抗真菌低毒1, 2, 3-三氮唑的构效关系及其与血液蛋白相关的靶标发现

The studys on biological activity, the structure-function relationship and the interaction mechanism of small drug molecules and blood protein are several core problems revealing pharmacokinetics in vivo. The l, 2, 3-triazoles have antibacterial, antiviral, antimicrobial and anti-HIV activity, yet we know very little about the antifungal activity related to the inhibition of CYP51, toxicity related to the inhibition of CYP3A4 and the target discovery related to blood protein. Our previous research indicated that six new synthetic l, 2, 3-triazoles exhibited different inhibitory activity against Staphylococcus albus、Staphylococcus aureus、Bacillus subtilis、Bacillus cereus、Micrococcus tetragenus and Moniliaalbican. And these compounds could interact strongly with two globulins (including human serum albumin and human gammaglobulin) used as model protein. In order to further reveal the structure-activity relationship and mechanism of action of l, 2, 3-triazoles on blood protein, the antifungal activity in vivo and in vitro and toxicity of these compounds related to the different target enzyme will be determined in the project. Several key scientific problems will be solved by applying two-dimensional fluorescence difference gel electrophoresis (2D-DIGE)、isobaric tags for relative and absolute quantization (iTRAQ)、surface plasmon resonance technology based on recombinant PCR site-directed mutagenes、multi-spectroscopy and imaging technology in combination with chemical and bioinformatics techniques. (I) To understand the antifungal mechanism and toxicity of these compounds and reveal the related structure-activity relationship. (II) To clarify on the mechanism of action of blood proteins and find new target proteins that resistance to fungal infection. (III) How to determine binding site on amino acid of these important differentially expressed proteins in order to explore the structural foundation of interaction. This will provide technical support and theoretical foundation for the further research, exploitation and reasonable utility of l, 2, 3-triazole drugs.

药物的生物活性、构效关系及其与血液蛋白的作用机理是揭示其体内动力学的几个核心问题。1,2,3-三氮唑具有抗菌、抗病毒、抗肿瘤及HIV等活性,但对其是否通过抑制CYP51酶的抗真菌、抑制CYP3A4酶的毒理机制及其与血液蛋白相互作用了解很少。我们前期对六种新合成1,2,3-三氮唑的抗典型菌活性及与血液中两种模型蛋白相互作用的研究表明,它们抗菌活性不同,与模型蛋白相互作用较强。为进一步揭示其构效关系及与血液蛋白的作用机理，本项目拟用双向荧光差异凝胶电泳及同位素标记相对和绝对定量技术、重组PCR定点突变的表面等离子共振法等多种策略, 通过靶标酶的体内外抗真菌活性及毒性实验, 了解1)抗真菌活性和毒性机制,揭示与此相关的构效关系;2)其对血液蛋白的作用机制,发现新靶标蛋白;3)明确与相关血液蛋白氨基酸的键合位点,探索相互作用结构基础。为深度开发利用1,2,3-三氮唑类药物提供技术支持及理论依据.

(1)对7种1,2,3-三氮唑化合物（简称：B, C, E, G, J, L及M），将水稻白叶枯病菌、香蕉镰刀枯萎病菌和芒果炭疽菌作为三个实验菌种，通过抑制生长速率法分别测试其毒力直线回归方程和E50值，筛选了七种化合物抗植物病原菌的活性，结果显示它们均有不同程度的抗菌效果。田间试验结果表明，对水稻白叶枯病菌，当化合物浓度为 100ppm，28℃培养48个小时对细菌目测的药剂效果均为100%，相当或优于对照品氯霉素、春雷霉素和噻菌铜；C药剂对芒果炭疽病菌的抑菌活性最高，为1.40，其次是B、G，分别为22.73和32.12；对香蕉弯孢霉叶斑病，J、M、Z的效果最好，25ppm的抑制率均在54%以上。(2)通过7种化合物对KM种小鼠灌胃给药急性毒性试验显示，供试品组动物给予5 g/kg条件下，将毒性反应涉及动物的组织、器官或系统，进行组织病理学检查，结果表明无明显可见的病变，表明它们的毒性很低。(3)利用GaussView5.0量化程序及含时密度泛函 TD-DFT方法对几种化合物进行了结构及理论光谱计算，揭示了它们的光电性质；并建立它们与生物大分子作用的定量结构-性质/活性关系的模型，获得化合物的多个物理化学参数。(4)应用多种光谱结合分子模拟，研究模拟生理条件下不同1，2，3-三氮唑与DNA、重要血液蛋白（人血清蛋白，免疫球蛋白及血红蛋白）的键合作用，表征1，2，3-三氮唑对蛋白二级结构的影响，获得相互作用的结合常数、热力学常数及结合区域，从分子水平阐释它们的作用机制。(5)利用2-DE技术分析了在模拟生理条件下它们和人体血浆蛋白相互作用后的蛋白图谱，通过ImageMaster软件找出差异显示蛋白，再使用MALDI-TOF-MS进行鉴定，共获得139个差异蛋白点，经质谱鉴定及蛋白质数据库搜库确定为43个差异蛋白，它们从属16种类型的蛋白，初步分析了这些差异蛋白的生物功能。