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Small-molecule inhibitor for polymyxin drug resistance proteins and application thereof

A small molecule inhibitor, polymyxin technology, applied in the field of biomedicine, can solve problems such as reducing the efficacy of polymyxin, and achieve the effect of inhibiting protein activity and effectively applying space

Inactive Publication Date: 2018-03-06
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since polymyxin was widely used in animal husbandry and agriculture in the past, there are not many reports of clinical resistance to polymyxin, so polymyxin is considered to be the last line against these "super bacteria" line of defense, however, the presence of MCR-1, especially its highly efficient transfer rate between bacteria, greatly reduces the efficacy of polymyxins

Method used

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  • Small-molecule inhibitor for polymyxin drug resistance proteins and application thereof
  • Small-molecule inhibitor for polymyxin drug resistance proteins and application thereof
  • Small-molecule inhibitor for polymyxin drug resistance proteins and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Detect the inhibitory effect of ethanolamine small molecule inhibitors on MCR-1 positive bacteria, comprising the following steps:

[0024] 1) According to the experimental design, first add different concentrations of polymyxin B (polymyxin B) into the MH (Muller Hinton) agar that has been heated and dissolved, and equilibrated in a water bath at 45-50°C, and then the diluted Small molecule inhibitor ethanolamine (ETA) solutions of different concentrations were added to the above-mentioned agar, mixed evenly and poured into a sterilized plate, the thickness of the agar was 3-4 mm;

[0025] 2) Prepare MCR-1-positive bacteria (wide type, WT) and MCR-1-negative bacteria (nagtive cotrol, NC) bacterial suspensions with concentrations equivalent to 0.5 McFarland standard turbidimetric tubes, and serially dilute to 10% of the mother liquor. -2 , 10 -3 ...10 -7 concentration;

[0026] 3) Inoculate the diluted bacterial solution onto the MH agar plate in step 1) in sequence,...

Embodiment 2

[0029] Detecting the inhibitory effect of small molecule inhibitors on MCR-1 protein activity, comprising the following steps:

[0030] 1) Construction of prokaryotic expression plasmid for MCR-1 protein: the gene sequence of MCR-1 was inserted into pET28a plasmid through NcoI and XhoI restriction sites to construct pET28a-MCR plasmid, which was verified by DNA sequencing; MCR-1 The gene sequence is shown in SEQID NO.1;

[0031] 2) Expression of MCR-1 protein: pass the above pET28a-MCR plasmid through CaCl 2 coli Roseta strain, screened by kanamycin and chloramphenicol, the strains grown on LB (Luria-Bertani) culture plates containing kanamycin and chloramphenicol were inoculated into In the LB liquid medium of kanamycin and chloramphenicol, culture at 37°C, 220rpm to the logarithmic growth phase, then add IPTG (isopropylthiogalactopyranoside) to 0.5mM, and induce at 20°C for 20 hours. Finally, the bacteria were collected by centrifugation;

[0032] 3) Purification of MCR-1...

Embodiment 3

[0038] The inhibitory effect of the MCR-1 inhibition site is detected by the method of determining the polymyxin minimum inhibitory concentration of the mutant strain, comprising the following steps:

[0039] 1) Using the pET28a-MCR plasmid obtained in Example 2, 8 possible MCR-1 inhibitory sites: the 246th glutamic acid (E246), the 285th threonine (T285), the 329th day Paragine (N329), Lysine 333 (K333), Histidine 395 (H395), Aspartic acid 465 (D465), Histidine 466 (H466) and 478 Histidine (H478) was subjected to site-directed mutation and mutated into alanine, respectively.

[0040] 2) Pass the above 8 kinds of mutated pET28a-MCR plasmids through CaCl 2 Transformed into Escherichia coli Roseta strain, screened by kanamycin and chloramphenicol, and verified by DNA sequencing.

[0041] 3) Add different concentrations of polymyxin B (polymyxin B) to the MH (Muller Hinton) agar that has been heated and dissolved, and equilibrated in a water bath at 45-50°C, mix well and pour i...

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Abstract

The invention belongs to the field of biomedicines and in particular relates to a small-molecule inhibitor for polymyxin drug resistance proteins and application thereof. The invention firstly discovers that ethanol amine serving as the small-molecule inhibitor is capable of inhibiting drug tolerance of polymyxin, inhibiting MCR-1 protein activity and inhibiting the activity of phosphotransferasewith a similar structure to the MCR-1. The ethanol amine serving as the small-molecule inhibitor is combined with the polymyxin to be medicated, so that the polymyxin can well achieve effects, and aneffective application space is provided for application of the polymyxin. The small-molecule inhibitor for polymyxin drug resistance proteins has significant application value in the fields of pharmaceuticals and animal breeding and has great significances at nowadays with wide existence of drug tolerance.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a small molecule inhibitor of polymyxin drug-resistant protein and application thereof. Background technique [0002] Polymyxin was discovered in the 1960s. It is a polypeptide antibiotic against Gram-negative bacteria. It was first discovered in the culture medium of Paenibacillus polymyxa. Polymyxin is mainly composed of a cationic polypeptide ring and a lipophilic fatty acid side chain in structure. There are five types of polymyxins, A, B, C, D, and E, and the three types of A, C, and D are more toxic. The toxicity is less, so B and E are used clinically, and B and E have only one amino acid difference, but they have basically the same biological activity and function. The clinical use of polymyxin is not much, and it is precisely because of this that bacteria have not developed enough resistance to it. Today, when carbapenem antibiotic resistance is becoming stronger ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/133A61K38/12A61P31/04A23K20/195
CPCA61K31/133A61K38/12A23K20/195
Inventor 殷雷魏鹏程宋广济
Owner WUHAN UNIV
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