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Drug screening method for detecting organic solvent induced protein aggregation based on fluorescence correlation spectrum

An organic solvent and screening method technology, applied in the field of biomedicine, can solve the problems of poor curative effect correlation, complex system, high cost, etc., and achieve the effect of less sample consumption, simplified test steps, and high repetition times

Active Publication Date: 2021-10-26
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • Drug screening method for detecting organic solvent induced protein aggregation based on fluorescence correlation spectrum
  • Drug screening method for detecting organic solvent induced protein aggregation based on fluorescence correlation spectrum
  • Drug screening method for detecting organic solvent induced protein aggregation based on fluorescence correlation spectrum

Examples

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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 for detecting protein aggregation induced by an organic solvent based on a fluorescence correlation spectrum, fluorescently-labeled protein generates an aggregation phenomenon in an aqueous solution containing the organic solvent, and when a to-be-screened compound interacts with the protein, the aggregation phenomenon of the protein in the aqueous solution containing the organic solvent can be inhibited; and the characteristic diffusion time (diffusion coefficient) of the fluorescently-labeled protein aggregate is detected through a fluorescence correlation spectrum, and the strength information of the interaction between the protein and the compound to be screened is obtained, so that the drug screening based on the organic solvent induced protein aggregation is realized, and the method is small in detection volume and high in sensitivity. Clinical drugs of common diseases and sudden diseases can be screened, the drug screening time is greatly shortened, the drug screening cost is greatly reduced, and the method which is simpler, rapider and high in universality is provided 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 Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/643G01N2021/6417
Inventor 任吉存黄香宜薛彩宁
Owner SHANGHAI JIAO TONG UNIV
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