CN111349608A - Cell screening model of unmarked histamine receptor H2 - Google Patents

Cell screening model of unmarked histamine receptor H2 Download PDF

Info

Publication number
CN111349608A
CN111349608A CN201811578003.8A CN201811578003A CN111349608A CN 111349608 A CN111349608 A CN 111349608A CN 201811578003 A CN201811578003 A CN 201811578003A CN 111349608 A CN111349608 A CN 111349608A
Authority
CN
China
Prior art keywords
sample
histamine
dmr
cells
receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201811578003.8A
Other languages
Chinese (zh)
Inventor
梁鑫淼
王志伟
王纪霞
单彩龙
薛珍珍
于广璞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Original Assignee
Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd filed Critical Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Priority to CN201811578003.8A priority Critical patent/CN111349608A/en
Priority to PCT/CN2019/087151 priority patent/WO2020124946A1/en
Publication of CN111349608A publication Critical patent/CN111349608A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a cell screening model of a marker-free histamine receptor H2. The invention discloses a method for screening agonists and antagonists of H2 receptor by using a cell line stably expressed by H2 based on a label-free cell integration pharmacological technology. This method can also be used to study modulators that affect pathways downstream of the H2 receptor. The H2 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 quantity, simplicity and convenience in operation and the like. It is used for searching agonists, antagonists and pathway regulators of the H2 receptor from natural product libraries, metabolite libraries and combined chemical libraries, and for screening drugs of acidic gastropathy such as hyperacidity, gastric ulcer and duodenal ulcer and neuropathic pain in which the H2 receptor participates.

Description

Cell screening model of unmarked histamine receptor H2
Technical Field
The invention relates to the field of cell screening, in particular to a cell screening model of a non-labeled histamine receptor H2.
Background
A G-protein-coupled receptor (GPCR) is the most important membrane receptor in cell signaling, and is one of the most interesting drug targets in small molecule drug development, and about 34% of modern drugs directly target the receptor family. Histamine is a biogenic amine widely present in human tissues and exerts a wide range of physiological effects by binding to its receptors. To date, 4 subtypes of histamine receptors have been discovered, namely: h1, H2, H3, H4 receptors. Histamine receptors belong to the family of G-protein coupled receptors, which transduce signals by coupling activation of specific G-proteins. Among them, the research on the H2 receptor and its receptor antagonist is started from 60 s in the 20 th century, the H2 receptor is mainly related to mediating gastric acid secretion, and the H2 receptor antagonist is widely used for treating acid stomach diseases such as gastric hyperacidity, gastric and duodenal ulcer, etc.; it has also been reported that histamine can increase primary sensory neuron Nav1.8 through H2 receptor and participate in neuropathic pain, which has great inspiration for developing drugs with H2 receptor-related targets; therefore, constructing a cell screening model of the H2 receptor to find high-activity agonists, antagonists and pathway modulators of the H2 receptor is of great significance for revealing the biological function and pharmacological characteristics of H2.
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 traditional radioligand receptor binding experiment method has 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 the rest GPCR detection methods mainly aim at the activation of a certain signal path, do not usually consider the activation of multiple paths, usually need fluorescent protein labeling or additionally add an indicator, so that the operation is complicated, and the addition of the indicator can damage cells to a 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 histamine receptor H2 by means of a novel non-labeled cell integration pharmacological technology, so as to screen the drug screening application of H2 receptor agonist, antagonist and channel regulator, and the drug screening application of H2 receptor involved in acid gastric diseases such as hyperacidity, gastric ulcer and duodenal ulcer and neuropathic pain in 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 the H2 receptor is established by using a cell line HEK-293-H2 which stably expresses H2 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 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.
A cell screening model of the unlabeled histamine receptor H2 is established by the following steps:
1) HEK-293-H2 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 × 104And each hole is filled with 40 mu L of cell culture solution, and the cell culture time after inoculation is 18-24 h.
2) Adding histamine agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the cimetidine antagonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 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 histamine agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 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-H2 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 the H2 antagonist cimetidine into a micropore plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 2), detecting the DMR signal of the sample, and judging the sample to be the agonist of the H2 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 histamine into a micropore plate inoculated with HEK-293-H2 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 MuM, and the concentration of the histamine 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 histamine 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 histamine in step 1), the sample to be tested is judged to be an antagonist of H2 receptor.
Further, the step of the activity of the sample to be tested on the H2 pathway is as follows:
1) respectively adding a sample to be detected and histamine into a micropore plate inoculated with HEK-293-H2 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 MuM, and the concentration of the histamine is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) continuously adding histamine 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 histamine in the step 1) in any one stage of ascending period (1-10 min), plateau period (10-20 min) and delay period (20-60 min);
3) adding the H2 antagonist cimetidine into a micropore plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 1), detecting the DMR signal of the sample, and judging that the sample to be detected is the regulator of the downstream signal path of the H2 receptor 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 H2 unmarked high-throughput screening model constructed by adopting the unmarked cell integration pharmacological technology can greatly improve the discovery efficiency of an agonist, an antagonist and a pathway regulator of H2, has great significance for explaining the pharmacological and physiological functions of H2, and simultaneously provides guidance for screening drugs for acidic stomach diseases such as hyperacidity, gastric ulcer, duodenal ulcer and the like and neuropathic pain in which the H2 receptor participates.
Drawings
FIG. 1(A) DMR signature spectra on HEK-293-H2 cells of histamine at various concentrations; (B) concentration-response dependence curves of various concentrations of histamine on HEK-293-H2 cells; wherein the concentration of histamine is in nM.
FIG. 2 DMR signature spectra of cimetidine on HEK-293-H2 cells; wherein the concentration of cimetidine is in nM.
FIG. 3(A) DMR signal spectra of histamine at fixed concentrations after 1H of different concentrations of histamine pre-treated HEK-293-H2 cells; (B) after HEK-293-H2 cells are pretreated by histamine with different concentrations for 1H, a concentration-response dependence curve corresponding to a DMR signal spectrum of histamine with fixed concentration is obtained; wherein the concentration of histamine is in nM.
FIG. 4(A) DMR signal spectra of histamine at fixed concentrations after 1H pretreatment of HEK-293-H2 cells with varying concentrations of cimetidine; (B) after HEK-293-H2 cells are pretreated by cimetidine with different concentrations for 1H, a concentration-response dependence curve corresponding to a DMR signal spectrum of histamine with fixed concentration is obtained; wherein the concentration of histamine and cimetidine is 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 histamine agonist on HEK-293-H2 cells
Human embryonic kidney cells HEK-293-H2 cells were obtained from a laboratory self-constructed cell bank, obtained under an inverted microscope from OLYMPUS, and histamine and cimetidine from carbofuran and AMQUAR, respectively. 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
Figure BDA0001914807910000041
An imager, the detected signal being a wavelength shift caused by a cell Dynamic Mass Reset (DMR).
HEK-293-H2 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 2.0 × 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. Will be fine in the microplateThe cell culture solution was changed to Hank's balanced salt solution (containing 20mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the mixture was allowed to stand
Figure BDA0001914807910000042
Balancing for 1h on the imager; rescanning the baseline for 2min, histamine was added to the microplate 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, 3 times in parallel, and DMR signals were monitored in real time on an Epic instrument for 1h, and the EC of histamine was calculated based on the maximal DMR response of the cells over 40min after histamine exposure50The values, results are shown in FIG. 1. The study shows that histamine is in a dose-dependent H2 receptor agonism, the dose response curve is in a monophasic S shape and all reach saturation response, the highest DMR response value reaches 250pm, and the corresponding EC is50The value was 0.56. + -. 0.12. mu.M.
Example 2: DMR characteristic signal spectrum of antagonist cimetidine on HEK-293-H2 cell
HEK-293-H2 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 2.0 × 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
Figure BDA0001914807910000051
Balancing for 1h on the imager; different concentrations of cimetidine were added to the plates 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 by rescanning the base line for 2min, and the DMR signal was monitored in real time on Epic instruments for 1h in parallel 3 times, the results are shown in FIG. 2. Studies have shown that the DMR response signal of different concentrations of cimetidineClose to zero.
Example 3: desensitization DMR signature spectra of HEK-293-H2 cells
HEK-293-H2 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 2.0 × 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
Figure BDA0001914807910000052
Balancing for 1h on the imager; adding histamine with different concentrations into pretreated HEK-293-H2 cells for 1H, adding 10 μ L of histamine 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, and paralleling for 3 times; rescanning the baseline for 2min, adding histamine at a fixed concentration into the microplate, adding a volume of 10 μ L per well, a concentration of 1000nM, and paralleling for 3 times, monitoring the DMR signal for 1h in real time on an Epic instrument, and calculating IC based on the maximum DMR response value of the cells under histamine action within 40min50The values, results are shown in FIG. 3. Studies have shown that histamine is a dose-dependent desensitized H2 receptor, with a monophasic "S" profile with all achieving a saturating response, corresponding to IC50The value was 0.27. + -. 0.06. mu.M.
Example 4: antagonistic DMR signature profiles of HEK-293-H2 cells
HEK-293-H2 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 2.0 × 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 medium in the plate was changed to Hank's Balanced salt solution (containing 20mM HEPES), and added to each wellThe mixture was added in a volume of 30. mu.L, and then placed in a flask
Figure BDA0001914807910000061
Balancing for 1h on the imager; the different concentrations of cimetidine were added to the microplate to pre-treat the cells for 1h, with a volume of 10 μ 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; rescanning the baseline for 2min, adding histamine at a fixed concentration into the microplate, adding a volume of 10 μ L per well, a concentration of 1000nM, and paralleling for 3 times, monitoring the DMR signal for 1h in real time on an Epic instrument, and calculating IC based on the maximum DMR response value of the cells under histamine action within 40min50The values, results are shown in FIG. 4. The study shows that cimetidine antagonizes H2 receptor in a dose-dependent manner, the dose response curve is in a single-phase S shape, the single-phase S shape and the single-phase S shape all reach saturation response, and the corresponding IC50The value was 0.48. + -. 0.1. mu.M.
The invention establishes an H2 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 a commercialized small molecule library, a self-prepared natural product extract, a self-prepared component or compound library and a chemical modifier are efficiently and reliably screened to obtain the H2 receptor agonist, antagonist and pathway regulator, and H2 receptor-regulated drugs for treating acid gastropathy such as hyperacidity, gastric ulcer, duodenal ulcer and the like and neuropathic pain diseases.

Claims (5)

1. A cell screening model of the unlabeled histamine receptor H2 is characterized in that the establishment process comprises the following steps:
1) HEK-293-H2 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 histamine agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the cimetidine antagonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 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 cell screening model of unlabeled histamine receptor H2 according to claim 1, wherein the screening step for the agonistic activity of the test sample is as follows:
1) adding histamine agonist dissolved in HBSS buffer salt containing 0.1% BSA into a 384 micro-well plate inoculated with HEK-293-H2 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-H2 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 H2 antagonist cimetidine into a micropore plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 2), detecting the DMR signal of the sample, and judging the sample to be the agonist of the H2 receptor if the DMR signal intensity is lower than that in the step 2).
3. The model of claim 1, wherein the step of screening for antagonistic activity in a test sample comprises:
1) respectively adding a sample to be detected and histamine into a micropore plate inoculated with HEK-293-H2 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of histamine 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 histamine 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 histamine in step 1), the sample to be tested is judged to be an antagonist of H2 receptor.
4. The model for cell screening of the unlabeled histamine receptor H2 according to claim 1, wherein the step of testing the sample for modulating activity on the H2 pathway comprises:
1) respectively adding a sample to be detected and histamine into a micropore plate inoculated with HEK-293-H2 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of histamine is 0.01-100000 nM, and detecting a DMR signal spectrum;
2) continuously adding histamine 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 histamine signal in step 1) at any stage of ascending phase, plateau phase and lag phase;
3) adding the H2 antagonist cimetidine into a micropore plate inoculated with HEK-293-H2 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 1), detecting the DMR signal of the sample, and judging that the sample to be detected is the regulator of the downstream signal path of the H2 receptor if the DMR signal spectrum is consistent with that of the sample in the step 1).
5. The model of claim 4, wherein the time period of the up phase is 1-10 min, the time period of the plateau phase is 10-20 min, and the time period of the lag phase is 20-60 min.
CN201811578003.8A 2018-12-21 2018-12-21 Cell screening model of unmarked histamine receptor H2 Pending CN111349608A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201811578003.8A CN111349608A (en) 2018-12-21 2018-12-21 Cell screening model of unmarked histamine receptor H2
PCT/CN2019/087151 WO2020124946A1 (en) 2018-12-21 2019-05-16 Cell screening model for unlabeled histamine h2 receptor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811578003.8A CN111349608A (en) 2018-12-21 2018-12-21 Cell screening model of unmarked histamine receptor H2

Publications (1)

Publication Number Publication Date
CN111349608A true CN111349608A (en) 2020-06-30

Family

ID=71100027

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811578003.8A Pending CN111349608A (en) 2018-12-21 2018-12-21 Cell screening model of unmarked histamine receptor H2

Country Status (2)

Country Link
CN (1) CN111349608A (en)
WO (1) WO2020124946A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102292636A (en) * 2008-11-24 2011-12-21 康宁股份有限公司 Methods for characterizing molecules
CN106539786A (en) * 2015-09-23 2017-03-29 中国科学院大连化学物理研究所 A kind of histamine H 1 receptor antagonist and its screening technique and application
CN106544392A (en) * 2015-09-23 2017-03-29 中国科学院大连化学物理研究所 A kind of screening technique of histamine H 1 receptor antagonist
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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120022116A1 (en) * 2010-07-20 2012-01-26 Huayun Deng Compositions and methods for the treatment of pathological condition(s) related to gpr35 and/or gpr35-herg complex

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102292636A (en) * 2008-11-24 2011-12-21 康宁股份有限公司 Methods for characterizing molecules
CN106539786A (en) * 2015-09-23 2017-03-29 中国科学院大连化学物理研究所 A kind of histamine H 1 receptor antagonist and its screening technique and application
CN106544392A (en) * 2015-09-23 2017-03-29 中国科学院大连化学物理研究所 A kind of screening technique of histamine H 1 receptor antagonist
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
S. LIEBA,等: "Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion", PHARMACOLOGICAL RESEARCH, vol. 108, pages 65 - 74 *
贾德军等: "西米替丁在儿科的临床应用", 包头医学, vol. 36, no. 1, pages 30 - 31 *

Also Published As

Publication number Publication date
WO2020124946A1 (en) 2020-06-25

Similar Documents

Publication Publication Date Title
Charlton et al. Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays
Zhang et al. Tools for GPCR drug discovery
JP2008536117A (en) Method and system for performing G protein-coupled receptor (GPCR) cell analysis using a waveguide grating sensor
Sleno et al. Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors
RU2008115455A (en) HIGH-PERFORMANCE TRPM5 ION CHANNEL SCREENING ANALYSIS
Trivedi et al. Cellular HTS assays for pharmacological characterization of NaV1. 7 modulators
Klein et al. Approaches for probing allosteric interactions at 7 transmembrane spanning receptors
Pottie et al. In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5‐HT2AR
Patrizio et al. Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques
CN111349613A (en) Cell screening model of unmarked muscarinic receptor M4
Wu et al. High-mass MALDI-MS unravels ligand-mediated G protein–coupling selectivity to GPCRs
Hoare et al. Biosensor assays for measuring the kinetics of G-protein and arrestin-mediated signaling in live cells
CN111349608A (en) Cell screening model of unmarked histamine receptor H2
CN111349607A (en) Cell screening model of unmarked bombesin receptor BB3
CN114107210A (en) Cell screening model of label-free membrane receptor GPR84 and application thereof
CN111349610A (en) Cell screening model of unmarked Delta receptor
CN111349612A (en) Cell screening model of unmarked opioid receptor NOP
Wirth et al. Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding
CN111349609A (en) Cell screening model of unmarked histamine receptor H3
CN111349611A (en) Cell screening model of unmarked Kappa receptor
CN112980797A (en) Cell screening model of unmarked melatonin membrane receptor MT1 and application
CN112746058A (en) Cell screening model of alpha 2A adrenergic receptor
CN113005089A (en) Cell screening model of unlabeled cell membrane receptor GPR88 and application
Tang et al. Real-time and high throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye
CN112575051A (en) Cell screening model of beta 1 adrenergic receptor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20200630