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A Drug Screening Method Based on Fluorescence Correlation Spectroscopy to Detect Protein Aggregation Induced by Organic Solvents

A technology related to organic solvents and fluorescence, applied in the field of biomedicine, can solve the problems of poor curative effect correlation, complex system, and long time consumption, and achieve the effect of less sample consumption, simplified test steps, and high repetition times

Active Publication Date: 2022-05-17
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve the problems of complex system and poor curative effect correlation of traditional in vitro drug screening methods, a drug screening model based on fluorescence correlation spectroscopy to detect protein aggregation induced by organic solvents was established
[0005] It also solves the time-consuming and high-cost problems of the traditional in vitro drug screening method. This method can be used for rapid and low-cost drug screening at the single-molecule level, providing a new method for preclinical anti-tumor drug evaluation

Method used

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  • A Drug Screening Method Based on Fluorescence Correlation Spectroscopy to Detect Protein Aggregation Induced by Organic Solvents
  • A Drug Screening Method Based on Fluorescence Correlation Spectroscopy to Detect Protein Aggregation Induced by Organic Solvents
  • A Drug Screening Method Based on Fluorescence Correlation Spectroscopy to Detect Protein Aggregation Induced by Organic Solvents

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Aggregation of β-amyloid precursor protein cleaving enzyme 1 (BACE-1) in different organic solvents and drug inhibition of aggregation

[0035] Such as figure 1 As shown, when an organic solvent is added to the solution, it will induce aggregation of the fluorescently labeled protein. Add the drug molecule to the fluorescently labeled protein solution, and then add the organic solvent. Due to the interaction between the drug molecule and the protein, the aggregation of the protein will be inhibited. The application of fluorescence correlation spectroscopy can monitor the characteristic diffusion time of proteins in different aggregation states, so as to monitor the strength of interaction between different drug molecules and proteins. Different organic solvents have different effects on protein aggregation. Such as figure 2 As shown, we used five organic solvents methanol, ethanol, acetonitrile, acetone and ethyl acetate, and found that when different or...

Embodiment 2

[0036] Example 2: Application of a Drug Screening Model of Beta-Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) Inhibitor

[0037] We used fluorescence correlation spectroscopy to study the inhibitory effect of different concentrations of the drug AZD3293 on the aggregation of BACE-1 protein in organic solvents, so as to evaluate the strength of the interaction between the drug and BACE-1 protein. By monitoring the characteristic diffusion time of protein molecules labeled with fluorescent dyes, information on the strength of interactions between different drug molecules and protein molecules can be obtained. The concentration of BODIPY-labeled BACE-1 was 20 nM, and the concentration of drug AZD3293 increased from 10 -12 increased to 10 -4 mol / L, the characteristic diffusion time of BACE-1 gradually decreased, indicating that the drug AZD3293 had a strong interaction with BACE-1, effectively inhibiting the aggregation of BACE-1 protein in organic solvents. We further st...

Embodiment 3

[0038] Example 3: A drug screening model for fluorescent protein EGFR-labeled dihydroreductase

[0039] We studied the inhibitor MTX's inhibitory effect on the aggregation of fluorescent protein EGFR-labeled dihydrofolate reductase in organic solvents by fluorescence correlation spectroscopy, so as to evaluate the strength of the drug's interaction with dihydrofolate reductase. We first constructed a plasmid, expressed the fluorescent protein EGFR-tagged dihydrofolate reductase by culturing Escherichia coli, and then purified the protein through a nickel column. We investigated the effect of the concentration of acetonitrile on the aggregation of fluorescent protein EGFR-labeled dihydrofolate reductase with and without drug addition. Such as Figure 6 As shown, when the concentration of acetonitrile is 10%, the characteristic diffusion time difference of the dihydrofolate reductase labeled with fluorescent protein EGFR before and after the action of the drug MTX is the larges...

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Abstract

The invention relates to a drug screening method based on fluorescence correlation spectrum detection of protein aggregation induced by organic solvents. Fluorescence-labeled proteins produce aggregation in aqueous solutions containing organic solvents. When the compound to be screened interacts with the protein, it will inhibit the protein from Aggregation occurs in aqueous solutions containing organic solvents, and the characteristic diffusion time (diffusion coefficient) of fluorescently labeled protein aggregates is detected by fluorescence correlation spectroscopy to obtain information on the strength of the interaction between the protein and the compound to be screened, thereby realizing the induction of protein aggregation based on organic solvents Drug screening, the method has a small detection volume and high sensitivity. The invention can screen clinical drugs for common diseases and sudden diseases, greatly reduce the time and cost of drug screening, and provide a simpler, faster and more universal method for drug research and development.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a drug screening method for detecting protein aggregation induced by organic solvents based on fluorescence correlation spectroscopy. Background technique [0002] The development of new drugs and their pharmacological research are crucial to improving human health. In pharmacological research, drug-target protein interaction analysis is a key step in promoting new drug discovery and developing precision medicine. The therapeutic effect of a drug largely depends on the combination of its target. Currently, various methods for studying drug-target protein interactions have been developed. Such as mass spectrometry, nuclear magnetic resonance, capillary electrophoresis, total internal reflection fluorescence, fluorescence polarization and fluorescence resonance energy transfer, etc. However, it is still a great challenge to develop low-sample, fast and highly sensitive metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/643G01N2021/6417
Inventor 任吉存黄香宜薛彩宁
Owner SHANGHAI JIAOTONG UNIV
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