Screening method for histamine H1 receptor antagonist

A technology of receptor antagonists and screening methods, applied in the field of drug screening research, can solve the problems of anticholinergic weight gain and other problems, and achieve high sensitivity and high throughput effects

Inactive Publication Date: 2017-03-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Clinically used histamine H1 receptor antagonists are important anti-allergic and antipruritic drugs, but

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  • Screening method for histamine H1 receptor antagonist
  • Screening method for histamine H1 receptor antagonist
  • Screening method for histamine H1 receptor antagonist

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[0052] Example 1 Establishment of screening method for histamine H1 receptor antagonists

[0053] 1. Method

[0054] 1.1 Cell culture

[0055] A-431 human epidermal cancer cells and A-549 human non-small cell lung cancer cells were obtained from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai, China). A-431 cells use DMEM medium containing 10% fetal bovine serum, 100U / ml ampicillin, 100μg / ml streptomycin sulfate (GIBCO, article number 12800017, containing D-Glucose 4.5g / L, supplemented with NaHCO 3 1.5g / L), at 37℃, 5% CO 2 A-549 cells are cultured in an incubator containing 10% fetal calf serum, 100U / ml ampicillin, 100μg / ml streptomycin sulfate in F12K medium, and cultured in an incubator at 37°C and 5% CO2.

[0056] 1.2 Pharmacological experiments on unlabeled cell targets

[0057] A-431 cells or A-549 cells at 2×10 4 Inoculated into 384-well biosensor microplate, placed at 37℃, 5% CO 2 Cultivate for 24h in the incubator. A-431 cel...

Example Embodiment

[0071] Example 2 Application of histamine H1 receptor antagonist screening method

[0072] Using label-free cell target pharmacology technology, 40 tested compounds were screened for histamine H1 receptor antagonistic activity. First, add 10 μL of the compound to be tested (maximum working concentration 64 μM, 2-fold dilution, 2 concentration gradients) into the inoculated A-431 cells or A-549 cells 384-well biosensor microplate, in Monitor the system for 30 minutes, and then add 10 μM histamine (8 μM) to continue monitoring for 30 minutes. According to the maximum DMR response signal antagonism within 30 minutes after adding histamine in the second step, the antagonistic activity of the test compound on histamine H1 receptor is judged. Among them, arctigenin showed H1 receptor antagonistic activity. After pretreatment of A-431 cells with arctigenin (maximum working concentration 64μM, 2-fold dilution, 7 concentration gradients), 16μM histamine (EC 80 ), the DMR response signal...

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Abstract

The invention discloses a screening method for a histamine H1 receptor antagonist. By adopting unmarked cell target point pharmacology technology, DMR response signals generated after histamine acts on A-431 cells or A-549 cells are monitored. Desensitization is conducted in a dose-dependent mode through the DMR response signals generated after the histamine acts on the A-431 cells or the A-549 cells, high-expression H1 receptors on the A-431 cells or the A-549 cells are certified, and the DMR response signals generated after the receptors are activated can be successfully monitored. The DMR response signals are not generated when the A-431 cells or the A-549 cells are preprocessed by a specificity histamine H1 receptor antagonist, but the DMR response signals of the histamine is antagonized in a dose-dependent mode after the histamine is added again. In this way, the screening method for the histamine H1 receptor antagonist is successfully established. The DMR response signals generated when to-be-screened compounds subject preprocessing to the A-431 cells or the A-549 cells are monitored firstly; and if the to-be-screened compounds do not generate the DMR response signals, the DMR response signals of the histamine are antagonized after the histamine is added again, and antagonistic activity of the compounds on the H1 receptors are judged according to the antagonized extent of the response signals.

Description

technical field [0001] The invention belongs to the field of drug screening research, in particular to a screening method for histamine H1 receptor antagonists. Background technique [0002] Histamine usually exists in mast cells and basophils in an inactive state. After external stimuli such as trauma and antigen-antibody reactions, histamine is released outside the cells in an activated form and participates in inflammatory reactions. [1] . Histamine exerts its physiological effects by binding to histamine receptors. There are four types of histamine receptors in the human body: H1, H2, H3 and H4 receptors. These four types of histamine receptors are all G protein-coupled receptors (GPCRs), which transduce extracellular signals to the second intracellular signal system through G proteins [1,2] . The allergic inflammatory effect mediated by histamine mainly binds to H1 receptors, causing hyperemia, edema and itching of the skin and mucous membranes. Asthma, anaphylacti...

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 梁鑫淼史丽颖张秀莉侯滔王纪霞何牮魏来
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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