| 光谱法联合分子对接研究人血清白蛋白与新的抗肿瘤活性小分子的体外结合 |
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| 基金项目:国家自然科学基金项目(81403177)；辽宁省教育厅一般项目(LQN201715)；辽宁大学大学生创新创业训练项目(x201610140243) |
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| 中文摘要:前期研究中合成了全新的以4-苯氧基喹啉环为母核结构的， 具有较好抗肿瘤活性的Type Ⅱ型小分子c-Met激酶抑制剂：N-［3-氟-4-［6-甲氧基-7-［3-(4-甲基-1-哌嗪基)丙氧基］喹啉-4-氧基］苯基］-1-(2-氟苯基)-4-甲基-5-氧代-4,5-二氢-1H-1,2,4-三氮唑-3-甲酰胺（LJC-116）。该文进一步通过荧光、同步荧光、三维荧光、紫外-可见吸收等光谱方法联合分子对接技术研究了在模拟生理条件下，LJC-116与人血清白蛋白 (HSA)的结合作用。研究表明LJC-116通过静态结合作用猝灭HSA的荧光，此静态作用方式同时被三维荧光以及紫外可见吸收光谱确证。在288、299、310 K温度下，计算LJC-116与HSA的表观结合常数的数量级均为104 L·mol-1，说明两者的结合是中等强度。热力学常数ΔG°为负值，表明两者的结合是自发的。此外，ΔS°为正值，同时ΔH°为负值表明疏水作用和氢键是形成HSA- LJC-116（1∶11）复合物的主要驱动力。在实验条件下，通过 Frster偶极-偶极非辐射能量转移理论计算重叠积分J=7.169 7×10-15 cm3·L·mol-1，R=1.28 nm，r=1.69 nm，E=15.6%。表明能量转移效率很高。位点探针试剂华法林和布洛芬的加入实验表明LJC-116与HSA结合部位处于HSA的疏水腔亚结构域ⅡA(siteⅠ)中。两者结合引起HSA的以下变化：内源性荧光猝灭、同步荧光 红移0.4 nm，三维荧光光谱红移 2 nm，HSA紫外吸收光谱改变以及HSA氨基酸残基微环境极性增加、疏水性下降。最后，利用分子对接对热力学计算得到的作用力和光谱方法中探讨的HSA构象变化进行了验证。该文全面阐述了两者在分子水平的作用机制，为Type Ⅱ型小分子c-Met激酶抑制剂的体内转运情况提供了有用的信息。 |
| 中文关键词:人血清白蛋白(HSA) c-Met激酶抑制剂 三维荧光光谱 结合位点 分子对接 |
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| Investigation on In Vitro Binding of a New Anti-tumor Compound and Human Serum Albumin by Spectroscopic Methods and Molecular Docking |
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| Abstract:N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-4-methyl -5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxamide(LJC-116) is a kind of new Type Ⅱ compounds,c-Met kinase inhibitors with 4-phenoxy quinolone.In vitro experiments showed that they have an excellent anti-tumor activity.In this paper,the in vitro molecular binding interaction between LJC-116 and human serum albumin(HSA) was further investigated by various methods,such as fluorescence spectrometry,synchronous fluorescence spectrometry,fluorescence probe experiments,three-dimensional(3-D) fluorescence spectrometry,ultraviolet-visible(UV-Vis) absorption spectroscopy and molecular docking.It was found that the intrinsic fluorescence of HSA could be quenched by LJC-116 through a static quenching process.The static quenching process was also proved by 3-D fluorescence spectrometry and ultraviolet-visible absorption spectroscopy.The apparent binding constants between LJC-116 and HSA at 288,299，310 K were estimated to be in the order of 104 L·mol-1,which indicated the binding between LJC-116 and HSA was moderate.The thermodynamic parameters ΔH°,ΔG° and ΔS° were calculated,in which the negative ΔG° suggested that the binding of LJC-116 to HSA was spontaneous.Moreover,the positive ΔS° and negative ΔH° revealed that hydrophobic interaction and hydrogen bonds were the major forces to stabilize the protein- LJC-116(1∶1) complex.Under the experimental conditions,the overlapping integral J=7.169 7×10-15 cm3·L·mol-1，R=1.28 nm，r=1.69 nm,E=15.6% were obtained.It indicated a high probability of energy transfer from HSA to LJC-116.Probe agent ibuprofen and warfarin were added in system of HSA-LJC-116,it showed that the primary binding site of LJC-116 was located at site Ⅰ(subdomain ⅡA) of HSA.The slight redshift of fluorescence(0.3 nm),synchronous fluorescence(0.4 nm) and 3-D fluorescence spectra(2 nm) all demonstrated that the polarity of microenvironment for amino acid residues in HSA increased and the hydrophobic property decreased.Finally,the forces deduced from the thermodynamic calculation,and the conformational changes of HSA were validated by the molecular docking.The interaction mechanism of HSA and LJC-116 was comprehensively expatiated at a molecular level in this paper,and some useful information for the transport of Type Ⅱ c-met kinase inhibitors in vivo was provided. |
| Key Words:human serum albumin(HSA) c-Met kinase inhibitors 3-D fluorescence spectrum binding site molecular docking |
| 引用本文：刘举,宫雪,徐亮,张力,宫平.光谱法联合分子对接研究人血清白蛋白与新的抗肿瘤活性小分子的体外结合[J].分析测试学报,2018,37(4):389-396. |
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