may be closely related to endocrine secretion. Recent studies have also found that the Delta opioid receptor plays an important role in the development, progression and treatment of hyperalgesia. Besides the analgesic effect, the function of the opioid receptor also relates to the effects of gastrointestinal motility, emotion, behavior, cardiovascular regulation and the like, and the opioid receptor agonist has the side effect of inducing epilepsy, so the application of the opioid receptor agonist in clinical work is limited, but the reinforcing effect of the Delta opioid receptor agonist in the aspect of analgesia and the non-addictive property make the development of the Delta opioid receptor agonist not neglected. If the anti-epileptic side effects of Delta opioid receptor agonists are addressed, Delta opioid receptor agonists are likely to be the most promising members of opioid analgesic drugs. Therefore, establishing a Delta opioid receptor cell model has important significance for finding Delta opioid receptor agonists and antagonists and further finding the physiological function and pharmacological characteristics of the Delta opioid receptor.
The existing high-throughput screening method of the receptor mainly comprises a traditional radioligand receptor binding experiment method, a GTP gamma S binding experiment method, a cyclic adenosine monophosphate (cAMP) analysis method, a calcium flux detection method, a reporter gene detection method, a receptor endocytosis detection method, a β -arrestin recruitment detection method and the like, wherein the methods have certain limitations, for example, the traditional radioligand receptor binding experiment method needs washing and filtering, the experiment period is long, the flux is low and the like, the technology cannot distinguish an agonist and an antagonist of the receptor, and other detection methods mainly aim at the activation of a certain signal path, do not consider the activation of multiple paths, often need fluorescent protein labeling or additionally add an indicator, so that the operation is complicated, and the addition of the indicator can also damage cells to certain extent and influence the reliability of a screening result.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a cell screening model of a non-labeled Delta receptor by means of a novel non-labeled cell integration pharmacological technology, so as to screen Delta receptor agonists, antagonists and pathway regulators at high throughput, and drug screening application of relevant diseases such as analgesia and gastrointestinal motility, emotion, behavior, cardiovascular and the like in which the Delta receptor participates.
The technical scheme of the invention is as follows:
based on a marker-free cell integration pharmacological technology, a cell screening model of Delta receptor is established by using a cell line HEK-293-Delta stably expressing Delta and by means of known agonist and antagonist. And judging the agonistic activity, the antagonistic activity or the regulation influence of a downstream passage of the sample to be detected according to the similarity of the DMR signal spectrum of the sample to be detected and the DMR characteristic signal spectrum of the known agonist and antagonist.
The label-free cell integration pharmacology technology is characterized in that a Resonance Waveguide Grating (RWG) biosensor is used for converting a dynamic redistribution phenomenon of intracellular components caused by a medicament into an integral and dynamic wavelength shift response signal, the signal is a response value (pm) of wavelength change, and the signal is realized through an Epic optical biosensor 384 micro-porous plate.
The establishment process of a cell screening model of a marker-free Delta receptor comprises the following steps:
1) HEK-293-Delta cells are inoculated in a 384 micro-porous plate which is compatible with cells and has an optical biosensing function, and the density of the inoculated cells is 1.0-4.5 × 104The cell culture solution is 40 mu L/hole, and the cell culture time after inoculation is 18-24 h;
2) adding the enkephalin agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the naloxone antagonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
4) all the obtained DMR characteristic signal spectrums have concentration-response dependence and have sensitivity, saturation and specificity.
Further, the screening step of the sample to be tested with the agonistic activity is as follows:
1) adding the enkephalin agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
2) adding a sample to be detected into a micropore plate inoculated with HEK-293-Delta cells at the speed of 0.01 nM-100 MuM, and detecting the DMR signal spectrum;
3) correlating and analyzing the DMR signal spectra in the step 1) and the step 2), wherein if the DMR signal spectrum in the step 2) has no similarity with the DMR characteristic spectrum in the step 1), the sample has no agonist activity; if the contour similarity exists, the next step is carried out;
4) adding Delta antagonist naloxone into a micropore plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected at the same concentration as that in the step 2), detecting a DMR signal of the sample, and judging the sample to be an agonist of a Delta receptor if the DMR signal intensity is lower than that in the step 2).
Further, the screening steps of the sample to be tested for having the antagonistic activity are as follows:
1) respectively adding a sample to be detected and enkephalin into a micropore plate inoculated with HEK-293-Delta cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the enkephalin is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) if the sample to be detected in the step 1) does not cause the DMR signal spectrum, continuously adding enkephalin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting the DMR signal spectrum; if the DMR signal is weaker than the signal of the enkephalin in the step 1), the sample to be detected can be judged to be the antagonist of the Delta receptor.
Further, the step that the sample to be tested has the activity of regulating the Delta passage is as follows:
1) respectively adding a sample to be detected and enkephalin into a micropore plate inoculated with HEK-293-Delta cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the enkephalin is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) adding the enkephalin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting a DMR signal spectrum for 1-60 min; if the DMR signal is different from the BA-1 signal in the step 1) in one stage of a rise period (1-10 min), a plateau period (10-20 min) and a delay period (20-60 min);
3) adding Delta antagonist naloxone into a micropore plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected at the same concentration as that in the step 1), detecting a DMR signal of the sample, and judging that the sample to be detected is a regulator of a Delta receptor downstream signal path if the DMR signal spectrum is consistent with that of the sample in the step 1).
The novel label-free cell integration pharmacological technology adopted by the invention is based on a label-free Resonance Waveguide Grating (RWG) biosensor to convert the dynamic redistribution process of intracellular components caused by a medicament into an integral and dynamic wavelength shift response signal, namely a Dynamic Mass Resetting (DMR) signal, has the characteristics of no damage, high space-time resolution, high sensitivity, high flux, capability of target point-path integration research and simple operation, short experimental period and the like, does not need to add labels and additional indicators in the detection process, and more truly responds to the action of the medicament on the integral level of living cells. Therefore, the method adopts the unmarked cell integrated pharmacological technology to construct the unmarked Delta high-throughput screening model, can greatly improve the discovery efficiency of Delta agonists, antagonists and pathway regulators, has great significance for explaining the pharmacological and physiological functions of Delta, and simultaneously provides guidance for the drug screening of relevant diseases such as analgesia participated by Delta receptors and regulation of gastrointestinal motility, emotion, behavior, cardiovascular and the like.
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FIG. 1(A) DMR signature signal profile of various concentrations of enkephalin on HEK-293-Delta cells; (B) concentration-response dependence curves of different concentrations of enkephalin on HEK-293-Delta cells; wherein the concentration of enkephalin is in nM.
FIG. 2 DMR signature spectra of naloxone on HEK-293-Delta cells; wherein the concentration of naloxone is in nM.
FIG. 3(A) DMR signal spectra of fixed concentrations of enkephalin after 1h of different concentrations of enkephalin pre-treatment of HEK-293-Delta cells; (B) after HEK-293-Delta cells are pretreated by enkephalin with different concentrations for 1h, fixing a concentration-response dependence curve corresponding to a DMR signal spectrum of the enkephalin with fixed concentration; wherein the concentration of enkephalin is in nM.
FIG. 4(A) DMR signal spectra of fixed concentrations of enkephalin after 1h pretreatment of HEK-293-Delta cells with different concentrations of naloxone; (B) after HEK-293-Delta cells are pretreated by naloxone with different concentrations for 1h, a concentration-response dependence curve corresponding to a DMR signal spectrum of the enkephalin with fixed concentration is obtained; the concentration of enkephalin and naloxone was in nM.
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: DMR characteristic signal profile of agonist enkephalins on HEK-293-Delta cells
Human embryonic kidney cell HEK-293-Delta cell is derived from a cell bank independently constructed in a laboratory and is subjected to an inverted microscopeObtained from OLYMPUS, enkephalin and naloxone from Tocris. The cell culture plate is an Epic optical biosensing 384 micro-porous plate purchased from Corning company, and the detection platform is the third generation of Corning
An imager, the detected signal being a wavelength shift caused by a cell Dynamic Mass Reset (DMR).
HEK-293-Delta cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (C11995503BT, GIBCO) in a seeding volume of 40. mu.L per well and a cell number of 2.0 × 10 per well4And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. The cell culture solution in the microplate was replaced with Hank's balanced salt solution (containing 20mM HEPES), and 30. mu.L of the solution was added to each well, followed by placing the well in the medium
Balancing for 1h on the imager; rescanning the baseline for 2min, adding enkephalin to the microplate in a volume of 10. mu.L per well at concentrations of 10000nM, 3333.33nM, 1111.11nM, 370.37nM, 123.46nM, 41.15nM, 13.71nM, 4.57nM, 1.52nM, 0.51nM, 0.17nM, 0.06nM, 0.02nM and 0.01nM, 3 times in parallel, monitoring the DMR signal in real time on an Epic instrument for 1h, calculating the EC for enkephalin based on the maximum DMR response of the cells within 20min of enkephalin action50The values, results are shown in FIG. 1. Research shows that enkephalin is in a dose-dependent Delta receptor, a dose response curve is in a single-phase S type and reaches saturation response, the highest DMR response value reaches 100pm, and the corresponding EC50The value was 5.5. + -. 1.2 nM.
Example 2: DMR characteristic signal profile of antagonist naloxone on HEK-293-Delta cells
HEK-293-Delta cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (C11995503BT, GIBCO) in a seeding volume of 40. mu.L per well and a cell number of 2.0 × 10 per well4Respectively, inoculating the cellsAnd (3) placing the plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. The cell culture solution in the microplate was replaced with Hank's balanced salt solution (containing 20mM HEPES), and 30. mu.L of the solution was added to each well, followed by placing the well in the medium
Balancing for 1h on the imager; the base line was rescanned for 2min and naloxone was added to the plates at different concentrations in a volume of 10. mu.L per well at concentrations of 100000nM, 33333.33nM, 11111.11nM, 3703.7nM, 1234.57nM, 411.52nM, 137.17nM, 45.72nM, 15.24nM, 5.08nM, 1.69nM, 0.56nM, 0.19nM and 0.06nM, in 3 replicates, and placed on Epic instruments to monitor the DMR signal for 1h in real time, as shown in FIG. 2. Studies have shown that the DMR response signal is close to zero for different concentrations of naloxone.
Example 3: desensitization DMR signature Spectrum of HEK-293-Delta cells
HEK-293-Delta cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (C11995503BT, GIBCO) in a seeding volume of 40. mu.L per well and a cell number of 2.0 × 10 per well4And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. The cell culture solution in the microplate was replaced with Hank's balanced salt solution (containing 20mM HEPES), and 30. mu.L of the solution was added to each well, followed by placing the well in the medium
Balancing for 1h on the imager; adding enkephalin with different concentrations into a microplate to pretreat HEK-293-Delta cells for 1h, adding the enkephalin with the volume of 10 muL in each well and the concentrations of 10000nM, 3333.33nM, 1111.11nM, 370.37nM, 123.46nM, 41.15nM, 13.71nM, 4.57nM, 1.52nM, 0.51nM, 0.17nM, 0.06nM, 0.02nM and 0.01nM in each well, paralleling for 3 times, rescanning the base line for 2min, adding the enkephalin with fixed concentration into the microplate, adding the enkephalin with the volume of 10 muL in each well and the concentration of 20nM in each well, paralleling for 3 times, placing the microplate on an Epic instrument to monitor a DMR signal for 1h in real time, and based on the fact that the cells are subjected to the enkephalin and DM withinCalculating IC at Rmax response value50The values, results are shown in FIG. 3. Research shows that enkephalin is in dosage-dependent desensitization Delta receptor, the dosage response curve is in single-phase S type and all reach saturation response, and corresponding IC50The value was 1.7. + -. 0.4 nM.
Example 4: antagonistic DMR signature profiles of HEK-293-Delta cells
HEK-293-Delta cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (C11995503BT, GIBCO) in a seeding volume of 40. mu.L per well and a cell number of 2.0 × 10 per well4And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. The cell culture solution in the microplate was replaced with Hank's balanced salt solution (containing 20mM HEPES), and 30. mu.L of the solution was added to each well, followed by placing the well in the medium
Balancing for 1h on the imager; adding naloxone with different concentrations into a microplate to pre-treat cells for 1h, adding naloxone with a volume of 10 muL per well, with concentrations of 100000nM, 33333.33nM, 11111.11nM, 3703.7nM, 1234.57nM, 411.52nM, 137.17nM, 45.72nM, 15.24nM, 5.08nM, 1.69nM, 0.56nM, 0.19nM and 0.06nM, paralleling for 3 times, rescanning the base line for 2min, adding enkephalin with a fixed concentration into the microplate with a volume of 10 muL per well, with a concentration of 20nM, paralleling for 3 times, placing on an Epic apparatus to monitor the DMR signal for 1h in real time, calculating the IC based on the maximum DMR response value of the cells within 10min after the action of enkephalin 10min50The values, results are shown in FIG. 4. Research shows that naloxone antagonizes Delta receptor in a dose-dependent manner, a dose response curve is in a single-phase S shape and reaches a saturation response, and corresponding IC50The value was 0.258. + -. 0.055. mu.M.
The invention establishes a Delta unmarked screening model based on the unmarked cell integrated pharmacological technology, the model has the advantages of no need of fluorescent marking and no need of adding an indicator in the detection process, and efficiently and reliably screens a commercialized small molecule library, an autonomously prepared natural product extract, a component or compound library and a chemical modifier so as to obtain the Delta receptor agonist, antagonist and pathway regulator and the Delta receptor-involved analgesic and medicines for regulating gastrointestinal motility, emotion, behavior, cardiovascular and other related diseases.
Claims (5)
1. A cell screening model of a marker-free Delta receptor is characterized in that the establishment process comprises the following steps:
1) HEK-293-Delta cells are inoculated in a 384 micro-porous plate which is compatible with cells and has an optical biosensing function, and the density of the inoculated cells is 1.0-4.5 × 104The number of the cells is one, the volume of a cell culture solution is 40 muL/hole, and the cell culture time after inoculation is 18-24 h;
2) adding the enkephalin agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the naloxone antagonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
4) all DMR signature spectra obtained have a concentration-response dependence.
2. The model for screening cells for the unlabeled Delta receptor according to claim 1, wherein the step of screening for the agonistic activity of the test sample is as follows:
1) adding the enkephalin agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
2) adding a sample to be detected into a micropore plate inoculated with HEK-293-Delta cells by 0.01 nM-100 mu M, and detecting a DMR signal spectrum;
3) correlating and analyzing the DMR signal spectra in the step 1) and the step 2), wherein if the DMR signal spectrum in the step 2) has no similarity with the DMR characteristic spectrum in the step 1), the sample has no agonist activity; if the contour similarity exists, the next step is carried out;
4) adding Delta antagonist naloxone into a micropore plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected at the same concentration as that in the step 2), detecting a DMR signal of the sample, and judging the sample to be an agonist of a Delta receptor if the DMR signal intensity is lower than that in the step 2).
3. The model for screening cells for unlabeled Delta receptor according to claim 1, wherein the step of screening for antagonistic activity in the test sample is as follows:
1) respectively adding a sample to be detected and enkephalin into a micropore plate inoculated with HEK-293-Delta cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the enkephalin is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) if the sample to be detected in the step 1) does not cause the DMR signal spectrum, continuously adding enkephalin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting the DMR signal spectrum; if the DMR signal is weaker than the signal of the enkephalin in the step 1), the sample to be detected can be judged to be the antagonist of the Delta receptor.
4. The model for cell screening of the unlabeled Delta receptor according to claim 1, wherein the step of determining the activity of the sample to modulate the Delta pathway comprises:
1) respectively adding a sample to be detected and enkephalin into a micropore plate inoculated with HEK-293-Delta cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the enkephalin is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) adding the enkephalin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting a DMR signal spectrum for 1-60 min; if the DMR signal is different from the BA-1 signal in the step 1) in a certain stage of ascending period, plateau period and delay period;
3) adding Delta antagonist naloxone into a micropore plate inoculated with HEK-293-Delta cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected at the same concentration as that in the step 1), detecting a DMR signal of the sample, and judging that the sample to be detected is a regulator of a Delta receptor downstream signal path if the DMR signal spectrum is consistent with that of the sample in the step 1).
5. The model for cell screening of unlabeled Delta receptor according to claim 4, wherein the rise phase is 1-10 min, the plateau phase is 10-20 min and the lag phase is 20-60 min.
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| CN102292636A (en) * | 2008-11-24 | 2011-12-21 | 康宁股份有限公司 | Methods for characterizing molecules |
| CN106645735A (en) * | 2015-11-02 | 2017-05-10 | 中国科学院大连化学物理研究所 | Unmarked neurotensin receptor (NTSR) ligand screening model |
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| CN102292636A (en) * | 2008-11-24 | 2011-12-21 | 康宁股份有限公司 | Methods for characterizing molecules |
| CN106645735A (en) * | 2015-11-02 | 2017-05-10 | 中国科学院大连化学物理研究所 | Unmarked neurotensin receptor (NTSR) ligand screening model |
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| MEGAN MORSE 等: "Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family.", BMC PHARMACOLOGY AND TOXICOLOGY., vol. 14, pages 17, XP021143642, DOI: 10.1186/2050-6511-14-17 * |
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| CN112391351A (en) * | 2020-11-18 | 2021-02-23 | 泰州医药城国科化物生物医药科技有限公司 | Opioid receptor allosteric modulator screening model and application |
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