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Compound for enhancing coupling degree of complex TRPV4-KCa2.3 and application thereof in resisting hypertension

A technology of trpv4-kca2.3 and compounds, applied in the field of antihypertensive drugs, can solve problems such as unclear interaction sites

Active Publication Date: 2017-08-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its specific interaction site has not been studied clearly. Finding its interaction site and discovering compounds acting on its site is of great significance for the development of hypertension drugs

Method used

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  • Compound for enhancing coupling degree of complex TRPV4-KCa2.3 and application thereof in resisting hypertension
  • Compound for enhancing coupling degree of complex TRPV4-KCa2.3 and application thereof in resisting hypertension
  • Compound for enhancing coupling degree of complex TRPV4-KCa2.3 and application thereof in resisting hypertension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: the preparation method of compound

[0058] (1) Dissolve compound 1-propylenediamine (30g, 405mmol, 1eq) in 150mL of dichloromethane, stir in an ice bath; dissolve and dilute 2-tert-butyl sodium bicarbonate (16.1g, 73mmol, 0.18eq) in 50mL of dichloromethane; slowly pour the mixture into a flask, and stir at room temperature for 3 hours; after the reaction is complete, use thin-layer chromatography (TLC) to detect, dilute with 50mL of dichloromethane and wash with water several times, then wash with saturated NaCl solution Wash, followed by anhydrous Na 2 SO 4 After drying and concentrating, Compound 2 (19g, 27%) was obtained;

[0059] (2) Compound 2 (10g, 57mmol, 1eq) and triethylamine (TEA) (8.7g, 86mmol, 1.5eq) were dissolved in 100mL of dichloromethane and stirred in an ice bath; diphenylacetyl chloride (13.1g, 57mmol ,1eq) was dissolved in 30mL of dichloromethane, and slowly poured into the flask, and stirred at room temperature for 2.5 hours; after...

Embodiment 2

[0062] Example 2: Find the structural domain of the interaction site between TRPV4 protein and KCa2.3 protein

[0063] Experimental method: select possible binding sites according to the three-dimensional structure and functional characteristics of TRPV4 protein and KCa2.3 protein (such as figure 1 shown), the selected domains are mainly used to regulate the relationship between protein-protein, which is a platform for protein interaction. The selected binding site was mutated to delete the binding site, and the primers used are shown in Table 1.

[0064] Table 1

[0065]

[0066]

[0067] The PCR reaction system used is: template (CFP-TRPV4 whole gene or YFP-KCa2.3 whole gene) 0.5 μl, PrimStar HS 25 μl, upstream and downstream primers 0.5 μl each, add H 2 O to make up to 50 μl. The PCR reaction program was: pre-denaturation at 95°C for 2min, denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 68°C for 5min, 30 cycles, and full extension at 68°C for ...

Embodiment 3

[0073] Example 3: The compound prepared in Example 1 targets the domain AR2 of TRPV4 and the domain 17C of KCa2.3.

[0074] experimental method:

[0075] The primary isolated C57BL / 6J mouse mesenteric endothelial cells were cultured in a cell constant temperature incubator, and biotinylated JNc-440 (10 μM / L) or short peptide and biotinylated JNc-440 (10 μM / L) were added to the cells. Incubate for 96 hours. The cell protein was lysed with RIPA, and the protein supernatant was added to 10 μL streptavidin-coated magnetic beads, and incubated overnight at 4°C. Adsorb the incubated suspension with a magnetic stand to obtain an avidinylated magnetic bead-biotinylated JNc-440-protein complex. Resuspend the complex with 50 μL 1xLoadingbuffer and boil in a boiling water bath for 5 minutes. The samples were separated by SDS-PAGE, the protein was transferred to PVDF membrane, blocked with 5% BSA at room temperature for 4 hours, the primary antibody was incubated overnight at 4°C, the ...

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Abstract

The invention provides a compound for enhancing space coupling degree of an endothelial cell ion channel complex TRPV4-KCa2.3 and application thereof in resisting hypertension. The compound has the advantages that by finding the structure domain of interaction sites of the endothelial cell ion channel complex TRPV4-KCa2.3, the specific compound which can simultaneously act on the two action sites is prepared; the space coupling degree of the complex TRPV4-KCa2.3 can be enhanced by the compound, and the important meaning is realized for the research and development of hypertension drugs.

Description

technical field [0001] The invention relates to the technical field of antihypertensive drugs, in particular to the application of a compound that enhances the spatial coupling degree of endothelial cell ion channel complex TRPV4-KCa2.3 in inhibiting hypertension. Background technique [0002] The "2014-2019 Research Report on China's Antihypertensive Drug Market Operation Monitoring and Development Prospects" released by China Industry Information Network pointed out that hypertension is a lifelong disease, and ordinary patients need to take medicine every day for life. The development of hypertension drugs has gone through several eras. Diuretics were introduced in the 1960s, β-blockers were introduced in the 1970s, calcium channel antagonists and angiotensin-converting enzyme inhibitors (ACEI) were introduced in the 1980s, and again in the 1990s. Developed more specific angiotensin II receptor antagonists (sartans), and then several single and compound preparations were a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D239/90A61K31/498A61P9/12
CPCA61K31/498C07D239/90A61K31/517A61P9/12
Inventor 马鑫何冬旭唐春雷张鹏陈震蔡燕飞
Owner JIANGNAN UNIV
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