【作者】 费燕；

【导师】 范国荣；

【作者基本信息】 第二军医大学 ， 药物分析学， 2008， 硕士

【摘要】 蛋白质、脱氧核糖核酸（DNA）是组成生命重要的生物大分子,具有各自的生物功能,对一切生命现象都起着至关重要的作用。在分子水平上阐明这些生物大分子与小分子、离子,特别是药物分子的相互作用,是当前生命科学、临床医学、药学及化学等众多领域的重要研究课题,引起了许多学者的关注。建立药物-生物大分子结合的体外模型,了解结合的模式、程度、结合部位、结合力等问题,不仅有助于在分子层次的水平上认识生物大分子与小分子的作用机理和规律,了解大分子结构与功能的关系,为生命科学研究提供有用的信息和数据,而且有助于认识药物药效作用的药理、毒物毒性作用的毒理之微观机制,为药物分子的设计与筛选及新药的开发提供有益的启发与重要指导,其中以蛋白质为作用靶和以核酸为作用靶的药物设计正是目前药物化学和生命科学的前沿研究课题。此外,这方面研究的许多成果还应用于临床医学检验和生命科学中的生物大分子分离检测。光谱法尤其是荧光光谱法是研究生物大分子与小分子、离子相互作用的重要手段。荧光测试中的发射峰特征、能量转移（即发生了荧光的加强或猝灭）等指标可以对分子中荧光生色基团的结构及其所处的微环境提供有用的信息。在生物大分子与有机小分子作用机理的研究中可以引入外源性荧光作为指示探针,也可利用大分子自身的内源性荧光作为探针（蛋白质中色氨酸、酪氨酸和苯丙氨酸的存在,使其具有内源荧光）。紫外-可见光谱法也是研究药物分子与生物分子相互作用机理常用的技术,因为有些药物分子与生物大分子结合后,它们的吸收光谱会有一定的改变,出现吸收峰位移或谱峰宽度变化。蛋白质中的某些芳香氨基酸如色氨酸、苯丙氨酸等在紫外区存在吸收峰,当所处的环境发生变化时,其紫外吸收光谱随之发生变化:吸收峰红移或者蓝移,吸光度及谱带亦有变化。DNA分子由于含有碱基生色团的双螺旋结构,在260nm附近处有强吸收,可根据相互作用前后DNA或其靶向分子的吸收谱带的变化对二者相互作用模式进行判断。因此,利用光谱方法通过蛋白质和DNA分子结合药物分子后结构上的变化来研究结合机理,是非常有效、简便且应用最广泛的方法。喹诺酮类药物以其高效、广谱、服用方便等优点在临床上发挥了越来越重要的作用。但是,因为仍存在一系列缺点,有关其药效作用机理、毒副作用等方面还需不断深入研究,开发高活性、低副作用的新型喹诺酮类药物也一直是研究热点。西那沙星（sinafloxacin）是我国自主研发的第四代喹诺酮类抗感染新药,其作用机理是通过抑制细菌DNA促旋酶和Ⅳ的活性,从而抑制细菌DNA的合成,导致细菌死亡。母核的7位具有氮杂螺环结构,扩大了抗菌谱、增强了抗菌活性,尤其对盐酸环丙沙星耐药金葡球菌、表葡球菌、大肠埃希菌等抗菌活力强,且细胞毒性显著低于同类产品中的加替沙星和左氧氟沙星,8位上的甲氧基能提高其光稳定性,减少光解产物带来的不良反应,为临床抗感染治疗提供了新的选择。为了探讨西那沙星的生物体内作用机制,本论文研究了西那沙星与生物大分子的相互作用,主要包括以下两大部分:第一部分以牛血清白蛋白（BSA）为研究对象,利用西那沙星对蛋白质分子的荧光具有猝灭作用这个特征,用荧光光谱和紫外光谱相结合的方法对BSA与西那沙星相互作用的机理进行了探讨。西那沙星能使BSA的内源荧光产生规律性的猝灭,根据Stern-Volmer方程,西那沙星对BSA的猝灭速率常数Kq远大于各类猝灭剂对于生物大分子的最大扩散碰撞猝灭速率常数2.0×10¹⁰L·mol·s^-1,且随温度升高,Stern-Volmer猝灭常数K_sv减小;紫外光谱亦显示BSA吸收峰发生了明显的红移（△λ=8nm）,因此其荧光猝灭机制属于因复合物的形成而引起的静态猝灭。利用荧光猝灭Lineweaver-Burk双倒数方程计算了不同温度与pH条件下西那沙星与BSA之间的结合常数K_D,基于F（?）rster非辐射能量转移理论分别将一定浓度BSA的荧光光谱数据和与BSA等物质的量的西那沙星的紫外吸收光谱数据输入Matlab软件,测定计算了两者之间的结合距离r和能量转移效率E。根据热力学参数由于△G＜0,结合反应可以自发进行,△S均为正值、△H均为负值确定了西那沙星与牛血清白蛋白之间的主要作用力类型为静电作用力,采用同步荧光、三维荧光技术考察了西那沙星对BSA构象的影响,结果显示色氨酸残基的最大发射波长略有蓝移,表明其所处环境的疏水性增加,从而揭示药物分子不仅可以和蛋白质分子结合,而且对蛋白质分子的构象都有一定的影响,使BSA形成一种新的无序结构。生物体内含有多种金属元素,它们不仅参与许多重要的生命过程,而且能与喹诺酮药物发生配位作用形成配合物,也可与蛋白质等生物大分子发生结合反应,成为相互作用研究的一个重要方面。为此,本文还进一步考察了金属离子(Cu²⁺,Fe³⁺,Zn²⁺,Mg²⁺)对西那沙星-BSA间结合的影响。研究表明,在金属离子存在的情况下,减小了药物与蛋白质间的结合常数,缩短了药物在血浆中的潴留时间,增大了药物的最大作用强度,这对希望短期提高药效的临床治疗有一定的作用。第二部分以小牛胸腺DNA为研究对象,采用光谱检测技术,结合离子强度考察、单双链DNA实验、偏振实验、KI猝灭实验、DNA热变性实验、粘度法等多种方法研究了西那沙星与DNA之间的相互作用模式。实验结果表明,加入DNA后西那沙星紫外吸收峰位置并未出现明显红移,Stern-Volmer方程计算荧光猝灭常数K_sv随温度增加而降低;随着以Na⁺表征的离子强度的增加,DNA对西那沙星的荧光猝灭程度变化甚小,说明西那沙星与DNA之间不存在非特异性的静电作用;单链DNA（ssDNA）对西那沙星的猝灭作用略大于双链DNA（dsDNA）;不同DNA浓度时,西那沙星的荧光偏振值没有明显改变;KI猝灭数据显示西那沙星并未受到DNA碱基对的有效保护,DNA热变性温度（T_m）、粘度也没有显著升高;以上结果可以判断西那沙星与DNA间的结合作用为沟槽结合,其荧光猝灭亦是由形成复合物的静态猝灭过程所致。根据荧光强度变化求得结合常数与结合位点数。同时考察了镁离子对相互作用的影响,镁离子和DNA均能使西那沙星的荧光强度出现降低现象,且镁离子的存在能使DNA对喹诺酮药物的猝灭作用显著增强。推测该三元体系形成了稳定的三元复合物,镁离子起了桥梁的作用。更多还原

【Abstract】 Protein and DNA are important bio-macromolecules in the life. They have respective biological function, and have been playing many vital roles for all kinds of biological phenomena. Exploring the interaction mechanisms on these bio-macromolecules with small molecules or ions, especially for those targeting-drug molecules, at the molecular level is of current interest in many research areas such as biology, clinical medicine, medicinal chemistry, chemistry and so on. Quinolones gets more and more use in the clinic for its advantages of high activity, broad spectrum and the convenience in the taking, et al. But its pharmacology and toxicology still have to be studied further. At the time, it also has some disadvantages, so develop new quinolones which has the high activity and low side-effect is the point of research. There are many metal ions in the organism, and they participate in many important vital action. Metal ions can compound with quinolone drug, and also can combine with bio-macromolecules such as protein. Sinafloxacin is a novel fourth-generation quinolone antimicrobial drug, which has in vitro and in vivo activities against a wide range of Gram-positive and Gram-negative bacteria. In a previous study, it was observed that sinafloxacin had more potent antibacterial activity but lower cytotoxicity than ciprofloxacin, levofloxacin and gatifloxacin. This paper studied the interaction mechanisms of sinafloxacin with bio-macromolecules, and it was consisted of two parts.In the first part, bovine serum albumin （BSA） has been selected for investigative object, and the binding characteristics and mechanism of sinafloxacin to BSA have been investigated by using fluorescence spectroscopy and absorbance spectra. Sinafloxacin can quench the endogenous fluorescence of BSA regularly and the quenching was not initiated by dynamic collision but from compound formation, it was a static quenching process. The interaction association constants of BSA and sinafloxacin were determined from the double reciprocal Lineweaver-Burk plot. The transfer efficiency of energy and distance are obtained in view of the theory of F（o|¨）rster energy transfer. From thermodynamic coordination it can be judged that the binding force between sinafloxacin and BSA is mainly electro-static force. The effect of sinafloxacin on the conformation of BSA was analyzed by synchronous fluorescence spectrometry and three-dimensional fluorescence spectrometry. The spectroscopy analysis shows that can sinafloxacin react with BSA and form a drug-protein compound. Furthermore, it can affect the configuration of the protein. The interaction between sinafloxacin and BSA in the presence of metal ions (Cu²⁺, Fe³⁺, Zn²⁺, Mg²⁺) was also studied. In the second part, calf thymus DNA has been selected for investigative object, and the binding characteristics and mechanism of sinafloxacin to DNA have been studied by fluorescence spectroscopy and UV-vis absorption spectroscopy. The variations in the spectroscopy characteristics of sinafloxacin in an aqueous medium upon addition of DNA were observed. The maximum absorption wavelength of sinafloxacin did not exhibit red-shift and the absorption has little change in intensity and shape after adding DNA into the system. The fluorescence intensity of sinafloxacin at 469nm is decreased dramatically, but there is no shift in the emission wavelength. The results show that the Stern-Volmer quenching constant K_SV is inversely correlated with temperature. It was proved that the fluorescence quenching of sinafloxacin by DNA is a result of the formation of sinafloxacin-DNA complex. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between sinafloxacin and DNA .Salt concentration effect, KI quenching, fluorescence polarization, DNA denaturation experiments, melting temperature （T_m） curves and viscosity measurements were also carried out to investigate the binding mechanism. In our experiments, the fluorescence of sinafloxacin -DNA system in a gradual increasing of NaCl concentration was studied and the results showed that the fluorescence intensity ratio had no significant change when the concentration of NaCl was within 0-0.2M range, which indicated that the interaction between sinafloxacin and DNA was not surface-binding mode. The experiments showed that single stranded DNA （ssDNA） could quench the fluorescence of sinafloxacin linearly, and the quenching of the fluorescence from sinafloxacin by double stranded DNA （dsDNA） was a little smaller than that by ssDNA under the same conditions. Fluorescence polarization measurements were performed on steady state sinafloxacin-DNA solutions. Only a slight increase was obtained with the addition of DNA. In aqueous solutions, iodide quenched the fluorescence of sinafloxacin efficiently. The K_SV values between sinafloxacin and iodide anions with the presence of DNA decreased slightly, which indicated that sinafloxacin could be partly protected. The addition of sinafloxacin caused no appreciable the melting temperature（T_m） and viscosity change in our experiments. From the experiment evidences, the major binding mode of sinafloxacin with DNA was evaluated to be the groove binding. The influence of Mg²⁺ on the binding between sinafloxacin and DNA was studied. The spectroscopy analysis shows that Mg²⁺ and DNA both can quench the endogenous fluorescence of sinafloxacin, and thefluorescence intensity of sinafloxacin quenched by DNA was aggravated in the presence of Mg²⁺. This means that there may be a ternary complex formed among Mg²⁺, sinafloxacin and DNA, Mg²⁺ plays a stimulative role as a bridge in the interaction between sinafloxacin and DNA.更多还原