Unmarked endothelin receptor cell model construction and screening method and application

A technology of endothelin receptor and screening method, which is applied in the field of ligand screening of ETA and ETB receptors, achieving the effect of simple operation and high throughput

Inactive Publication Date: 2020-06-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no small molecule ETA receptor-specific agonist has been found. Therefore, the establishment of this model and screening method is of great help to the discovery of specific ligands and the pharmacology of receptors ( A.P. Davenport, et al., Endothelin, Pharmacological Reviews, 2016, 68, 357-418; J.J. Maguire and A.P. Davenport, Endothelin receptors and their antagonists, Semin Nephrol, 2015, 35, 125-36; M. Burnier, Update on Endothelin Receptor Antagonists, in H Current Hypertension Reports, 2018, 20)

Method used

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  • Unmarked endothelin receptor cell model construction and screening method and application
  • Unmarked endothelin receptor cell model construction and screening method and application
  • Unmarked endothelin receptor cell model construction and screening method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Characterization of ETA receptor on SH-SY5Y cell line

[0046] Seed SH-SY5Y cells in Epic optical biosensor 384 microwell plate, about 4.5×10 per well 4 After culturing for 48 hours, discard the medium, add 30 μL of HBSS buffer after incubation, and let stand for 1 hour until the cell state is stable. After establishing a baseline of 2 minutes, add a gradient ETA receptor agonist (such as endothelin-1, starting from the final concentration of 60nM, 1 / 2 times decreasing to 0.007324nM, a total of 14 concentration points) or antagonist (such as CI 1020, Among them, CI 1020 started from the final concentration of 3000nM, 1 / 2 times decreased to 0.366211nM, a total of 14 concentration points), 10μL per well, and continued to monitor the cell response signal for 1h, which was recorded as step 1. Re-establish the baseline, add endothelin-1 (10 μL) at a final concentration of 10 nM to each well, and continue to monitor the cell response signal for 1 h, which is recorded as step...

Embodiment 2

[0050] Characterization of ETA receptor on PC3 cell line

[0051] Seed PC3 cells in Epic optical biosensor 384 microwell plate, about 2.5×10 per well 4 After culturing for 24 hours, discard the medium, add 30 μL of HBSS buffer after incubation, and let stand for 1 hour until the cell state is stable. After establishing a baseline of 2 minutes, add a gradient ETA receptor agonist (such as endothelin-1, from the final concentration of 60nM, 1 / 2-fold decrease to 0.007324nM, a total of 14 concentration points) or antagonist (such as ambesentan, Starting from the final concentration of 1000nM, 1 / 2-fold decrease to 0.12207nM, a total of 14 concentration points), 10 μL per well, continue to monitor the cell response signal for 1h, which is recorded as step 1. Re-establish the baseline, add endothelin-1 (10 μL) at a final concentration of 10 nM to each well, and continue to monitor the cell response signal for 1 h, which is recorded as step 2.

[0052] Imager Beta v3.7 software was ...

Embodiment 3

[0055] Characterization of ETB receptor on U251 cell line

[0056] Seed U251 cells in Epic optical biosensor 384 microwell plate, about 2.0×10 per well 4 After culturing for 24 hours, replace the serum-free medium to starve the cells for 24 hours, discard the medium, add 30 μL of HBSS buffer after incubation, and let stand for 1 hour until the cell state is stable. After establishing the baseline for 2 minutes, add a gradient ETB receptor agonist (such as endothelin-1 or IRL 1620, in which endothelin-1 is gradually decreased from the final concentration of 60nM to 0.007324nM by 1 / 2 times, and IRL 1620 is gradually reduced from the final concentration of 200nM, 1 / 2-fold decrease to 0.024414nM, each with 14 concentration points) or antagonist (such as Posentan, starting from the final concentration of 40μM, 1 / 2-fold decrease to 4.88nM, a total of 14 concentration points), per well 10 μL. If it is an agonist, continue to monitor the cell response signal for 1 hour; if it is an ...

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Abstract

The invention relates to a model building method and application of a G protein coupled receptor (GPCR) on a new platform, in particular to model building and application of two subtypes, namely an Atype (ETA receptor) and a B type (ETB receptor), of an endothelin receptor on different cell lines by using an unmarked cell dynamic mass reset technology. The ETA receptor model is constructed on anSH-SY5Y cell line and a PC3 cell line for endogenously expressing ETA receptors respectively, and the ETB receptor model is constructed on a U251 cell line for endogenously expressing ETB receptors. The method has the characteristics of being close to the real environment in vivo, unmarked, real-time in monitoring, high in flux and simple to operate, can screen the ETA receptor and ETB receptor ligands (including agonists and antagonists) with subtype selectivity, and can compare the results of the same compound on the two receptors in the same ratio.

Description

technical field [0001] The invention belongs to the field of pharmacological research and drug screening research, and specifically relates to the endogenous endothelin receptors of two different subtypes of endothelin receptors, namely endothelin receptor type A (ETA) and endothelin receptor type B (ETB). The model establishment on the cell, the present invention also relates to the ligand screening method and analysis of ETA and ETB two kinds of receptors. Background technique [0002] Endothelin receptors belong to the rhodopsin A family of G protein-coupled receptors. According to their amino acid sequences, they can be divided into two subtypes, endothelin receptor type A (ETA receptor) and endothelin receptor Type B (ETB receptors), these two receptors differ in distribution and function. Among them, ETA receptors are mainly distributed in the pulmonary artery, renal artery, coronary artery, heart, etc., and ETB receptors are mainly distributed in the kidney, brain, l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/84G01N33/543G01N33/68
CPCG01N21/84G01N33/543G01N33/68
Inventor 梁鑫淼曲腊腊张秀莉王纪霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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