CN102370633A - Bacterial YycG histidine kinase inhibitor - Google Patents
Bacterial YycG histidine kinase inhibitorDownload PDFInfo
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明属生物技术领域，涉及细菌抑制剂，具体涉及一种细菌YycG组氨酸激酶抑制剂，尤其是对革兰氏阳性菌的信号转导系统YycG蛋白的抑制剂。本发明的细菌YycG组氨酸激酶抑制剂，对表皮葡萄球菌信号转导系统中YyCG蛋白具有抑制作用，能抑制革兰氏阳性球菌生长，并抑制葡萄球菌生物膜的形成。The invention belongs to the field of biological technology and relates to a bacterial inhibitor, in particular to a bacterial YycG histidine kinase inhibitor, especially an inhibitor to the signal transduction system YycG protein of Gram-positive bacteria. The bacterial YycG histidine kinase inhibitor of the invention has inhibitory effect on the YyCG protein in the signal transduction system of staphylococcus epidermidis, can inhibit the growth of Gram-positive cocci, and inhibit the formation of staphylococcus biofilm.
背景技术Background technique
对全基因组测序的表皮葡萄球菌ATCC12228和ATCC35984菌株分析，发现其具有16或17对双组分信号转导系统。双组分信号转导系统主要存在于各种原核生物细胞，调控生物生长、毒力等多种生物学功能，而真核细胞中未能发现有同源蛋白。细菌的双组分信号转导系统包括组氨酸蛋白激酶和反应调节蛋白，组氨酸蛋白激酶在受到外界刺激后激活，将信号转递给下游蛋白以调控细菌的相关生物学活性。我们对表皮葡萄球菌双组分信号转导系统中的YycG蛋白（YycG组氨酸蛋白激酶）进行了蛋白质结构三维模拟，并根据此模型，利用对接软件进行虚拟，获得能够抑制表皮葡萄球菌等革兰阳性细菌生长和抑制生物膜形成的小分子抑制物。Analysis of Staphylococcus epidermidis ATCC12228 and ATCC35984 strains with whole-genome sequencing revealed that they have 16 or 17 pairs of two-component signal transduction systems. The two-component signal transduction system mainly exists in various prokaryotic cells, and regulates various biological functions such as biological growth and virulence, while no homologous proteins have been found in eukaryotic cells. The two-component signal transduction system of bacteria includes histidine protein kinase and response regulator protein. Histidine protein kinase is activated after being stimulated by the outside world, and transmits the signal to downstream proteins to regulate the relevant biological activities of bacteria. We performed a three-dimensional simulation of the protein structure of the YycG protein (YycG histidine protein kinase) in the two-component signal transduction system of Staphylococcus epidermidis. Small molecule inhibitors of blue-positive bacterial growth and inhibition of biofilm formation.
S. epidernidis已成为移植手术中发生医源性感染的最主要因素。除此之外，由于抗生素的滥用，出现了更严重的多抗性和抗万古霉素的S. epidernidis。而S. epidernidis产生抗药性的主要原因是附着在医疗器械表面的细菌分泌保护性的生物膜，该生物膜可以阻碍传统的抗生素穿过作用于细菌，发挥杀菌效果。除了医疗器械上的S. epidernidis，感染宿主体内的S. epidernidis分泌的生物膜可以阻碍宿主免疫系统的攻击。因此特异性具有破坏生物膜和杀伤细菌的新型抗生素在发挥杀菌作用上具有更广泛的前景。S. epidernidis has become the most important factor of iatrogenic infection in transplant surgery. In addition, due to the overuse of antibiotics, more severe polyresistant and vancomycin-resistantS. epidernidis emerged. The main reason for the drug resistance ofS. epidernidis is that bacteria attached to the surface of medical devices secrete a protective biofilm, which can prevent traditional antibiotics from passing through and acting on bacteria to exert a bactericidal effect. In addition toS. epidernidis on medical devices, the biofilm secreted byS. epidernidis in infected hosts can hinder the attack of the host immune system. Therefore, novel antibiotics that specifically destroy biofilms and kill bacteria have broader prospects in exerting bactericidal effects.
发明内容Contents of the invention
本发明的目的是提供一种细菌YycG组氨酸激酶抑制剂，尤其是对革兰氏阳性菌的信号转导系统YycG蛋白的抑制剂。本发明所述的革兰氏阳性菌包括临床常见的革兰阳性球菌，如表皮葡萄球菌、金黄色葡萄球菌或化脓性链球菌。The object of the present invention is to provide a bacterial YycG histidine kinase inhibitor, especially an inhibitor to the signal transduction system YycG protein of Gram-positive bacteria. Gram-positive bacteria described in the present invention include common clinical Gram-positive cocci, such as Staphylococcus epidermidis, Staphylococcus aureus or Streptococcus pyogenes.
本发明的细菌YycG组氨酸激酶抑制剂，其特征在于，由式（Ⅰ）结构的小分子化合物4-甲基-3-（5-（2-（萘并[2,1-b]呋喃）乙酰胺基）氨基亚甲基-2-呋喃基）苯乙酸（简称FKK051103-51）和0.1- 2%的DMSO（二甲亚砜）组成。The bacterial YycG histidine kinase inhibitor of the present invention is characterized in that the small molecule compound 4-methyl-3-(5-(2-(naphtho[2,1-b]furan ) acetamido) aminomethylene-2-furyl) phenylacetic acid (referred to as FKK051103-51) and 0.1-2% DMSO (dimethyl sulfoxide).
（Ⅰ）(I)
本发明的小分子化合物FKK051103-51具有破坏生物膜和杀伤细菌的双重功能，能有效抑制几种临床常见的革兰阳性球菌生长和抑制表皮葡萄球菌生物膜的形成，且对哺乳动物细胞无明显毒性。。The small molecular compoundFKK051103-51 of the present invention has dual functions of destroying biofilm and killing bacteria, can effectively inhibit the growth of several common clinical Gram-positive cocci and inhibit the formation of Staphylococcus epidermidis biofilm, and has no obvious effect on mammalian cells. toxicity. .
本发明的小分子化合物FKK051103-51可以通过市购（荷兰SPECS）获得。The small molecular compoundFKK051103-51 of the present invention can be obtained from the market (SPECS, Netherlands).
本发明采用最小抑菌浓度试验、微量细菌生物膜检测试验、酶活抑制试验、MTT试验等方法，检测了所述小分子化合物的抑菌活性、抗生物膜活性、酶抑制活性以及细胞毒性。The present invention uses methods such as minimum inhibitory concentration test, trace bacterial biofilm detection test, enzyme activity inhibition test, MTT test, etc. to detect the antibacterial activity, anti-biofilm activity, enzyme inhibitory activity and cytotoxicity of the small molecule compound.
用美国的NCCLS（the National Committee for Clinical Laboratory Standards）推荐的标准试管稀释法检测小分子化合物的最小抑菌浓度（MIC）。结果表明，该小分子化合物能明显抑制几种临床常见的革兰阳性球菌生长(表皮葡萄球菌、金黄色葡萄球菌、化脓性链球菌)，如表1所示。The minimum inhibitory concentration (MIC) of small molecular compounds was detected by the standard tube dilution method recommended by NCCLS (the National Committee for Clinical Laboratory Standards) in the United States. The results showed that the small molecular compound could significantly inhibit the growth of several common clinical Gram-positive cocci (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes), as shown in Table 1.
采用微量细菌生物膜检测试验（96孔板生物膜形成试验）进行抗生物膜活性检测，用表皮葡萄球菌生物膜形成阳性株ATCC35984作为测试菌株（购于美国模式培养集存库，American type culture collection），将小分子化合物接与细菌混合（终浓度200μM），接种于96孔板上，培养后根据细菌在孔内形成生物膜的强弱（结晶紫染色判断）。结果显示，该小分子化合物能明显抑制表皮葡萄球菌生物膜的形成，与不加化合物的对照组相比降低生物膜形成30％以上，如表2所示。The anti-biofilm activity was detected by the micro-bacteria biofilm detection test (96-well plate biofilm formation test), and the biofilm-forming positive strain ATCC35984 of Staphylococcus epidermidis was used as the test strain (purchased from the American type culture collection bank, American type culture collection ), the small molecule compound was mixed with bacteria (final concentration 200 μM), inoculated on a 96-well plate, and cultured according to the strength of the bacteria forming a biofilm in the well (judged by crystal violet staining). The results showed that the small molecular compound could significantly inhibit the formation of Staphylococcus epidermidis biofilm, and compared with the control group without compound, the biofilm formation was reduced by more than 30%, as shown in Table 2.
用MTT法检测小分子化合物对哺乳动物细胞的毒性作用，结果说明该小分子化合物对Vero细胞生长的半数抑制率在200μM以上，在最小抑菌浓度该小分子化合物对Vero细胞的生长抑制率为2％，基本上无毒性，如表3所示。The toxicity of the small molecular compound to mammalian cells was detected by the MTT method, and the results showed that the half inhibitory rate of the small molecular compound on the growth of Vero cells was above 200 μM, and the growth inhibitory rate of the small molecular compound on the Vero cells at the minimum inhibitory concentration was 2%, basically non-toxic, as shown in Table 3.
用96孔板法检测小分子化合物对人红细胞的溶血作用，结果说明该小分子化合物与目的蛋白表皮葡萄球菌组氨酸激酶YycG保守功能域片段有很强的结合力（结合平衡常数KD＜10-5M，且能明显抑制蛋白组氨酸激酶YycG的自身磷酸化活性（半数抑制浓度＜50μM。The hemolysis effect of the small molecular compound on human erythrocytes was detected by the 96-well plate method, and the results showed that the small molecular compound had a strong binding force to the fragment of the conserved functional domain of the target protein Staphylococcus epidermidis histidine kinase YycG (binding equilibrium constant KD < 10-5 M, and can significantly inhibit the autophosphorylation activity of protein histidine kinase YycG (half inhibitory concentration <50μM.
本发明具有如下优点：The present invention has the following advantages:
1．小分子化合物FKK051103-51能明显抑制表皮葡萄球菌生物膜的形成，同时能显著抑制几种临床常见的革兰阳性球菌的生长。1. The small molecule compoundFKK051103-51 can significantly inhibit the formation of Staphylococcus epidermidis biofilm, and can significantly inhibit the growth of several clinically common Gram-positive cocci.
2．该小分子化合物具有破坏生物膜和杀伤细菌的双重功能。2. The small molecular compound has dual functions of destroying biofilm and killing bacteria.
3．该小分子化合物在最小抑菌浓度时对哺乳动物细胞基本无毒性，且对人红细胞无明显的溶血作用。3. The small molecule compound has basically no toxicity to mammalian cells at the minimum inhibitory concentration, and has no obvious hemolysis effect on human red blood cells.
4.该小分子化合物与目的蛋白表皮葡萄球菌组氨酸激酶YycG保守功能域片段有很强的结合力，且能明显抑制蛋白组氨酸激酶YycG的自身磷酸化活性。4. The small molecular compound has a strong binding force to the conserved functional domain fragment of the target protein Staphylococcus epidermidis histidine kinase YycG, and can significantly inhibit the autophosphorylation activity of the protein histidine kinase YycG.
本发明所述的小分子化合物可以制备细菌YycG组氨酸激酶抑制剂。The small molecular compound of the invention can be used to prepare bacterial YycG histidine kinase inhibitors.
本发明所述的小分子化合物可以制备医疗器械或医疗器具消毒液。The small molecular compound of the present invention can be used to prepare medical equipment or medical equipment disinfectant.
本发明所述的小分子化合物可进一步进行化学结构的改造，制备抗革兰阳性球菌感染疾病的新药物。The small molecular compound of the present invention can be further modified in chemical structure to prepare new drugs against Gram-positive coccus infection diseases.
附图说明Description of drawings
图1 是小分子化合物FKK051103-51对人红细胞的溶血作用结果，Figure 1 is the result of the hemolysis of the small molecule compound FKK051103-51 on human red blood cells,
其中，以1％DMSO＋红细胞为阴性对照，1％细胞穿透液Triton-100＋细胞为阳性对照；小分子化合物的MIC值为4μg/ml；Tet：四环素，参考文献报道其MIC值取0.3μg/ml；Cip：环丙沙星，参考文献报道其MIC值取0.25μg/ml；Among them, 1% DMSO + red blood cells were used as negative control, and 1% cell penetration solution Triton-100 + cells were used as positive control; the MIC value of small molecule compounds was 4 μg/ml; Tet: tetracycline, its MIC value was 0.3 μg/ml as reported in the literature. ml; Cip: ciprofloxacin, the MIC value reported in the literature is 0.25 μg/ml;
图1显示了该小分子化合物在最小抑菌浓度（MIC）和4倍MIC浓度时，对人红细胞没有明显的溶血作用。Figure 1 shows that the small molecule compound has no obvious hemolytic effect on human red blood cells at the minimum inhibitory concentration (MIC) and 4 times the MIC concentration.
具体实施方式Detailed ways
下面结合实验例对本发明进一步详细描述但不限制本发明。The present invention will be further described in detail below in conjunction with experimental examples, but the present invention is not limited.
实施例1 FKK051087-51的最小抑菌浓度检测The minimum inhibitory concentration detection of
用美国的NCCLS（the National Committee for Clinical Laboratory Standards）推荐的标准试管稀释法检测小分子化合物的最小抑菌浓度（MIC），实验方法如下：Use the standard tube dilution method recommended by NCCLS (the National Committee for Clinical Laboratory Standards) in the United States to detect the minimum inhibitory concentration (MIC) of small molecule compounds. The experimental method is as follows:
1.细菌接种于新鲜的MH培养基，37℃培养过夜。1. Bacteria were inoculated in fresh MH medium and cultured overnight at 37°C.
2.液1:100接种于新鲜的MH培养基，37℃培养至对数生长早期，用新鲜的MH培养基稀释至OD600＝，每管1ml，加入不同浓度梯度的小分子化合物（溶剂DMSO终浓度保持1％），37℃培养18小时。因小分子化合物溶解于DMSO中，因此设1％DMSO＋细菌为对照，以无菌培养基为空白对照。2. Liquid 1:100 was inoculated in fresh MH medium, cultured at 37°C until early logarithmic growth, diluted with fresh MH medium to OD600 =, 1ml per tube, added different concentration gradients of small molecule compounds (solvent DMSO The final concentration was maintained at 1%) and incubated at 37°C for 18 hours. Since small molecule compounds are dissolved in DMSO, 1% DMSO + bacteria was used as the control, and sterile medium was used as the blank control.
3.与空白对照比较，细菌不生长的浓度最低的一管即为该小分子化合物的最小抑菌浓度。3. Compared with the blank control, the tube with the lowest concentration where bacteria do not grow is the minimum inhibitory concentration of the small molecule compound.
结果显示该小分子化合物对几种临床常见的革兰阳性球菌(表皮葡萄球菌、金黄色葡萄球菌、化脓性链球菌)生长有明显的抑制作用，如表1所示。The results showed that the small molecular compound had obvious inhibitory effect on the growth of several common clinical Gram-positive cocci (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes), as shown in Table 1.
表1 小分子化合物FKK051103-51对几种临床常见的革兰阳性球菌生长的抑制作用Table 1 Inhibitory effect of the small molecule compound FKK051103-51 on the growth of several common clinical Gram-positive cocci
实施例2 FKK051087-51抑制表皮葡萄球菌生物膜的形成Example 2 FKK051087-51 inhibits the formation of Staphylococcus epidermidis biofilm
采用微量细菌生物膜检测试验（96孔板生物膜形成试验），将表皮葡萄球菌生物膜形成阳性株ATCC35984与小分子化合物混合（终浓度200μM），接种于96孔板上，同时以表皮葡萄球菌生物膜形成阴性株ATCC12228为阴性对照，37℃培养20小时后，细菌在孔内形成生物膜可用结晶紫染色法检测，结晶紫染色水洗后，根据OD570读数判断细菌生物膜形成情况的强弱。实验方法如下：Using trace bacterial biofilm detection test (96-well plate biofilm formation test), the Staphylococcus epidermidis biofilm-forming positive strain ATCC35984 was mixed with a small molecule compound (final concentration 200 μM), inoculated on a 96-well plate, and Staphylococcus epidermidis The biofilm-forming negative strain ATCC12228 was used as a negative control. After 20 hours of incubation at 37°C, the formation of biofilm in the wells could be detected by crystal violet staining. After washing with crystal violet staining, the strength of bacterial biofilm formation was judged according to the OD570 reading. . The experimental method is as follows:
1.表皮葡萄球菌接种于新鲜的TSB培养基中，37℃培养过夜。1. Staphylococcus epidermidis was inoculated in fresh TSB medium and cultured overnight at 37°C.
2. 菌1:200用新鲜的TSB培养基稀释接种于96孔板，每孔200μl，37℃静置培养6小时后，加入小分子化合物（终浓度200μM，溶剂DMSO终浓度为1％），每个样品采用三复孔上样。因小分子化合物溶解于DMSO中，因此设1％DMSO＋表皮葡萄球菌生物膜阳性株为阳性对照，设1％DMSO＋表皮葡萄球菌生物膜阴性株为阴性对照。2. Bacteria 1:200 were diluted with fresh TSB medium and inoculated in a 96-well plate, 200 μl per well, and incubated at 37°C for 6 hours, then added small molecule compounds (final concentration 200 μM, solvent DMSO
3.菌液，用PBS洗板3次，晾干，每孔加200μl 2％结晶紫室温染色5分钟。3. For bacterial solution, wash the plate 3 times with PBS, dry it in the air, add 200 μl 2% crystal violet to each well and stain for 5 minutes at room temperature.
4.多余染液，将板晾干，酶标仪OD570读数，每个样品读数取三复孔的均值。4. For excess dye solution, dry the plate, read OD570 on a microplate reader, and take the average value of triplicate wells for each sample reading.
5.小分子化合物对表皮葡萄球菌生物膜的抑制率：5. Inhibition rate of small molecule compounds on Staphylococcus epidermidis biofilm:
阳性对照OD570均值－样品OD570均值Positive control OD570 average - sample OD570 average
阳性对照OD570均值Positive control OD570 mean
结果说明该小分子化合物在体外能明显抑制表皮葡萄球菌生物膜的形成，与不加化合物的阳性对照相比，能降低生物膜形成30％以上,如表2所示。The results show that the small molecular compound can significantly inhibit the formation of Staphylococcus epidermidis biofilm in vitro, and can reduce the formation of biofilm by more than 30% compared with the positive control without compound, as shown in Table 2.
表2 小分子化合物FKK051103-51对表皮葡萄球菌生物膜形成的影响Table 2 Effect of small molecule compound FKK051103-51 on biofilm formation of Staphylococcus epidermidis
a阳性对照指1％DMSO＋表皮葡萄球菌生物膜形成阳性株a Positive control refers to 1% DMSO + Staphylococcus epidermidis biofilm-forming positive strain
b阴性对照指1％DMSO＋表皮葡萄球菌生物膜形成阴性株b Negative control refers to 1% DMSO + Staphylococcus epidermidis biofilm negative strain
实施例3 FKK051087-51的细胞毒性检测Example 3 Cytotoxicity detection of FKK051087-51
用MTT法检测小分子化合物对哺乳动物细胞的毒性作用，实验方法如下：The MTT method is used to detect the toxic effect of small molecular compounds on mammalian cells, and the experimental method is as follows:
1.新鲜培养的Vero细胞接种于96孔板，每孔100ul细胞（约5×104细胞），37℃，5％CO2条件下培养24小时，使细胞长成单层。1. Freshly cultured Vero cells were inoculated in a 96-well plate, 100ul cells per well (about 5×104 cells), and cultured at 37°C and 5% CO2 for 24 hours to make the cells grow into a monolayer.
2.去培养基，加入100ul／每孔新鲜的MEM培养基，其中含有不同浓度的小分子化合物（溶剂DMSO终浓度保持1％），每个样品采用四复孔上样，37℃，5％CO2条件下继续培养24小时。因小分子化合物溶解于DMSO中，因此设1％DMSO＋细胞为对照。2. Remove the medium, add 100ul/well of fresh MEM medium, which contains different concentrations of small molecule compounds (the final concentration of solvent DMSO is kept at 1%), each sample is loaded in quadruple wells, 37°C, 5% Culture was continued for 24 hours under CO2 conditions. Since the small molecule compounds were dissolved in DMSO, 1% DMSO + cells were used as the control.
3.加入10ul MTT标记物，37℃，5％CO2条件下培养4小时。3. Add 10ul of MTT markers and culture for 4 hours at 37°C and 5% CO2 .
4.6孔板取出读取OD570值，每个样品读数取四复孔的均值，计算不同浓度的小分子化合物对Vero细胞生长的抑制率：4. Take out the 6-well plate and read the OD570 value, take the average value of the quadruple wells for each sample reading, and calculate the inhibition rate of small molecule compounds at different concentrations on Vero cell growth:
对照OD570均值－样品OD570均值Control OD570 average - sample OD570 average
对照OD570均值Control OD570 mean
半数抑制量IC50值采用Logit法计算。The half inhibitory dose IC50 value was calculated by Logit method.
结果说明在细胞水平，该小分子化合物的半数抑制率在200μM以上，在最小抑菌浓度时对Vero细胞的生长抑制率为1％，基本没有毒性，如表3所示。The results show that at the cellular level, the half inhibitory rate of the small molecule compound is above 200 μM, and the growth inhibitory rate to Vero cells is 1% at the minimum inhibitory concentration, and there is basically no toxicity, as shown in Table 3.
表3 小分子化合物FKK051103-51对Vero细胞的毒性作用Table 3 Toxic effect of small molecule compound FKK051103-51 on Vero cells
a括号中为小分子化合物FKK051103-51对表皮葡萄球菌的最小抑菌浓度a The minimum inhibitory concentration of the small molecule compound FKK051103-51 to Staphylococcus epidermidis in brackets
实施例4 FKK051087-51与目的蛋白结合及抑制磷酸化Example 4 FKK051087-51 binds to target protein and inhibits phosphorylation
利用能量共振生物传感芯片检测FKK051087-52与表皮葡萄球菌组氨酸激酶YycG保守功能域片段的结合力；用Kinase-GloTM Luminescent Kinase Assay Kit检测酶活及FKK051087-52抑制酶活性的作用，实验方法如下：The energy resonance biosensor chip was used to detect the binding force between FKK051087-52 and the conserved functional domain fragment of Staphylococcus epidermidis histidine kinase YycG; the Kinase-GloTM Luminescent Kinase Assay Kit was used to detect the enzyme activity and the inhibitory effect of FKK051087-52 on enzyme activity, The experimental method is as follows:
1．将重组表达、纯化的目的蛋白表皮葡萄球菌组氨酸激酶YycG保守功能域片段偶联在能量共振生物传感芯片（Biacore CM5型芯片）上，将待测的小分子化合物按一定的梯度稀释，让其流过芯片与蛋白相互作用，观察小分子化合物与蛋白结合的情况，计算出结合平衡常数KD。1. The recombinantly expressed and purified fragment of the conserved functional domain of Staphylococcus epidermidis histidine kinase YycG of the target protein was coupled to an energy resonance biosensing chip (Biacore CM5 type chip), and the small molecular compound to be tested was diluted in a certain gradient. Let it flow through the chip to interact with the protein, observe the combination of the small molecular compound and the protein, and calculate the binding equilibrium constant KD .
2．小分子化合物抑制组氨酸激酶YycG自身磷酸化的实验用美国PROMEGA公司的Kinase-GloTM Luminescent Kinase Assay Kit (Cat. No.V6711, Promega, Madison, USA)完成。将4μg重组表达、纯化的目的蛋白组氨酸激酶YycG保守功能域片段与梯度稀释的小分子化合物在25℃作用20分钟，然后加入100μM ATP（反应总体积50ul）在25℃作用20分钟后将反应混合物加入96孔酶标板，每孔50ul，加入Kinase-GloTM Reagent每孔50 ul室温静置10分钟，读取化学发光值表示反应剩余的ATP的量，实验设不加小分子化合物组为对照组。最后按下式计算小分子化合物对目的蛋白磷酸化的抑制率：2. The experiment of small molecule compounds inhibiting the autophosphorylation of histidine kinase YycG was completed with Kinase-GloTM Luminescent Kinase Assay Kit (Cat. No.V6711, Promega, Madison, USA) from PROMEGA, USA. React 4 μg recombinantly expressed and purified target protein histidine kinase YycG conserved functional domain fragment with serially diluted small molecule compounds at 25°C for 20 minutes, then add 100 μM ATP (total reaction volume 50ul) and react at 25°C for 20 minutes. Add the reaction mixture to a 96-well ELISA plate, 50 ul per well, add Kinase-GloTM Reagent 50 ul per well, let it stand at room temperature for 10 minutes, read the chemiluminescence value to indicate the amount of ATP remaining in the reaction, the experiment is set without adding small molecule compound group for the control group. Finally, the inhibition rate of the small molecule compound on the phosphorylation of the target protein was calculated according to the following formula:
（蛋白＋小分子＋Reagent）组化学发光均值－（蛋白＋Reagent）组化学发光均值(Protein + small molecule + Reagent) group chemiluminescence average - (protein + Reagent) group chemiluminescence average
×100％ ×100%
（Reagent）组化学发光均值－（蛋白＋Reagent）化学发光均值(Reagent) group chemiluminescence mean - (protein + Reagent) chemiluminescence mean
半数抑制浓度IC50值采用Logit法计算。The half inhibitory concentration IC50 value was calculated by Logit method.
结果说明该小分子化合物与目的蛋白表皮葡萄球菌组氨酸激酶YycG保守功能域片段有很强的结合力（结合平衡常数KD＜10-5M），且能明显抑制蛋白组氨酸激酶YycG的自身磷酸化活性（半数抑制浓度＜50μM，如表4所示。The results show that the small molecular compound has a strong binding force to the conserved functional domain fragment of the target protein Staphylococcus epidermidis histidine kinase YycG (binding equilibrium constant KD < 10-5 M), and can significantly inhibit the protein histidine kinase YycG The autophosphorylation activity (half inhibitory concentration <50 μM, as shown in Table 4.
表4. FKK051103-51与目的蛋白的结合力及抑制磷酸化的作用Table 4. The binding ability of FKK051103-51 to the target protein and the effect of inhibiting phosphorylation
a KD值为结合平衡常数，代表小分子化合物与目的蛋白表皮葡萄球菌组氨酸激酶YycG保守功能域片段结合力，其值越小表示结合力越强a KD value is the binding equilibrium constant, which represents the binding force between the small molecule compound and the conserved functional domain fragment of Staphylococcus epidermidis epidermidis histidine kinase YycG. The smaller the value, the stronger the binding force
b IC50 值即半数抑制浓度，代表小分子化合物抑制反应体系中半数组氨酸激酶YycG保守功能域片段自身磷酸化时的浓度。b The IC50 value is the half inhibitory concentration, which represents the concentration when the small molecule compound inhibits the self-phosphorylation of the conserved functional domain fragment of cysteine kinase YycG in the reaction system.
the
实施例5 FKK051087-51对人红细胞的溶血作用Example 5 Hemolysis of FKK051087-51 on human erythrocytes
用96孔板法检测小分子化合物对人红细胞的溶血作用，具体试验过程如下：The 96-well plate method was used to detect the hemolysis of small molecule compounds on human red blood cells, and the specific test process was as follows:
1.分离的健康人红细胞用无菌生理盐水洗3遍，并稀释至5％。1. The isolated healthy human erythrocytes were washed 3 times with sterile saline and diluted to 5%.
2.不同浓度的小分子化合物（溶剂DMSO终浓度保持1％）于5％红细胞悬液中，每孔200ul接种于96孔板上，每个样品采用三复孔。因小分子化合物溶解于DMSO中，因此设1％DMSO＋细胞为阴性对照，设1％细胞穿透液Triton-100＋细胞为阳性对照，并设两种常用抗生素四环素和环丙沙星作对照。2. Different concentrations of small molecule compounds (the final concentration of the solvent DMSO is maintained at 1%) are inoculated in 5% red blood cell suspension, 200ul per well is seeded on a 96-well plate, and each sample is used in triplicate wells. Since the small molecule compounds were dissolved in DMSO, 1% DMSO + cells were set as negative control, 1% cell penetration solution Triton-100 + cells were set as positive control, and two commonly used antibiotics tetracycline and ciprofloxacin were set as controls.
3.置37℃培养箱培养1小时，离心后取100ul上清移入另一块干净的96孔板，OD570读数，每个样品读数取三复孔的均值。3. Place in a 37°C incubator and incubate for 1 hour. After centrifugation, take 100ul of the supernatant and transfer it to another clean 96-well plate, read the OD570 , and take the average value of triplicate wells for each sample reading.
结果说明与阴性对照、阳性对照及两种常用抗生素对照相比，该小分子化合物在最小抑菌浓度（MIC）和4倍MIC浓度时，对人红细胞没有明显溶血作用，如图1所示。The results showed that compared with the negative control, positive control and two commonly used antibiotic controls, the small molecule compound had no obvious hemolytic effect on human red blood cells at the minimum inhibitory concentration (MIC) and 4 times the MIC concentration, as shown in Figure 1.
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