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Screening and fishing method for active pharmaceutical ingredients with function of blocking uPA-uPAR interaction

A drug activity, upa-fitc technology, applied in the field of biomedicine, can solve the problems of cumbersome steps, unknown biological effects, unable to explain target agonism or antagonism, etc.

Inactive Publication Date: 2018-06-29
CHINA PHARM UNIV
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Problems solved by technology

However, the shortcomings of these current methods are that the steps are cumbersome, and the proteins used for target fishing often cannot confirm their in vitro activity. The active substances that are fished out are only able to bind to the target protein. The effect is unknown, let alone the agonistic or antagonistic effect on the target, and subsequent verification and evaluation of pharmacological efficacy are still needed

Method used

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  • Screening and fishing method for active pharmaceutical ingredients with function of blocking uPA-uPAR interaction
  • Screening and fishing method for active pharmaceutical ingredients with function of blocking uPA-uPAR interaction
  • Screening and fishing method for active pharmaceutical ingredients with function of blocking uPA-uPAR interaction

Examples

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Embodiment 1

[0032] Such as figure 1 As shown, the application's active ingredient screening and fishing method based on blocking uPA-uPAR interaction comprises the following steps:

[0033] Step 1: Construction of FITC-labeled uPA probe:

[0034] A. Using the FITC Fast Conjugation kit, according to the method provided by the kit, FITC was coupled to human uPA protein to obtain a FITC-labeled uPA ligand probe (uPA-FITC);

[0035] B. Add the labeled uPA-FITC probe to the endogenous uPAR high-expressing cell line MDA-MB-231 (ATCC) or uPAR-deficient cell line HEK-293 (ATCC), and incubate at 37°C in the dark for 30min , washed twice with PBS, and excited at 488nm under a fluorescence microscope, the labeled uPA-FITC probe can specifically bind to uPAR high-expressing cell MDA-MB-231 and distribute on the cell surface, indicating that FITC labeling has no effect on uPA and Binding of uPAR.

[0036] Step 2: Construct the yeast surface display cell line KM71-uPAR that induces the expression of...

Embodiment 2

[0046] Example 2 Screening of active ingredients that block the interaction between uPA and uPAR in the extract of thistle

[0047] The traditional Chinese medicine Daji Cirsium japonicum Fisch. ex DC. The dry above ground part has the effects of cooling blood and stopping bleeding, reducing swelling and astringing sores. In order to verify the feasibility and effectiveness of the screening method provided in Example 1, this example further selected the primary extraction mixture of the traditional Chinese medicine Daji, from which the active ingredients based on blocking the interaction between uPA and uPAR were screened. The specific process is as follows:

[0048] 1) Preparation of the extract mixture of the traditional Chinese medicine thistle: Take 100g of thistle medicinal material, crush it to about 50 mesh, extract it twice with 80% ethanol, and concentrate the extract under reduced pressure to dry powder for storage for later use. Before use, it was dissolved in an ...

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Abstract

The invention discloses a screening and fishing method for active pharmaceutical ingredients with a function of blocking uPA-uPAR interaction. The method comprises steps as follows: constructing an FITC marked uPA probe; constructing a yeast surface display cell line KM71-uPAR for induced expression of uPAR; constructing a traditional Chinese medicine mixture screening system with competitive inhibition for uPA-uPAR combination; constructing a drug lead compound system for fishing targeted uPAR from a traditional Chinese medicine mixture. With adoption of the fishing method for further separating the lead compound, the method has characteristics of high sensitivity and high flux, trace constituents in the traditional Chinese medicine mixture can be obtained massively by fishing, and the drug lead compound with obvious molecular action targets can be screened and obtained in the receptor level with one step.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a method for identifying and separating active components capable of blocking uPA-uPAR interaction from a mixture of traditional Chinese medicines, and obtaining a drug lead compound, in particular to a drug based on blocking uPA-uPAR interaction Active ingredient screening and fishing methods. technical background [0002] Existing studies have shown that traditional Chinese medicine contains a large number of compounds with diverse structures and low toxic and side effects, which is an important source for screening new drug lead compounds. The traditional compound separation-oriented method of screening and isolating active ingredients from traditional Chinese medicine mixtures is often time-consuming and laborious, with a long experimental cycle, and finally needs to be verified at the cell, organ, and animal levels. Especially for the identification and separation of trace active i...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12N15/81C12N15/87
CPCC12N15/815C12N15/87G01N21/6428
Inventor 刘秀峰刘吉华李丽黄雅璇
Owner CHINA PHARM UNIV
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