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Application of actin-binding protein 2 in screening smooth muscle dysfunction disease therapeutic drugs

A technology of actin and binding protein, which is applied in the direction of drug combinations, respiratory diseases, medical preparations containing active ingredients, etc., to achieve high screening efficiency and practical and effective screening

Active Publication Date: 2019-08-27
SHANGHAI UNIV OF T C M +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is no research on the relationship between MT-2 protein and Transgelin2 protein, and the involvement of Transgelin2 in the development of asthma drugs

Method used

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  • Application of actin-binding protein 2 in screening smooth muscle dysfunction disease therapeutic drugs
  • Application of actin-binding protein 2 in screening smooth muscle dysfunction disease therapeutic drugs
  • Application of actin-binding protein 2 in screening smooth muscle dysfunction disease therapeutic drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Detection of Transgelin2 protein and its specific ligand MT-2 protein interaction

[0049] 1. Experimental materials

[0050] 1.1 MT-2 protein with His tag

[0051] According to the NCBI tissue distribution information, normal rat lung tissue was selected, total RNA was extracted by one-step method of guanidine isothiocyanate, and cDNA was synthesized by AMV reverse transcriptase, which was used as a template for PCR amplification of MT-2 gene under the action of DNA taq enzyme. PCR amplification (see Table 1 for the amplification primers, and the sequence of the amplified product is shown in SEQ ID NO: 2), the PCR amplification program is: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30s, 58°C annealing for 30s, 72°C Extended for 60s, a total of 35 cycles, and finally extended at 72°C for 5min.

[0052] After the amplified product was digested with EcoR I enzyme and Xho I enzyme, T4 DNA ligase was connected to the corresponding restriction sit...

Embodiment 2

[0075] Example 2 Influence of Intracellular Ligand MT-2 Protein on Contraction and Relaxation of Tracheal Smooth Muscle Cells with Transgelin2 Low Expression

[0076] 1. Experimental method

[0077] Add the suspension of Transgelin2 low-expression tracheal smooth muscle cells into a special glass-bottomed culture dish, and add different concentrations of MT-2 protein (0, 100, 200 and 400 ng / ml) to the culture supernatant after the cells adhere to the wall, using Olympus The polarized light stereoscopic imaging method of the xcellence living cell workstation was used to observe the axial length changes of 30 cells in the field of view before and after 10 minutes (axial length change = axial length after intervention - axial length before intervention, the unit is μm), the experimental results are shown in Figure 8 .

[0078] Add the suspension of Transgelin2 low-expression tracheal smooth muscle cells into the xCELLigence real-time cell analyzer. After the cells adhere to the...

Embodiment 3

[0086] Example 3 Transgelin2 Screening Application Research of Asthma Therapeutic Drugs

[0087] The purpose of this example is to use Transgelin2 protein as a drug to act on receptors on the surface of airway smooth muscle cells, design small molecule drugs targeting the target protein, and screen out effective drugs for asthma treatment.

[0088] 1. Experimental method

[0089] 1) Design of small molecule compounds

[0090] Based on the three-dimensional structure of Transgelin2 protein (PDB number: 1WYM), the Transgelin2 antibody (Proteintech Group company) sequence (a46-a63, QPGRENFQNWLKDGTVLC, a total of 18 amino acids, SEQ ID NO: 1) as the main target site, based on molecular docking small molecule virtual screening. The software used for virtual screening is maestro2010 software (main modules include glide module, ligperp, protein preparation wizard, etc.), small molecule preparation parameters (default parameters): pH control at 5-9 during protonation; use epik posit...

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Abstract

The invention relates to the field of drug research and development, and specifically discloses the use of actin-binding protein 2 in screening drugs for treating smooth muscle dysfunction diseases. The present invention uses Transgelin 2 as the target protein for drug action, designs and initially screens 22 possible small molecule compounds, and then screens out 2 small molecule drugs that do have the function of relaxing airway smooth muscle through further cell experiments, which can be used for Preparation of airway smooth muscle relaxation drugs; for the use of Transgelin 2 protein in the treatment of various diseases that may be caused by smooth muscle dysfunction, including vascular smooth muscle spasm, intestinal smooth muscle spasm, tracheal smooth muscle spasm, and endocrine diseases Therapy provides guidance and reference.

Description

technical field [0001] The invention relates to the field of drug research and development, and specifically discloses a new application of actin-binding protein 2 for screening drugs for treating smooth muscle dysfunction diseases. Background technique [0002] Actin-binding protein 2 (Transgelin2, SM22-Alpha Homolog) was first discovered in 1998, and its gene is located on chromosome 1q21-q25, but so far, the function and mechanism of this protein are still unclear. The expression of Transgelin2 protein is located in the cell membrane, actin microfilament, cytoplasm and other parts, and contains a calmodulin homolog domain (calponin homolog domain), which can bind to cytoskeleton proteins. Existing literature suggests that this protein is related to smooth muscle cell morphology and B cell immune function, and may be a tumor marker such as colorectal cancer. [0003] MT-2 (Metallothionein, MT) is one of the important members of the metallothionein family. Its main physiol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/62G01N27/04A61K31/7048A61P11/08
Inventor 杨永清尹磊淼王宇徐玉东朱维良陈照强张勇
Owner SHANGHAI UNIV OF T C M
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