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Drug target point based on mycobacterium tuberculosis ubiquitin ligase PafA and application thereof

A technology of Mycobacterium tuberculosis and Mycobacterium tuberculosis, which is applied in the field of biomedicine to achieve the effects of growth inhibition, small side effects, and increased success rate

Active Publication Date: 2018-09-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far there is a lack of research on the inhibitors of the Mycobacterium tuberculosis ubiquitin-like ligase PafA and the related inhibitory mechanism

Method used

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  • Drug target point based on mycobacterium tuberculosis ubiquitin ligase PafA and application thereof
  • Drug target point based on mycobacterium tuberculosis ubiquitin ligase PafA and application thereof
  • Drug target point based on mycobacterium tuberculosis ubiquitin ligase PafA and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1. Screening of inhibitors of ubiquitin-like ligation reaction against Mycobacterium tuberculosis ubiquitin-like ligase

[0028] In order to screen for inhibitors against the ubiquitin-like ligation reaction of Mycobacterium tuberculosis ubiquitin-like ligase, a ubiquitin-like ligation reaction system was constructed in vitro to facilitate screening. The specific implementation is as follows:

[0029] 1) The Mycobacterium tuberculosis ubiquitin-like ligase PafA required for the construction of the enzymatic reaction (the amino acid sequence is shown in SEQ ID No. 1), the substrate protein PanB, and the ubiquitin-like protein Pup E. That is, the corresponding fragment was obtained by PCR from the genome of Mycobacterium tuberculosis H37Rv strain (gifted by Wuhan Institute of Virology, Chinese Academy of Sciences), cloned and connected to PET28a, and a Flag tag was introduced into the C-terminus of the substrate protein PanB to facilitate subsequent detection. ...

Embodiment 2

[0041] Example 2. The enzymatic mechanism of the small molecule inhibitor AEBSF inhibiting the enzymatic activity of the ubiquitin-like ligase PafA

[0042] In order to analyze the enzymatic mechanism of the small molecule inhibitor AEBSF inhibiting the ubiquitin-like ligase PafA, taking into account that AEBSF, as a serine protease inhibitor, can be covalently linked to the serine of the enzyme to inhibit the enzyme activity, we put the ubiquitin-like ligase The ubiquitin ligase PafA (0.5μM) was co-incubated with the small molecule AEBSF (1mM), and then identified by mass spectrometry, it was found that the serine 119 position of the ubiquitin ligase was linked to AEBSF, and the results were as follows image 3 shown.

[0043] In order to prove that serine 119 is involved in the enzymatic mechanism of the small molecule inhibitor AEBSF to inhibit the ubiquitin-like ligase PafA, we mutated serine 119 into alanine and phenyl ring family amino acids, and the mutation to alanin...

Embodiment 3

[0044] Example 3. Screening of small molecule inhibitors that non-covalently bind to the serine region at position 119 of PafA

[0045] To screen small-molecule inhibitors that non-covalently bind to the Ser119 region of PafA, we first performed a local sequence alignment of the ubiquitin-like ligases from Mycobacterium tuberculosis and Corynebacterium glutamicum , the result is as Image 6 shown. We found that the sequence of serine 119 was highly conserved in the two strains, and this region happened to be located near the pocket of ubiquitin-like ligase binding ubiquitin-like protein Pup. Because the ubiquitin-like ligase structure currently available is only that of Corynebacterium glutamicum, and it is highly conserved with the ubiquitin-like ligase of Mycobacterium tuberculosis in this region, we take the use of Corynebacterium glutamicum The ubiquitin-like ligase PafA was docked with small molecule compounds, and we obtained three small molecules with the best compu...

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Abstract

The invention provides a drug target point based on mycobacterium tuberculosis ubiquitin ligase PafA and an application thereof, and belongs to the technical field of biomedicine. Specifically, the invention relates to a use of a 119-site serine of ubiquitin ligase PafA as a target point of an anti-tuberculosis drug. The serine site at the 119 site of the protein can specifically bind to a small molecule inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride so that the activity of the mycobacterium tuberculosis ubiquitin ligase PafA is inhibited.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a new drug target based on Mycobacterium tuberculosis ubiquitin-like ligase PafA and its application, in particular to the 119-position serine of the ubiquitin-like ligase PafA as an anti-tuberculosis drug The target also involves 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), an inhibitor that specifically binds to the 119-position serine site of the protein. Background technique [0002] It is imperative to stop tuberculosis: tuberculosis, caused by infection with Mycobacterium tuberculosis, has plagued mankind for 7,000 years. The advent of the BCG vaccine and antibiotics brought tuberculosis under control for a time. However, the emergence of drug-resistant Mycobacterium tuberculosis and co-infection with HIV have led to a resurgence of tuberculosis worldwide. In 1993, WHO declared an unprecedented "global emergency for tuberculosis", and in 1998, it reiterated tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/25C12Q1/04G01N33/53C12R1/32
CPCC12Q1/04C12Q1/25G01N33/53
Inventor 陶生策江河伟
Owner SHANGHAI JIAO TONG UNIV
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