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Staphylococcus tryptophanyl-tRNA synthetase inhibitor

A technology of staphylococcus and staphylococcus epidermidis, applied in the direction of active ingredients of heterocyclic compounds, antibacterial drugs, etc., can solve the problem of severe infection and easy development of chronic infection

Inactive Publication Date: 2007-12-12
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the frequent use of the above-mentioned medical materials and the abuse of antibiotics, Staphylococcus epidermidis that is resistant to vancomycin and multi-drug resistance has emerged, leading to increasingly serious problems of such nosocomial infections, which can easily develop into chronic infections, and new drugs are urgently needed research and development

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1 Construction of tryptophanyl-tRNA synthetase prokaryotic expression plasmid

[0015] (1) Construction of the prokaryotic expression plasmid of Staphylococcus epidermidis tryptophanyl-tRNA synthetase

[0016] Staphylococcus epidermidis tryptophanyl-tRNA synthetase gene (NC_004461, locus tag: SE0685) acquisition method is as follows:

[0017] Design an Nco I restriction site at the 5' end of the sense strand primer, and an Nhe I restriction site at the 5' end of the antisense strand primer:

[0018] sense:

[0019] 5'-CC CCA TGG AAA CTT TAT TCT CAG GA-3’

[0020] anti-sense:

[0021] 5’-CG GCT AGC TTA TCT TTT TCG ACC TAA GCC C-3’

[0022] The underlined sequence is the restriction site of Nco I, and the sequence in italics is the restriction site of Nhe I.

[0023] The coding gene of Staphylococcus epidermidis tryptophanyl-tRNA synthetase was amplified by PCR using the above primers. Digestion by restriction endonucleases (NcoI and NheI) and T 4 DNA lig...

Embodiment 2

[0031] Example 2 Expression and purification of tryptophanyl-tRNA synthetase protein

[0032] Host bacteria: Escherichia coli BL21-DE3

[0033] 1) Pick a single clone colony and inoculate into 5ml LB medium, 37℃×250rpm to OD 600 =1~2.

[0034] 2) Inoculate 10ul to 5ml LBK medium, add 1M IPTG to a final concentration of 1mM, and induce expression for 4h.

[0035] 3) The bacterial cells were collected by centrifugation, resuspended in PBS, ultrasonically disrupted, centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was obtained. with Invitrogen ProBond TM Purification System purifies protein. The distribution of the target protein was determined by 12% SDS-PAGE protein electrophoresis analysis. Proteins were quantified by the Bradford method.

Embodiment 3

[0036] The influence of embodiment 3 compound FDDDC02 on tryptophanyl-tRNA synthetase activity:

[0037] 1. The effect of compound FDDDC02 on the activity of staphylococcal tryptophanyl-tRNA synthetase and the activity of human tryptophanyl-tRNA synthetase (using two methods for detection):

[0038] 1) Detection of the reduction of the substrate ATP - Kinase-Glo TM Luminescent Kinase Assay Kit (Promega, USA): 2 μg recombinantly expressed and purified tryptophanyl-tRNA synthetase from Staphylococcus epidermidis, 2 μg tryptophanyl-tRNA synthetase from Staphylococcus aureus and 3 μg human tryptophanyl-tRNA synthetase Respectively react with the compound FDDDC02 of gradient dilution at 25°C for 20 minutes, then add 100 μM ATP (total reaction volume 50 μL), and act at 25°C for 20 minutes, add the reaction mixture to a 96-well microtiter plate, 50 μL per well, add Kinase- Glo TM 50 μL of Reagent per well was allowed to stand at room temperature for 10 minutes, and the chemilumin...

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Abstract

The invention belongs to biological technique field, relates to small molecular inhibitor against staphylococci tryptophanyl-tRNA synthetase (WRS), molecular structure of the inhibitor shown in formula I, method of the inhibitor as medicament for preventive or treating relevant bacterial disease. Biochemical and biological experiments have shown that the inhibitor has strong binding force with target protein staphylococcus epidermidis or Staphylococcus aureus tryptophanyl-tRNA synthetase, can inhibit the activity of the synthetase, inhibit the growth of staphylococcus epidermidis Staphylococcus aureus. It has no toxicity to mammalian cell. The inventive inhibitor can be used for preparing medicament for treating disease caused by staphylococci, and preparing into disinfectant liquid.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a small molecule inhibitor acting on staphylococcal tryptophanyl-tRNA synthetase (WRS); the molecular structure of the inhibitor; the inhibitor is used as a pharmaceutical compound to prevent or treat related bacterial infectious diseases Methods. Background technique [0002] Staphylococcus is associated with nosocomial infections, especially Staphylococcus aureus and Staphylococcus epidermidis. Staphylococcus epidermidis has become the most important factor of iatrogenic infection in transplant surgery, and the infection caused by it is related to the indwelling of some polymer medical devices in the body, such as intravenous catheters, pacemakers, cerebrospinal fluid drainage devices, artificial organs, etc. With the frequent use of the above-mentioned medical materials and the abuse of antibiotics, Staphylococcus epidermidis that is resistant to vancomycin and multi-drug r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/47A61P31/04
Inventor 瞿涤蒋华良沈旭吴旸于坤千
Owner FUDAN UNIV
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