Method for screening protease inhibitor

A technology for protease inhibitors and screening methods, applied in the field of screening protease inhibitors, can solve the problems of artificial synthesis of substrates, unfavorable reactions, increase costs, etc., and achieve the effects of small impact, shortening operation time, and saving time.

Inactive Publication Date: 2013-01-16
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the fluorescence method (or spectrophotometry) has been used in the screening of matrix metalloproteinase inhibitors, which has high sensitivity and large-throughput screening, there are still some shortcomings: 1) Due to oversensitivity, artificial false phases are common in experiments In the process, a reasonable control group should be designed; 2) If the inhibitor has fluorescence or can quench the fluorescence, it cannot be used; 3) It needs artificially synthesized substrates, which is expensi...

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  • Method for screening protease inhibitor
  • Method for screening protease inhibitor
  • Method for screening protease inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The preparation of embodiment 1 solution (1)

[0068] 1. Ninhydrin reaction solution

[0069] Solution A: stannous chloride-sodium citrate solution, containing 0.2M sodium citrate, 7mM stannous chloride, pH 5.0.

[0070] Solution B: ninhydrin-dimethyl sulfoxide solution, comprising dissolving ninhydrin in DMSO solution so that the final concentration of ninhydrin is 50.0 g / L.

[0071] Mix solution A and solution B in equal volumes to obtain a ninhydrin reaction solution.

[0072] 2. Reaction buffer

[0073] 50mM Tris-HCl, 150mM NaCl, 3mM CaCl 2 , 1 μM ZnCl 2 ,pH=7.5

[0074] 3. The reaction termination solution is

[0075] 50mM EDTA-Tris-HCl buffer containing 12% polyethylene glycol 6000.

Embodiment 2

[0076] The preparation of embodiment 2 solution (2)

[0077] 1. Ninhydrin reaction solution

[0078] Solution A: stannous chloride-sodium citrate solution, containing 0.1M sodium citrate, 8mM stannous chloride, pH 5.0.

[0079] Solution B: ninhydrin-DMF solution, which is dissolving ninhydrin in N,N-dimethylformamide (DMF) solution, so that the final concentration of ninhydrin is 40.0 g / L.

[0080] Mix solution A and solution B in equal volumes to obtain a ninhydrin reaction solution.

[0081] 2. Reaction buffer

[0082] 50mM Tris-HCl, 100mM NaCl, 1mM CaCl 2 , 1 μM ZnCl 2 ,pH=7.5

[0083] 3. Reaction termination solution

[0084] 25mM EDTA-Tris-HCl buffer containing 18% polyethylene glycol 4000.

Embodiment 3

[0085] The preparation of embodiment 3 solution (3)

[0086] 1. Ninhydrin reaction solution

[0087] Solution A: stannous chloride-sodium citrate solution, containing 0.2M sodium citrate, 8mM stannous chloride, pH 5.0.

[0088] Solution B: ninhydrin-ethylene glycol solution, which is dissolving ninhydrin in ethylene glycol solution so that the final concentration of ninhydrin is 60.0 g / L.

[0089] Mix solution A and solution B in equal volumes to obtain a ninhydrin reaction solution.

[0090] 2. Reaction buffer

[0091] 50mM Tris-HCl, 250mM NaCl, 5mM CaCl 2 , 1 μM ZnCl 2 ,pH=7.5,

[0092] Contains 3% polyethylene glycol 6000

[0093] 3. The reaction termination solution is 100 mM EDTA-Tris-HCl buffer.

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Abstract

The invention provides a method for screening a protease inhibitor. The method comprises the following steps of: (1) adding a substance to be tested in protease solution to be uniformly mixed and meanwhile setting the protease solution in which the substance to be tested is not added as a blank control; (2) adding substrate protein solution into the protease solution to react so as to obtain reaction solution; (3) adding reaction end solution in the reaction solution; (4) adding ninhydrin solution to react; and (5) adding double distilled water, judging a result according to an absorbance value OD570 nm, and ensuring that a sample to be tested is positive if the absorbance of the solution of the matter to be tested is less than that of the blank control. By using the method, the protease inhibitor can be conveniently and quickly screened, a substrate is not required to be manually synthesized, the cost is low, the flexibility is high, and a new approach is provided for finding a medicament for inhibiting tumor invasiveness and metastasis.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for screening protease inhibitors. Background technique [0002] Cancer is one of the major diseases that seriously affects the quality of life and survival of human beings. There is no feasible way for human beings to prevent cancer cells from infiltrating and metastasizing to other tissues and organs. Matrix metalloproteinases (MMP) are one of the main enzymes involved in cancer metastasis, which can degrade the tissues around cancer cells, allowing cancer cells to spread to a larger area or enter the blood circulation to metastasize to other organs. Inhibiting the activity of matrix metalloproteinases can effectively control the metastasis of cancer cells, so screening inhibitors that can inhibit the activity of matrix metalloproteinases from known or newly synthesized compounds is a hot spot in the current anticancer drug research. For this reason, it is necessary to estab...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 李长正张艳芳付云周素凤康丽霞孙芳
Owner XINXIANG MEDICAL UNIV
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