建立筛选尼克酰胺磷酸核糖转移酶抑制剂的方法
发布时间：2018-07-14 22:16
【摘要】：目的：建立筛选尼克酰胺磷酸核糖转移酶(NAMPT)抑制剂的方法。 方法：以1,4-二马来酰亚胺基丁烷(1,4-Bismaleimidobutane, BMB)交联G355C/D393C双突变NAMPT,封堵其催化活性中心,以NAMPT野生型及交联型NAMPT蛋白的自发荧光变化(280nm激发,333nm发射),来检测化合物与蛋白的结合,以核磁共振方法体外检测化合物对NAMPT催化作用的影响,以MTT方法检测化合物对细胞活性的影响。 结果：FK8660.1-1.0μM能够浓度依赖的抑制野生型NAMPT自发荧光,但对BMB-NAMPT的自发荧光没有影响。迷迭香酸、洋蓟素、1,3二咖啡奎宁酸0.5-50μM能够浓度依赖的抑制野生型NAMPT和BMB-NAMPT的自发荧光,对两种蛋白自发荧光的抑制率无显著差异。FK866分子比1:1(化合物:NAMPT蛋白)能够显著抑制NAMPT的催化作用,但迷迭香酸、洋蓟素、1,3二咖啡奎宁酸分子比100:1(化合物:NAMPT蛋白)对NAMPT的催化作用无抑制作用。10-7M FK866时间依赖的抑制A549细胞的活性,10-8～10-5M迷迭香酸、洋蓟素、1,3二咖啡奎宁酸对A549的细胞活性无抑制作用,反而在高浓度下对A549细胞的细胞活性有一定的增强作用。 结论：基于NAMPT和BMB-NAMPT蛋白的内源性荧光检测法可以筛选与NAMPT蛋白结合的化合物,并分析化合物是否结合在NAMPT的催化活性部位,迷迭香酸、洋蓟素、1,3二咖啡奎宁酸可结合在NAMPT催化活性位点以外的部位。 目的：建立一种筛选尼克酰胺磷酸核糖转移酶(NAMPT)非酶作用阻断剂的细胞模型。 方法：通过突变,获得酶活作用完全消失的NAMPT蛋白,以荧光光谱法检测无ATP时NAMPT的催化作用；以Real time-PCR方法检测IL-6mRNA的表达,观察各种NAMPT蛋白诱导BV2细胞和THP-1细胞IL-6mRNA表达的作用；获得稳定转染pIL-6-promoter-luc质粒的BV2细胞株,用于IL-6mRNA表达的高通量筛选。 结果：H247A、R311D、R392E、K423E突变均能够显著减弱JAMPT蛋白的催化作用,其中R392E突变使NAMPT的催化作用完全消失。NAMPT蛋白能够诱导BV2细胞和THP-1细胞表达IL-6mRNA,其作用与催化作用的强度呈负相关。基于IL-6报告基因的细胞模型可以用于NAMPT非酶作用的检测。 结论：R392E突变的NAMPT蛋白攻击BV2细胞或THP-1细胞,检测IL-6mRNA的表达水平,可以用于筛选NAMPT非酶作用阻断剂,而稳定表达IL-6报告基因的BV2细胞可以作为高通量筛选的细胞模型。
[Abstract]:Objective: to establish a method for screening nicotinamide phosphate ribonucleosyltransferase (NAMPT) inhibitors. Methods: G355C- / D393C double mutation was used to block the catalytic active sites, and the spontaneous fluorescence changes (280nm excited 333nm emission) of NAMPT proteins were used to detect the binding of the compounds to the proteins, and the catalytic activity sites of NAMPT were blocked by the cross-linked G355C / D393C double mutated NAMPTs (14-Bismaleimidobutane (BMB), and the spontaneous fluorescence changes of NAMPT proteins (excited by 333nm emission) were used to detect the binding of the compounds to the proteins. The effects of the compounds on the catalytic activity of NAMPT were detected by nuclear magnetic resonance (NMR) in vitro, and the effects of the compounds on the cell activity were detected by MTT method. Results: FK8660.1-1.0 渭 M could inhibit wild-type NAMPT autofluorescence in a concentration-dependent manner, but had no effect on BMB-NAMPT autofluorescence. Rosemary acid, artichoidin 1, dicoffee quinic acid 0.5-50 渭 M could inhibit the spontaneous fluorescence of wild type NAMPT and BMB-NAMPT in a concentration-dependent manner. There was no significant difference in the inhibition rate of autofluorescence between the two proteins. FK866 molecule could significantly inhibit the catalytic action of Nmpt compared with 1:1, but rosemary acid, The catalytic effect of 100: 1 (compound: NAMPT protein) on NAMPT was not inhibited. 10-7M FK866 inhibited the activity of A549 cells in a time-dependent manner. The activity of A549 cells was not inhibited by A549, but increased at high concentration. Conclusion: the endogenous fluorescence detection method based on NAMPT and BMB-NAMPT protein can be used to screen the compounds bound to NAMPT protein and analyze whether the compounds are bound to the catalytic active site of NAMPT, rosmarinic acid, or not. Artichokes can bind to a site other than the catalytic activity site of NAMPT. Aim: to establish a cell model for screening nonenzymatic blockers of nicotinamide phosphate ribonucleosyltransferase (NAMPT). Methods: the NAMPT protein whose enzyme activity disappeared completely was obtained by mutagenesis. The catalytic effect of NAMPT without ATP was detected by fluorescence spectrometry, the expression of IL-6 mRNA was detected by Real time-PCR method, and the expression of IL-6 mRNA in BV2 cells and THP-1 cells was observed by various NAMPT proteins. BV2 cell lines transfected stably with pIL-6-promoter-luc plasmid were obtained for high throughput screening of IL-6 mRNA expression. Results the mutation of R311DN R392EK423E significantly attenuated the catalytic effect of JAMPT protein. R392E mutation completely disappeared the catalytic effect of NAMPT. NAMPT protein could induce the expression of IL-6mRNAs in BV2 cells and THP-1 cells, which was negatively correlated with the intensity of catalytic action. The cell model based on IL-6 reporter gene can be used to detect the non-enzyme action of NAMPT. Conclusion the expression level of IL-6 mRNA in BV2 cells or THP-1 cells was assayed by the NAMPT protein of 1: R392E mutation, which could be used to screen non-enzymatic blockers of NAPT, while BV2 cells with stable expression of IL-6 reporter gene could be used as a high throughput screening cell model.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
本文编号：2123130
[Abstract]:Objective: to establish a method for screening nicotinamide phosphate ribonucleosyltransferase (NAMPT) inhibitors. Methods: G355C- / D393C double mutation was used to block the catalytic active sites, and the spontaneous fluorescence changes (280nm excited 333nm emission) of NAMPT proteins were used to detect the binding of the compounds to the proteins, and the catalytic activity sites of NAMPT were blocked by the cross-linked G355C / D393C double mutated NAMPTs (14-Bismaleimidobutane (BMB), and the spontaneous fluorescence changes of NAMPT proteins (excited by 333nm emission) were used to detect the binding of the compounds to the proteins. The effects of the compounds on the catalytic activity of NAMPT were detected by nuclear magnetic resonance (NMR) in vitro, and the effects of the compounds on the cell activity were detected by MTT method. Results: FK8660.1-1.0 渭 M could inhibit wild-type NAMPT autofluorescence in a concentration-dependent manner, but had no effect on BMB-NAMPT autofluorescence. Rosemary acid, artichoidin 1, dicoffee quinic acid 0.5-50 渭 M could inhibit the spontaneous fluorescence of wild type NAMPT and BMB-NAMPT in a concentration-dependent manner. There was no significant difference in the inhibition rate of autofluorescence between the two proteins. FK866 molecule could significantly inhibit the catalytic action of Nmpt compared with 1:1, but rosemary acid, The catalytic effect of 100: 1 (compound: NAMPT protein) on NAMPT was not inhibited. 10-7M FK866 inhibited the activity of A549 cells in a time-dependent manner. The activity of A549 cells was not inhibited by A549, but increased at high concentration. Conclusion: the endogenous fluorescence detection method based on NAMPT and BMB-NAMPT protein can be used to screen the compounds bound to NAMPT protein and analyze whether the compounds are bound to the catalytic active site of NAMPT, rosmarinic acid, or not. Artichokes can bind to a site other than the catalytic activity site of NAMPT. Aim: to establish a cell model for screening nonenzymatic blockers of nicotinamide phosphate ribonucleosyltransferase (NAMPT). Methods: the NAMPT protein whose enzyme activity disappeared completely was obtained by mutagenesis. The catalytic effect of NAMPT without ATP was detected by fluorescence spectrometry, the expression of IL-6 mRNA was detected by Real time-PCR method, and the expression of IL-6 mRNA in BV2 cells and THP-1 cells was observed by various NAMPT proteins. BV2 cell lines transfected stably with pIL-6-promoter-luc plasmid were obtained for high throughput screening of IL-6 mRNA expression. Results the mutation of R311DN R392EK423E significantly attenuated the catalytic effect of JAMPT protein. R392E mutation completely disappeared the catalytic effect of NAMPT. NAMPT protein could induce the expression of IL-6mRNAs in BV2 cells and THP-1 cells, which was negatively correlated with the intensity of catalytic action. The cell model based on IL-6 reporter gene can be used to detect the non-enzyme action of NAMPT. Conclusion the expression level of IL-6 mRNA in BV2 cells or THP-1 cells was assayed by the NAMPT protein of 1: R392E mutation, which could be used to screen non-enzymatic blockers of NAPT, while BV2 cells with stable expression of IL-6 reporter gene could be used as a high throughput screening cell model.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
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1 韩雪;建立筛选尼克酰胺磷酸核糖转移酶抑制剂的方法[D];浙江大学;2014年
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