CN112746058A

CN112746058A - Cell screening model of alpha 2A adrenergic receptor

Info

Publication number: CN112746058A
Application number: CN201911051053.5A
Authority: CN
Inventors: 梁鑫淼; 张鹏宇; 王纪霞; 王志伟; 侯滔; 单彩龙; 薛珍珍
Original assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Current assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2021-05-04

Abstract

The invention provides a cell screening model of an alpha 2A adrenergic receptor. The invention is based on the unmarked cell integration pharmacology technology, and utilizes a cell line stably expressed by an alpha 2A adrenergic receptor to establish a method for screening agonists and antagonists of the alpha 2A adrenergic receptor. The method can also be used to study modulators that affect the downstream pathway of the α 2A adrenergic receptor. The alpha 2A adrenergic receptor 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 no label, no harm, high flux, high sensitivity and the like. It is used for searching agonist, antagonist and pathway active molecules or active compounds of alpha 2A adrenergic receptors from natural product libraries, metabolite libraries and combinatorial chemistry libraries, and drug screening of alpha 2A adrenergic receptor participation for analgesia, sedation, ischemic heart disease, septic heart disease and blood pressure regulation.

Description

Cell screening model of alpha 2A adrenergic receptor

Technical Field

The invention relates to the field of cell screening, in particular to a cell screening model of an alpha 2A adrenergic receptor.

Background

Adrenergic receptors are a class of tissue receptors that mediate the action of catecholamines, and are G-protein coupled. G-protein-coupled receptors (GPCRs) are the most important class of membrane receptors in cell signaling. They are classified into adrenergic alpha receptors and beta receptors according to their response to norepinephrine. Norepinephrine is relatively more sensitive to the action of alpha receptors than epinephrine, which is more sensitive to the action of beta receptors. Alpha receptors are classified into alpha 1 receptors and alpha 2 receptors. The alpha 2 receptor is an important GPCR receptor, and is divided into 3 subtypes of alpha 2A, alpha 2B and alpha 2C according to different terminal amino acids.

α 2A is an important GPCR, and studies have shown that it is associated with a number of diseases. The populus anthropogonis and the like prove that an alpha 2A receptor plays an important role in analgesia, sedation and blood pressure regulation by constructing CHO-alpha 2A cells and gene knockout model animals; wang Huadong et al found that blocking the alpha 2A receptor of cecal ligation perforated mice could significantly improve mouse survival and cardiac function, and reduce cardiac apoptosis, demonstrating that the alpha 2A receptor plays an important role in septic heart disease. However, a highly effective model for detecting α 2A receptors at high throughput is currently lacking. Therefore, a selective alpha 2A receptor screening model is constructed to find endogenous ligands, high-activity agonists, antagonists and pathway modulators of alpha 2A receptors, and the method has potential value in finding high-selectivity alpha 2A drugs.

The current high-throughput screening methods for receptors mainly comprise the traditional radioligand binding technology, FLIPR, Western Blotting and the like. These methods all have certain limitations, for example, although FLIPR can detect components at high throughput, its dye is expensive and the steps are complicated; western Blotting has the defects of low flux, complicated steps, long period, unstable experimental results, difficulty in quantification and the like, and influences the reliability of screening results.

Disclosure of Invention

The present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a cell screening model for α 2A adrenoceptor.

In order to achieve the above objects, a cell screening model for α 2A adrenergic receptors is provided by means of a novel label-free cell integration pharmacological technique to screen α 2A receptor agonists, antagonists and pathway modulators at high throughput, and drug screening applications involving α 2A receptors for analgesia, sedation, ischemic heart disease, septic heart disease and regulation of blood pressure.

The technical scheme of the invention is as follows:

based on a marker-free cell integration pharmacological technology, a cell screening model of an alpha 2A receptor is established by using a cell line HEK-293-alpha 2A which stably expresses the alpha 2A and by means of known agonists and antagonists. 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 non-labeling cell integration pharmacology technology is characterized in that Dynamic Mass Redistribution (DMR) generated by stimulation of a cell by a drug is recorded as a visual spectral line by a resonant waveguide grating biosensor, so that the action target and the path of the drug are reflected. The label-free cell integration pharmacology technology confirms the activity result mainly through three experiments of excitation, desensitization and antagonism.

The establishment process of the cell screening model of the alpha 2A adrenergic receptor comprises the following steps:

1) HEK-293-alpha 2A cells are inoculated in a 384 microporous plate with the function of a resonant waveguide grating biosensor, and the density of the inoculated cells is 2.0-2.5 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 non-selective adrenergic receptor agonist adrenalin with the concentration of 1.2-10000 nM into a 384 micro-well plate inoculated with HEK-293-alpha 2A cells, and detecting the DMR characteristic signal spectrum;

3) adding a selective alpha 2A agonist Guanfacine into a 384 micro-porous plate inoculated with HEK-293-alpha 2A cells at the concentration of 1.2-10000 nM, and detecting the DMR characteristic signal spectrum;

4) adding the selective alpha 2A antagonist BRL44408 into a 384 micro-perforated plate inoculated with HEK-293-alpha 2A cells at the concentration of 0.2-25000 nM, and detecting the DMR characteristic signal spectrum;

5) 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 adrenalin or Guanfacine into a 384 micro-perforated plate inoculated with HEK-293-alpha 2A cells at the concentration of 1.2-10000 nM, and detecting the DMR characteristic signal spectrum;

2) adding a sample to be detected into a micropore plate inoculated with HEK-293-alpha 2A cells by 0.01 nM-100 mu M, 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 the alpha 2A receptor antagonist BRL44408 into a micropore plate inoculated with HEK-293-alpha 2A cells at the concentration of 0.2-25000 nM, pretreating for 5-60 min, adding a sample to be detected with 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 the alpha 2A 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 epinephrine or Guanfacine into a micropore plate inoculated with HEK-293-alpha 2A cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the epinephrine or Guanfacine is 1.2-10000 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 adrenaline or Guanfacine 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 adrenalin or Guanfacine in the step 1), the sample to be tested can be judged to be the antagonist of the alpha 2A receptor.

Further, the step that the sample to be detected has the regulation activity on the alpha 2A pathway is as follows:

2) continuously adding adrenalin or Guanfacine 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 1-60 min; if the DMR signal is different from the signal of the adrenalin or the Guanfacine in the step 1) in a certain stage of ascending period, plateau period and delay period;

3) adding the alpha 2A receptor antagonist BRL44408 into a micropore plate inoculated with HEK-293-alpha 2A cells at the concentration of 0.2-25000 nM, pretreating for 5-60 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 an alpha 2A 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 1-40 min, the plateau period is 40-50 min and the lag period is 50-60 min.

The technical method used in the invention is a label-free cell integration pharmacology technology, and Dynamic Mass Redistribution (DMR) generated by the stimulation of the cell by the drug is recorded as a visual spectral line by a resonant waveguide grating biosensor, so that the action target and the channel of the drug are reflected, and the label-free cell integration pharmacology technology has the characteristics of no label, no harm, high flux, high sensitivity and the like. Therefore, the construction of an alpha 2A label-free high-throughput screening model by adopting a label-free cell integration pharmacological technology can greatly improve the discovery efficiency of an alpha 2A agonist, an antagonist and a pathway regulator, has great significance for explaining the pharmacological and physiological functions of the alpha 2A, and simultaneously provides guidance for the alpha 2A receptor participating in the screening of drugs for analgesia, sedation, ischemic heart disease, septic heart disease and blood pressure regulation.

Drawings

FIG. 1 (A) DMR signature spectra on HEK-293-alpha 2A cells at various concentrations of epinephrine. (B) Concentration-response dependence curves of various concentrations of epinephrine on HEK-293- α 2A cells.

FIG. 2 (A) DMR signature of Guanfacine on HEK-293-alpha 2A cells at various concentrations.

(B) Concentration-response dependence curves of different concentrations of Guanfacine on HEK-293- α 2A cells.

FIG. 3 DMR signature spectra of BRL44408 on HEK-293- α 2A cells.

FIG. 4 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h pretreatment of HEK-293-alpha 2A cells with various concentrations of epinephrine; (B) after HEK-293-alpha 2A cells are pretreated for 1h by adrenaline with different concentrations, the DMR signal spectrum of adrenaline with fixed concentration corresponds to a concentration-response dependence curve.

FIG. 5 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h pretreatment of HEK-293- α 2A cells with different concentrations of Guanfacine; (B) after different concentrations of Guanfacine pretreat HEK-293-alpha 2A cells for 1h, the DMR signal spectrum of the epinephrine with fixed concentration corresponds to a concentration-response dependence curve.

FIG. 6 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h of pretreatment of HEK-293- α 2A cells with different concentrations of BRL 44408; (B) after HEK-293-alpha 2A cells are pretreated for 1h by BRL44408 with different concentrations, the DMR signal spectrum of adrenaline with fixed concentration corresponds 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 signature spectra on HEK-293-alpha 2A cells without selective adrenoceptor agonist epinephrine

Human embryonic kidney HEK-293-alpha 2A cells were obtained from a laboratory self-constructed cell bank, purchased from OLYMPUS under an inverted microscope, epinephrine, Guanfacine and BRL44408 from Tocris. 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).

HEK-293-alpha 2A cells in logarithmic growth phase were seeded in 384-well plates using DMEM (# SH30022.01B, Thermo) in a seeding volume of 40 μ L per well and a number of cells seeded per well of 2.0X 10⁴And (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 plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; rescanning the baseline for 2 min, adding epinephrine at different concentrations to the microplate, 10. mu.L per well, 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.3 nM, 78.1 nM, 39.1 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM, 3 times in parallel, monitoring the DMR signal in real time on an Epic instrument for 1h, calculating the EC for epinephrine based on the maximum DMR response over 40 min for epinephrine on cells₅₀The values, results are shown in FIG. 1.

The research shows that adrenalin is in dosage-dependent alpha 2A receptor stimulation, the dosage response curve is in a single-phase S shape and reaches the saturation response, the highest DMR response value reaches 330 pm, and the corresponding EC₅₀The value was 116.31. + -. 6.28 nM.

Example 2: DMR characteristic signal profile of selective alpha 2A agonist Guanfacine on HEK-293-alpha 2A cells

HEK-293-alpha 2A cells in logarithmic growth phase are inoculated in 384 micro-porous plates compatible with the cells,the culture medium used was DMEM (# SH30022.01B, Thermo), the inoculation volume per well was 40 μ L, and the number of cells inoculated per well was 2.0X 10⁴And (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 plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; rescanning the baseline for 2 min, adding different concentrations of Guanfacine to the microplate, each well being 10. mu.L, at concentrations of 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.3 nM, 78.1 nM, 39.1 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM, 3 times in parallel, monitoring the DMR signal in Epic instruments for 1h in real time, calculating the EC for Guanfacine based on the maximal DMR response for 40 min of Guanfacine on the cells₅₀The values, results are shown in FIG. 2.

The study shows that the Guanfacine stimulates the alpha 2A receptor in a dose-dependent manner, the dose response curve is in a monophasic S type but does not reach a saturation response, and the highest DMR response value reaches 40 pm.

Example 3: DMR signature Signaling Profile of antagonist BRL44408 on HEK-293-alpha 2A cells

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; the base line was rescanned for 2 min and different concentrations of BRL44408 were added to the plates in a volume of 10. mu.L per well at 25000 nM, 8333.33 nM, 2777.78 nM, 925.93 nM, 308.64 nM, 102.88 nM, 34.29 nM, 11.43 nM, 3.81 nM, 1.27 nM, 0.42 nM, 0.14nM,0.05 nM and 0.02 nM, 3 replicates, were placed on Epic instruments to monitor the DMR signal for 1h in real time, and the results are shown in FIG. 3.

Studies have shown that the DMR response signal is close to zero for different concentrations of BRL 44408.

Example 4: desensitizing DMR signature spectra for epinephrine

HEK-293-alpha 2A 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 2.0X 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 plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; adding epinephrine with different concentrations into a microplate to pretreat HEK-293-alpha 2A cells for 1h, adding epinephrine with a volume of 10 muL, a concentration of 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.3 nM, 78.1 nM, 39.1 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM into each well, paralleling for 3 times, rescanning the base line for 2 min, adding epinephrine with a fixed concentration into the microplate with a volume of 10 muL, a concentration of 625 nM, paralleling for 3 times, placing the microplate on an Epic instrument to monitor the DMR signal for 1h in real time, and calculating IC based on the maximum response value of the DMR within 40 min of the epinephrine on the cells₅₀The values, results are shown in FIG. 4.

Studies have shown that adrenalin presents dose-dependent desensitized alpha 2A receptors, dose response curves are monophasic "S" type and all reach saturation response, corresponding IC₅₀The value was 141.54. + -. 9.05 nM.

Example 5: desensitization DMR signature spectra of Guanfacine

HEK-293-alpha 2A 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 2.0X 10 per well⁴Respectively, placing the inoculated cell plate in a cell cultureCulturing in a culture box for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment.

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; adding Guanfacine with different concentrations into a microplate to pretreat HEK-293-alpha 2A cells for 1h, adding the Guanfacine with the volume of 10 muL into each well, adding the Guanfacine with the concentration of 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.3 nM, 78.1 nM, 39.1 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM in parallel for 3 times, rescanning the base line for 2 min, adding epinephrine with fixed concentration into the microplate with the volume of 10 muL into each well, adding the epinephrine with the concentration of 625 nM in parallel for 3 times, monitoring the DMR signal for 1h in real time on an Epic instrument, and calculating the IC based on the maximum response value of the DMR within 40 min of the epinephrine on the cells₅₀The values, results are shown in FIG. 5.

The research shows that the Guanfacine presents dosage-dependent desensitization alpha 2A receptor, the dosage response curve presents a monophasic S-type and all reach saturation response, and the corresponding IC₅₀Value of IC₅₀It was 957.9. + -. 98.5 nM.

Example 6: antagonistic DMR signature spectra for BRL44408

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; different concentrations of BRL44408 were added to the microplate in a volume of 10. mu.L per well for 1h, at concentrations of 25000 nM, 8333.33 nM, 2777.78 nM, 925.93 nM, 308.64 nM, 102.88 nM, 34.29 nM, 11.43 nM, 3.81 nM, 1.27 nM, 0.42 nM, 0.14nM, 0.05 nM and 0.02 nM, in parallel 3 times; to resumeScanning baseline for 2 min, adding epinephrine with fixed concentration into microporous plate, adding adrenaline with volume of 10 μ L and concentration of 625 nM into each well, paralleling for 3 times, monitoring DMR signal for 1h in real time on Epic instrument, and calculating IC based on maximum response value of DMR within 40 min of epinephrine acting on cells₅₀The values, results are shown in FIG. 6.

The study showed that BRL44408 antagonized alpha 2A receptor dose-dependently, and the dose response curve was monophasic "S" type and all reached saturation response, corresponding to IC₅₀The value was 1.87. + -. 0.43. mu.M.

The invention establishes an alpha 2A receptor unmarked screening model based on unmarked cell integration pharmacological technology, the model has the characteristics of no mark, no harm, high flux, high sensitivity and the like, and can efficiently screen a commercialized small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier so as to obtain an agonist, an antagonist and a pathway regulator of the alpha 2A receptor, and analgesia, sedation, ischemic heart disease, septic heart disease and blood pressure regulation regulated by the alpha 2A receptor.

Claims

1. A cell screening model for the α 2A adrenoceptor, characterized by: establishing a cell screening model of an alpha 2A receptor by utilizing a cell line HEK-293-alpha 2A for stably expressing alpha 2A and the aid of known agonists and antagonists based on a label-free cell integration pharmacological technology;

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 establishing process comprises the following steps:

1) HEK-293-alpha 2A cells are inoculated in an Epic 384-hole biosensor microplate, and the density of the inoculated cells is 2.0-2.5 multiplied by 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;

3) adding a selective alpha 2A adrenergic receptor agonist Guanfacine into a 384 micro-porous plate inoculated with HEK-293-alpha 2A cells at the concentration of 1.2-10000 nM, and detecting the DMR characteristic signal spectrum;

4) adding the selective alpha 2A receptor antagonist BRL44408 into a 384 micro-porous plate inoculated with HEK-293-alpha 2A cells at the concentration of 0.2-25000 nM, and detecting the DMR characteristic signal spectrum;

5) all DMR signature spectra obtained have a concentration-response dependence.

2. The model of claim 1, wherein the step of screening for agonist activity of the test sample comprises:

3. The model of claim 1, wherein the step of screening for antagonistic activity in a test sample comprises the steps of:

4. The model for screening α 2A adrenoceptor cells according to claim 1, wherein the step of testing the sample for α 2A adrenoceptor modulating activity comprises:

5. The model for cell screening of α 2A adrenoceptor according to claim 4, wherein the up phase is 1-40 min, the plateau phase is 40-50 min and the lag phase is 50-60 min.