CN113005089A - Cell screening model of unlabeled cell membrane receptor GPR88 and application - Google Patents
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Abstract
The invention relates to a construction method and application of a cell screening model of a non-labeled G protein-coupled receptor (GPCR), in particular to a cell screening model of a non-labeled cell membrane receptor GPR 88. The invention is based on a label-free cell integration pharmacological technology, and establishes a method for screening GPR88 receptor agonist and antagonist by using a cell line stably expressing GPR 88. This method can also be used to study modulators that affect pathways downstream of the receptor. The GPR88 cell screening model constructed by the invention does not need fluorescent labeling, does not need an additional indicator in the detection process, and has the characteristics of target spot-channel integration response, no damage to cells, reliable detection result, high sensitivity, high screening flux, short period, simplicity and convenience in operation and the like. The protein is used for searching agonists, antagonists and pathway modulators of GPR88 receptor and drug screening of central nervous system diseases closely related to GPR88 receptor, such as schizophrenia, Parkinson's disease, anxiety, depression, drug addiction and the like from natural product libraries, metabolite libraries and combined chemical libraries.
Description
Technical Field
The invention relates to a construction method of a cell screening model of a non-labeled GPCR, in particular to a cell screening model of a non-labeled cell membrane receptor GPR88 and application thereof.
Background
G protein-coupled receptors (GPCRs) are seven transmembrane receptors, up to 800 in number, that mediate regulation from blood pressure to a myriad of important physiological and pathological processes, and are therefore the most interesting drug targets in the development of small molecule drugs [ Allen, j. a., et al, Annual Review of Pharmacology and Toxicology 2011, 51, 117-. About 40% of the Drug targets approved by FDA are GPCRs, and these Drug targets only account for 20% of the total GPCRs, and there are a large number of GPCRs, especially orphan G protein-coupled receptors (opgpcrs), that have not yet been developed and utilized [ Rask-Andersen, m., et al, Nature Reviews Drug Discovery 2011, 10, 579-; wold, e.a., et al, Journal of Medicinal Chemistry 2019, 62(1), 88-127; lu, s. et al, Journal of Medicinal Chemistry 2019, 62(1), 24-45; topiol, s., Methods in Molecular Biology 2018, 1705, 1-21; hauser, a, s, et al, Nature Reviews Drug Discovery 2017, 16, 829-; chung, s., et al, British Journal of Pharmacology 2008, 153 (S1), S339; fang, y., et al, Frontiers in Pharmacology 2015, 6, 295; kroeze, W.K., et al., Nature Structural & Molecular Biology 2015, 22, 362-369 ]. G protein-coupled receptor-88 (GPR 88), a member of the GPCRs family, is expressed in the striatum, caudate nucleus, nucleus accumbens, and olfactory tubercle, with central nervous system expression in the striatum being particularly strong, comparable to that of the dopamine D2 receptor [ Mizushima, k., et al, Genomics 2000, 69, 314 ]. Studies have shown that GPR88 is involved in the regulation of various brain and behavioral functions, including cognition, mood, motor control, and reward-based learning, and is therefore becoming a novel drug target for the treatment of central nervous system disorders (including psychiatric disorders such as schizophrenia, parkinson's disease, anxiety, depression, and addiction), but the endogenous ligands for GPR88 are not yet known at present [ Massart, r., et al, European Journal of Neuroscience 2009, 30, 397-; jin, c.y., et al, Acs Chemical Neuroscience 2014, 5(7), 576-587; jin, c.y., et al, Acs Chemical Neuroscience 2016, 7(10), 1418-; ye, N., et al, Acs Chemical Neuroscience 2019, 10(1), 190-. In view of the huge treatment potential of GPR88 for central nervous system diseases, a cell screening model of GPR88 receptor is constructed, so that endogenous ligands, high-activity agonists, antagonists and pathway modulators of GPR88 receptor can be found, and the cell screening model has great significance for disclosing the biological function and pharmacological characteristics of GPR88 and promoting the development of novel target drugs for central nervous system diseases.
At present, the high-throughput screening method for the receptor mainly comprises the traditional radioligand receptor binding assay, GTP γ S binding assay, cyclic adenosine monophosphate (cAMP) assay, calcium flux assay, reporter gene assay, receptor endocytosis assay and β -arrestin recruitment assay [ Bylund, D.B., et al, American Journal of Physiology 1993, 265, L421-L429; harrison, C., et al, Life Sciences 2003, 74, 489-; zhang, r., et al, Acta pharmacolitica Sinica 2012, 33, 372-384; emkey, R., et al, in: Janzen, W.P., et al (Eds.), High Throughput Screening: Methods and protocols, Second Edition; fan, F., et al, Assay and Drug Development Technologies 2007, 5, 127-; zanella, f., et al, Trends in Biotechnology 2010, 28, 237-; luttrell, L.M., et al, Journal of Cell Science 2002, 115, 455-465 ]. However, these methods have certain limitations, for example, the traditional radioligand receptor binding assay requires washing and filtration, the assay cycle is long and the flux is low, and the technology can not distinguish the receptor agonist and antagonist; the other GPCR detection methods mainly aim at the activation of a certain signal path, can not distinguish the activation of a plurality of paths, require fluorescent protein labeling or additional addition of an indicator, are complex to operate, and cause certain damage to cells due to the addition of the indicator, thereby influencing the reliability of a screening result.
Disclosure of Invention
The object of the present invention is to provide a cell screening model of the unlabeled cell membrane receptor GPR88, which solves the above-mentioned problems of the background art.
In order to achieve the aim, by means of novel label-free cell integration pharmacological technology, a cell screening model of a label-free cell membrane receptor GPR88 is provided, and the drug screening application of the cell screening model for screening endogenous ligands, agonists, antagonists and pathway modulators of the GPR88 receptor and central nervous system diseases closely related to the GPR88 receptor, such as mental diseases such as schizophrenia, Parkinson's disease, anxiety, depression and addiction, is provided at high throughput.
The technical scheme of the invention is as follows:
based on a marker-free cell integration pharmacological technology, a cell screening model of a GPR88 receptor is established by using a cell line HEK293T-GPR88 stably expressing GPR88 and a known agonist. And judging the agonistic activity and antagonistic activity of the sample to be detected or the regulation influence on a downstream passage according to the similarity and specificity of the DMR signal spectrum of the sample to be detected and the DMR characteristic signal spectrum of the known agonist.
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 establishing process of the model is as follows:
1) HEK293T-GPR88 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 multiplied by 104The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.
2) Adding GPR88 receptor agonist 2-PCCA dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the lowest concentration (EC) corresponding to the highest response intensity into the 384 micro-well plate of the HEK293T-GPR88 cells added with the GPR88 receptor agonist 2-PCCA in the step 2)100) Detecting the characteristic DMR signal spectrum of the 2-PCCA of (1);
4) all the obtained DMR characteristic signal spectrums have concentration-response dependence and have sensitivity, saturation and specificity.
Wherein, the screening steps of the sample to be tested with the agonistic activity are as follows:
1) adding GPR88 receptor agonist 2-PCCA dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, and detecting the DMR characteristic signal spectrum;
2) adding a sample to be detected into a micropore plate inoculated with HEK293T-GPR88 cells at a concentration of 0.01 nM-100 mu M, and detecting a DMR signal spectrum of the sample;
3) correlating and analyzing the DMR signal spectrums in the step 1) and the step 2), and if the DMR signal spectrum in the step 2) has contour similarity with the DMR characteristic spectrum in the step 1), carrying out the next step;
4) continuously adding 2-PCCA with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 2), and detecting a DMR signal spectrum; if the DMR signal is weaker than the 2-PCCA signal in step 1).
5) Adding a sample to be detected with the same concentration as that in the step 2) into a 384 micro-well plate inoculated with HEK293T-GPR88 cells and added with GPR88 receptor agonist 2-PCCA in the step 1), detecting a DMR signal of the sample, and judging the sample to be the GPR88 receptor agonist if the DMR signal intensity is lower than that in the step 2).
Wherein, the screening steps of the sample to be tested with antagonistic activity are as follows:
1) respectively adding a sample to be detected and 2-PCCA into a micropore plate inoculated with HEK293T-GPR88 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the 2-PCCA is 2.4-20000 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 2-PCCA 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 2-PCCA in step 1), the sample to be tested can be judged to be an antagonist of GPR88 receptor.
Wherein, the step that the sample to be tested has the activity of regulating the GPR88 pathway is as follows:
1) respectively adding a sample to be detected and 2-PCCA into a micropore plate inoculated with HEK293T-GPR88 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the 2-PCCA is 2.4-20000 nM, and detecting a DMR signal spectrum;
2) adding 2-PCCA 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 for 1-120 min; if the DMR signal is different from the 2-PCCA signal in the step 1) in a certain stage of ascending period, plateau period and delay period;
3) adding GPR88 receptor agonist 2-PCCA into a microplate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, pretreating for 1-120 min, adding a sample to be detected with 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 GPR88 receptor downstream signal channel if the DMR signal spectrum is consistent with that of the sample in the step 1).
Wherein the rise period is 1-30 min, the plateau period is 30-60 min and the lag period is 60-120 min.
The cell screening model of the non-labeled cell membrane receptor GPR88, which is established by the invention, can be used for carrying out high-throughput screening on a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain an agonist, an antagonist and a pathway regulator of the GPR88 receptor. In addition, according to the relevance of the target and the diseases, the GPR88 receptor is found to play an important role in nervous system diseases, such as mental diseases of schizophrenia, Parkinson's disease, anxiety, depression, addiction and the like, and the drug screening of the relevant diseases can also be carried out.
Drawings
FIG. 1 (A) DMR signature signal profile of different concentrations of 2-PCCA on HEK293T-GPR88 cells; (B) concentration-response dependence curves of different concentrations of 2-PCCA on HEK293T-GPR88 cells.
FIG. 2 (A) DMR signal spectra of fixed concentrations of 2-PCCA after pretreatment of HEK293T-GPR88 cells for 2 h with different concentrations of 2-PCCA; (B) after HEK293T-GPR88 cells were pretreated with different concentrations of 2-PCCA for 2 h, the DMR signal spectrum of 2-PCCA at fixed concentration corresponded to a concentration-response dependence curve.
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 GPR88 receptor agonist 2-PCCA on HEK293T-GPR88 cells
Human embryonic kidney HEK293T-GPR88 cells were obtained from the institute of chemical and physical, university of Chinese academy of sciences, and were purchased from OLYMPUS under an inverted microscope and MedChemexpress under 2-PCCA (Cat: HY-100013C). The cell culture plate is an Epic optical biosensing 384 micro porous plate purchased from Corning company, the detection platform is a Corning third generation Epic imager, and the detected signal is wavelength shift caused by cell Dynamic Mass Resetting (DMR).
HEK293T-GPR88 cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (# SH30022.01B, Thermo) in a seeding volume of 40 μ L per well and a cell number of 3.0X 10 per well4And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-24 h until the cell fusion degree reaches about 95%, and carrying out activity detection. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 mu L of cell culture solution into each hole, and placing the cells on an Epic imager for balancing for 90 min after adding the cells; rescanning the baseline for 2 min, adding 2-PCCA to the microplate with a volume of 10. mu.L per well at a concentration of 20000nM, 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156 nM, 78 nM, 39 nM, 20 nM, 10 nM, 5 nM and 2.4 nM, in parallel 4 times, placing on an Epic instrument to monitor the DMR signal in real time for 120min, calculating the EC for 2-PCCA based on the maximum DMR response value of the cells over 120min of 2-PCCA exposure50The values, results are shown in FIG. 1.
Experiment knotThe result shows that 2-PCCA activates GPR88 receptor to generate DMR signal response and presents dose dependence, the dose response curve presents monophasic S type and reaches saturation response, the highest DMR response value reaches 300 pm, and the EC thereof50The value was 1.52. + -. 0.26. mu.M.
Example 2: desensitization DMR signature profile of HEK293T-GPR88 cells
HEK293T-GPR88 cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (# SH30022.01B, Thermo) in a seeding volume of 40 μ L per well and a cell number of 3.0X 10 per well4And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-24 h until the cell fusion degree reaches about 95%, and carrying out activity detection. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 mu L of cell culture solution into each hole, and placing the cells on an Epic imager for balancing for 90 min after adding the cells; adding different concentrations of 2-PCCA into a microplate to pretreat HEK293T-GPR88 cells for 120min, wherein the volume of each well is 10 muL, and the concentrations are 20000nM, 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156 nM, 78 nM, 39 nM, 20 nM, 10 nM, 5 nM and 2.4 nM, and are paralleled for 4 times; rescanning the baseline for 2 min, adding 2-PCCA with fixed concentration into the microplate, adding volume of 10 μ L and concentration of 5000 nM into each well, paralleling for 4 times, monitoring DMR signal in real time on Epic instrument for 120min, and calculating IC based on the maximum response value of DMR within 120min of 2-PCCA action of cells50The values, results are shown in FIG. 2.
The experimental results show that 2-PCCA desensitizes GPR88 receptor in a dose-dependent manner, the dose response curve is monophasic 'S' type and all reach saturation response, and the IC thereof50The value was 2.61. + -. 0.44. mu.M.
The invention establishes a GPR88 unmarked screening model based on the unmarked cell integration pharmacological technology, the model has the advantages of no need of fluorescent labeling and no need of adding an indicator in the detection process, and efficiently and reliably screens 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 medicaments for treating central nervous system diseases closely related to GPR88 receptor agonists, antagonists and pathway regulators and GPR88 receptor, such as schizophrenia, Parkinson's disease, anxiety, addiction and other mental diseases.
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 (7)
1. A cell screening model for the unlabeled cell membrane receptor GPR88, characterized in that: based on the marker-free cell integration pharmacological technology, a cell screening model of the GPR88 receptor is established by using a cell line HEK293T-GPR88 stably expressing GPR88 and using a known ligand of the GPR88 receptor.
2. The cell screening model of the unlabeled cell membrane receptor GPR88 according to claim 1, characterized in that: HEK293T-GPR88 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 multiplied by 104The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.
3. The cell screening model of the unlabeled cell membrane receptor GPR88 according to claim 1, characterized in that: the method specifically comprises the following steps:
[1] adding GPR88 receptor agonist 2-PCCA dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, and detecting the DMR characteristic signal spectrum;
[2]then to step [1]Addition of the lowest concentration (EC) corresponding to the highest response intensity was continued in the cell plate to which GPR88 receptor agonist 2-PCCA was added100) Detecting a characteristic signal spectrum of the desensitized DMR;
[3] all the obtained DMR characteristic signal spectrums have the characteristics of concentration-response dependency relationship, sensitivity, saturation and specificity.
4. The cell screening model of the unlabeled cell membrane receptor GPR88 according to claim 1, characterized in that: the screening steps of the sample to be tested for having the agonistic activity are as follows:
[1] adding GPR88 receptor agonist 2-PCCA dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, and detecting the DMR characteristic signal spectrum;
[2] adding a sample to be detected into a micropore plate inoculated with HEK293T-GPR88 cells by 0.01 nM-100 mu M, and detecting a DMR signal spectrum;
[3] performing correlation analysis on the DMR signal spectrums in the step [1] and the step [2], if the DMR signal spectrum in the step [2] has contour similarity with the DMR characteristic spectrum in the step [1 ];
[4] continuously adding 2-PCCA with the same concentration as that in the step (1) into the cell plate added with the sample to be detected in the step (2), and detecting a DMR signal spectrum; if the DMR signal is weaker than the signal of 2-PCCA in step [1 ];
[5] and (3) adding the sample to be detected with the same concentration as that in the step [2] into the 384 micro-porous plate added with the GPR88 receptor agonist 2-PCCA in the step [1], detecting the DMR signal of the sample, and if the DMR signal intensity is lower than that in the step [2], judging the sample to be the GPR88 receptor agonist.
5. The cell screening model of the unlabeled cell membrane receptor GPR88 according to claim 1, characterized in that: the screening steps of the samples to be tested for antagonistic activity are as follows:
[1] respectively adding a sample to be detected and a GPR88 receptor agonist 2-PCCA into a micropore plate inoculated with HEK293T-GPR88 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the 2-PCCA is 2.4-20000 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 2-PCCA 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) to detect the DMR signal spectrum; if the DMR signal is weaker than the signal of 2-PCCA in step [1], the sample to be tested is judged to be an antagonist of GPR88 receptor.
6. The cell screening model of the unlabeled cell membrane receptor GPR88 according to claim 1, characterized in that: the steps for determining the modulatory activity of a test sample on the GPR88 pathway are as follows:
[1] respectively adding a sample to be detected and a GPR88 receptor agonist 2-PCCA into a micropore plate inoculated with HEK293T-GPR88 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the 2-PCCA is 2.4-20000 nM, and detecting a DMR signal spectrum;
[2] continuously adding 2-PCCA 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 for 1-120 min; if the DMR signal is different from the 2-PCCA signal in the step [1] in a certain stage of ascending period, plateau period and delay period;
[3] adding GPR88 receptor agonist 2-PCCA into a microplate inoculated with HEK293T-GPR88 cells at the concentration of 2.4-20000 nM, pretreating for 5-120 min, adding a sample to be detected with 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 GPR88 receptor downstream signal channel if the DMR signal spectrum is consistent with that of the sample in the step [1 ].
7. The model of claim 6, wherein the time period of the ascending phase is 1-30 min, the time period of the plateau phase is 30-60 min, and the time period of the lag phase is 60-120 min.
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