Preparation method of rubiaceous cyclopeptide and application of rubiaceous cyclopeptide as Hippo-YAP signal pathway inhibitor

A signaling pathway, Rubiaceae technology, applied in the field of medicine, can solve the problems of expensive aminopropyl bonded silica materials, poor controllability and reproducibility of methanol recrystallization technology, and not commonly used

Inactive Publication Date: 2014-12-10
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage is that activated carbon has strong adsorption to samples, aminopropyl bonded silica gel material is more expensive, and is not commonly used in laboratories and industries, and the controllability and reproducibility of methanol recrystallization technology are not good.

Method used

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  • Preparation method of rubiaceous cyclopeptide and application of rubiaceous cyclopeptide as Hippo-YAP signal pathway inhibitor
  • Preparation method of rubiaceous cyclopeptide and application of rubiaceous cyclopeptide as Hippo-YAP signal pathway inhibitor
  • Preparation method of rubiaceous cyclopeptide and application of rubiaceous cyclopeptide as Hippo-YAP signal pathway inhibitor

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation method of Rubiaceae type cyclic peptide RA-V (1):

[0032] Take the roots and rhizomes (1kg) of Rubia multiflora, dry and pulverize them, and reflux extract with methanol for 3 times (3L×3 times), the time is 3h, 3h and 2h, and the extract is concentrated under reduced pressure to obtain the total extract 180g; the total extract was chromatographed on D101 macroporous resin, eluted with methanol / water gradient (100:0, 50:50, 0:100), combined with cyclic peptide TLC detection method to combine 100% methanol containing cyclic peptide The eluted part; each of the following steps must be separated and purified in combination with the cyclic peptide TLC detection method. The combined cyclic peptide fraction (11g) was chromatographed on a silica gel column, eluted with petroleum ether / acetone (3:1-1:1) gradient, and combined with the cyclic peptide RA-V (1) part to obtain the total RA-V fraction (120 mg); the total fraction of the cyclic peptide was further s...

Embodiment 2

[0036] The Rubiaceae type cyclic peptide RA-V(1) of the present invention was initially screened on HEK293 cells stably transfected with the Myc-YAP / TEAD / CTGF promoter-luciferase luciferase activity detection system, and then transiently transfected with beta galact Re-screening in HEK293 cells with the glycosidase / Flag-YAP / Gal4-TEAD4 / 9XUAS-luciferase activity detection system revealed that RA-V(1) can inhibit the activity of the Hippo-YAP pathway. The experimental principles, methods and results are as follows:

[0037] Experimental principle: luciferase reporter gene is a commonly used experimental method to detect the activity of signaling pathways. Luciferin emits fluorescence under the catalysis of luciferase, and the intensity of the detected fluorescence reflects the strength of the signaling pathway activity.

[0038] In HEK293 stable cells, the highly expressed Myc-YAP protein in the Myc-YAP / TEAD / CTGF promoter-luciferase luciferase activity detection system acts on th...

Embodiment 3

[0047] The Rubiaceae type cyclic peptide RA-V(1) of the present invention is used to detect its inhibitory activity on the expression levels of the downstream target genes CTGF and Cyr61 of the Hippo-YAP pathway in HeLa cells by Western blot technique. The experimental principles, methods and results are as follows:

[0048]Experimental principle: Western blotting, also known as immunoblotting, is an immunochemical technique that can detect proteins immobilized on a solid-phase carrier. Western blotting uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe" is an antibody, and the "color development" is labeled with a secondary antibody. SDS is an anionic surfactant that is a detergent, it can break the intramolecular and intermolecular hydrogen bonds, destroy the secondary and tertiary structure of the protein molecule, and combine with the hydrophobic part of the protein to destroy its folding structure , so that the proteins can be separated i...

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Abstract

The invention provides an application of rubiaceous cyclopeptide RA-V (1) as a Hippo-YAP signal pathway inhibitor, a preparation method of the rubiaceous cyclopeptide and an application of the rubiaceous cyclopeptide to preparation of drugs for treating and preventing YAP abnormal activation related cancers. The rubiaceous cyclopeptide provided by the invention is formed by condensation of peptide bonds of one alpha-D-alanine, one alpha-L-alanine, three substituted N-methyl-alpha-L-tyrosine and one alpha-L-amino acid of other types, and six amino acids are condensed to form an eighteen-membered ring, wherein a benzene ring between two vicinal tyrosines is connected through an oxygen bridge to form a fourteen-membered ring. Compared with the prior art, the preparation method provided by the invention can be used for rapidly separating and purifying the target compound RA-V (1) and has the advantages of high efficiency, strong purposiveness, few separation step, convenience in operation, good controllability and repeatability, recyclable solvent, low cost and suitability for industrial production.

Description

Technical field: [0001] The invention belongs to the technical field of medicines, and in particular relates to a series of Rubiaceae type cyclic peptides used as Hippo-YAP signaling pathway inhibitors, a pharmaceutical composition using it as an active ingredient, its preparation method and its application in cancer treatment and prevention application. Background technique: [0002] Hippo pathway is a new cell signaling pathway discovered in Drosophila in recent years, which plays an important role in organ size regulation, cancer occurrence, tissue regeneration, and stem cell function. The Hippo pathway is highly conserved, and its core consists of four proteins, Hippo (Hpo), Salvador (Sav), Wts and Mats in Drosophila; and Mst1 / 2, Sav1, Lats1 / 2 and Mob in mammals. Among them, Wts is the first member of the Hippo pathway discovered. In the genetic screening experiment of Drosophila, it was found that the mutation of wts gene can lead to excessive growth of tissues. Upstr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/64C07K1/36C07K1/16A61K38/12A61P35/00
Inventor 谭宁华赵斌赵思蒙吉新彦曾广智李佳
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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