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Screening the Histidine Kinase (1358) Inhibitors of Streptococcus Suis Serotype 2

Author: HuJin
Tutor: ChenHuanChun；BeiWeiCheng
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Streptococcus suis type 2 Enzyme kinetics analysis Homology modeling Virtual Screening
CLC: S852.611
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract


Bacterial two-component regulatory system (Two-component regulatory system, TCS) the bacterial sensor outside the complex environment, and maintain their own survival and the growth and reproduction of induction and regulation system. 1358hk/rr two-component regulatory system of Streptococcus suis type 2 (S.suis2, SS2) 15 one of TCS is also prevalent in other bacteria. Streptococcus suis type 2 1358hk/rr pneumoniae VicK / R high homology and 81% amino acid sequence identity was found by sequence alignment. The current study found that in Streptococcus pneumoniae in VicK histidine kinase gene deletion found its growth, biofilm formation, the bacteria chain of growth slows down and virulence reduce pneumoniae growth required. Gene deletion mutants of 1358hk/rr TCS screened through a lot of work, but has not been obtained mutant, the speculated 1358hk/rr may also affect suis type 2 growth or survival. Currently, there are homology modeling the histidine kinase YycG against Streptococcus pneumoniae and Staphylococcus epidermidis, and compound library was screened effective inhibitors article, while at home and abroad have not Streptococcus suis type 2 reported. Therefore, we would like to histidine kinase 1358 (1358hk) Streptococcus suis type 2 homology modeling, virtual screening out the targets can inhibit Streptococcus suis type 2 pilot of growth inhibitors. The main contents are as follows: 1. Streptococcus suis type 2 histidine kinase (1358hk) homology modeling and construction of analysis is constructed on the basis of the 1358 three-dimensional structure of the protein in the use of homology modeling method software this with Procheck to verify the reliability of the structure of the model, the results show that the histidine kinase of Streptococcus suis serotype 2 1358 PDB ID 3DGE the Thermotoga maritima X crystal diffraction structure (resolution 2.8?) has a 31% consistency; the mold built after the 1358 The structure can be good template superimposed; Show: modeling of Streptococcus suis type 2 histidine kinase 1358 to achieve the requirements of the model can be used for molecular docking, thereby inhibitor lead compound screening. 2 inhibitors of Streptococcus suis type 2 histidine kinase (1358hk) pilot virtual screening through the use of our choice; FlexX SPECS database preliminary screening of small molecule compounds, scoring again to rate using Xscore program to obtain certain number of compounds; Next 108 compounds through the drug-like analysis of screening; Finally FlexX and experience of artificial selection. Through four different filters in the compound database, we eventually get from the SPECS database of 40 small molecule compounds to be tested, and through the purchase. 3. Streptococcus suis functional domains of the histidine kinase type 2 (1358hk) fragment (1358 '), cloning, expression, purification and identification of Streptococcus suis type 2 histidine kinase 1358 is a transmembrane protein, so we through short its HATPase c intracellular kinase function domain fragment (1358 ') for expression. Streptococcus suis type 2 1358, with the ATP-binding of that part of the DNA sequence amplified by PCR, and the recombinant plasmid of the present experiment is built from pET28a vector. The target protein, recombinant plasmids were transformed into the expression strain BL21 expression by addition of IPTG induction 4h, crushed and expression identified by SDS-PAGE, the purified 1358 'protein with the enzymatic activity of the fluorescent kinase assay kit. Finally, we get to 1358 'of target protein of about 35kDa, the purified target protein can be reacted with ATP. 4 lead compounds inhibit enzyme test analysis through the establishment of enzyme kinetics, initially on the butt virtual screening and in the SPECS small molecule compound libraries purchase access to 40 compounds kinase kit preliminary screening, each compound to inhibit the kinase protein The inhibition rate of the decomposition of ATP; results show that compound 1 in the compound concentration in 500μM / L, inhibition rate reached 66%, the compound concentration down to 100 μm / L of the inhibition rate was 50%, but did not inhibit the growth effect in Streptococcus suis type 2. Other compounds inhibition rate of the compound 1 relative lower, some of the compounds in the reduced concentration inhibition rate but increased, the solubility of these compounds in DMSO is not very good. To arrive at our screening compounds in lower concentrations can inhibit the 1358 'enzyme protein activity, provided the experimental basis for further screening new antibacterial drugs.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria > Cocci
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