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High-throughput screening method of ALKBH5 small molecule inhibitor

A small-molecule inhibitor and targeted technology, applied in biochemical equipment and methods, material stimulation analysis, microbial measurement/testing, etc., can solve the problems of unclear molecular mechanism and biological function, and facilitate high-throughput screening , High sensitivity, high throughput effect

Inactive Publication Date: 2018-01-09
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above studies have shown that the RNA m 6 A modification affects many biological processes, but its specific molecular mechanism and biological function are still unclear

Method used

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  • High-throughput screening method of ALKBH5 small molecule inhibitor
  • High-throughput screening method of ALKBH5 small molecule inhibitor
  • High-throughput screening method of ALKBH5 small molecule inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0045] 1. Expression and purification of pET-28b-FDH recombinant protein

[0046] Reagents and methods:

[0047] Preparation of kanapenicillin: Weigh 1g of ampicillin, dissolve it in ultrapure water and dilute it to 10ml, filter it with a sterilized 0.22um filter head in an ultra-clean bench, and store it at -20°C;

[0048] Preparation of 100mM IPTG (isopropyl-β-D-thiogalactopyranoside): Weigh 2.383g IPTG, dissolve it with ultrapure water and set the volume to 100ml, and use a sterilized 0.22um filter head in the ultra-clean bench Filter and store at -20°C;

[0049] Preparation of Competent Cells: Insert the expression strain BL21(DE3) into sterilized liquid medium at a rate of 1‰, culture on a shaker at 37°C until logarithmic growth phase, take 1ml of the bacterial solution into a 1.5ml sterilized EP tube , centrifuge at 3000rpm for 5min to collect the bacteria, add 100ul 0.1M sterilized and pre-cooled CaCl to each tube 2 solution, pipetting evenly, and then adding 25ul 50...

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Abstract

The invention relates to the field of medicinal chemistry and in particular discloses a method for screening an ALKBH5 small molecule inhibitor by utilizing an FDH (Familial Dysalbuminemic Hyperthyroxinemia) enzyme-linked fluorescent principle and an Alpha Screen technology. The method comprises the following steps: successfully constructing and carrying out prokaryotic expression and purificationto obtain ALKBH5 recombinant protein with relatively high purity, and synthesizing m6A modified ssRNA (single-stranded Ribonucleic Acid) used as a substrate; detecting the activity of the ALKBH5 by utilizing the FDH enzyme-linked fluorescent principle and the Alpha Screen technology and establishing a small molecule inhibitor screening platform. An FDH enzyme-linked fluorescent reaction system isstable and a utilized reagent is cheap and easy to obtain and has a low background; an Alpha Screen reaction system is stable and interference, caused by a sample, on the system is very small; the Alpha Screen reaction system has low false positives and false negatives, can be used for removing the interference of spontaneous fluorescence of a natural product and has high sensitivity; the miniaturization is easy to realize by the two methods and the two methods have high throughput and are mutually complemented; the screening of the ALKBH5 small molecule inhibitor can be realized.

Description

technical field [0001] The invention relates to the field of medicinal chemistry, in particular to a method for high-throughput screening of ALKBH5 small molecule inhibitors by using the FDH enzyme-linked fluorescence principle and AlphaScreen technology respectively. Background technique [0002] So far, hundreds of post-transcriptional modifications of eukaryotic RNA, such as mRNA, tRNA, rRNA and snRNA, have been discovered, and these modifications have a great impact on the structure and function of RNA. Methylation modification of RNA adenine 6-position N (m 6 A) is the most common and highest methylation modification in eukaryotes. It mainly exists in the sequence coding region (CDS), transcription termination sites and 3'untranslated region (3'UTR) of mRNA ), affecting RNA splicing, nuclear export, translation efficiency and stability, and plays a key role in sperm development, stem cell differentiation, metabolism and tumorigenesis and development. [0003] m in mRN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C12Q1/26G01N21/64
Inventor 刘宏民申丹丹郑一超
Owner ZHENGZHOU UNIV
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