Understanding the Stereoselective Mechanism of Diniconazole Enantiomers Interacting with Serum Albumins
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摘要: 手性药物与血清蛋白的结合通常表现出立体选择性。采用UV-Vis吸收光谱、荧光光谱和分子对接技术研究了R-烯唑醇和S-烯唑醇与人血清蛋白(HSA)/牛血清蛋白(BSA)的结合差异。结果表明：血清蛋白与R-烯唑醇的结合能力强于S-烯唑醇；烯唑醇对血清蛋白的荧光猝灭机制为静态猝灭；R-烯唑醇和S-烯唑醇与HSA相互作用的总能量分别为-26.4 kJ/mol和-23.6 kJ/mol，与BSA的对接能量分别为-27.6 kJ/mol和-23.3 kJ/mol，说明R-烯唑醇与血清蛋白形成的复合物更稳定。研究结果可为后续开展烯唑醇的立体选择性作用机制研究提供依据。Abstract: Interactions of chiral pharmaceuticals and serum albumins show enantioselectivity. Herein, UV-Vis absorption spectroscopy, fluorescent spectroscopy, and molecular docking technology were applied in investigation of enantioselective interactions between diniconazole enantiomers and bovine/human serum albumins (BSA/HSA). The results showed that serum albumins possessed stronger binding affinity for R-diniconazole than S-diniconazole; fluorescent quenching of serum albumins induced by diniconazole enantiomers was ascribed to static quenching mechanism; the docking energies between R-diniconazole and S-diniconazole with HSA were -26.4 kJ/mol and -23.6 kJ/mol, and the docking energies with BSA were -27.6 kJ/mol and -23.3 kJ/mol, respectively, which indicates that binding of serum albumin with R-diniconazole was more stable than that with S-enantiomer. Therefore, this study would provide useful information for the stereoselective mechanism of diniconazole in biological system.
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图 2 不同烯唑醇对映体的浓度对HSA和BSA紫外吸收光谱的影响
注：曲线编号0→8代表血清蛋白溶液中烯唑醇的浓度从0逐步增加为1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0 μmol/L。
Figure 2. The effect of different concentrations of diniconazole enantiomers on the UV-Vis spectra of HSA and BSA
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图 3 HSA和BSA在加入烯唑醇对映体前后的荧光光谱
注：曲线编号0→5代表血清蛋白溶液中烯唑醇的浓度从0逐步增加为6.1、15.3、30.7、46.0和61.3 μmol/L。
Figure 3. The fluorescent spectra of HSA and BSA without/with diniconazole enantiomers
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图 4 不同温度下烯唑醇与HSA、BSA的Stern-Volmer图
Figure 4. The Stern-Volmer plots of diniconazole with HSA and BSA at different temperatures
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图 5 HSA-烯唑醇和BSA-烯唑醇复合物的分子对接结果
注：图A~D分别显示烯唑醇在HSA或BSA中的结合位点，放大图给出了烯唑醇与相邻氨基酸残基结合力的详细说明。
Figure 5. The molecular docking results of the HSA- and BSA-diniconazole complexes
表 1 不同温度下HSA和BSA与烯唑醇对映体的结合常数(Ka)、结合位点数(n)和热力学参数
Table 1 The binding constant (Ka), number of binding sites (n) and relative thermodynamic parameters of HSA and BSA with diniconazole enantiomers at different temperatures
作用体系 T/K Stern-Volmer方程 双对数方程 范特霍夫方程 KSV/
(L·mol-1)kq/
(L·mol-1·s-1)R2 Ka/
(L·mol-1)n R2 ΔH/
(kJ·mol-1)ΔG/
(kJ·mol-1)ΔS/
(J·mol-1·K-1)R2 HSA与R-烯唑醇 293 0.95×105 0.95×1013 0.945 7 74.31×105 1.60 0.994 8 -91.4 -13.5 -265.90 0.996 4 303 1.31×105 1.31×1013 0.993 2 3.51×105 1.21 0.997 5 -9.8 -269.44 313 1.62×105 1.62×1013 0.988 9 0.30×105 1.07 0.965 5 -1.2 -254.86 HSA与S-烯唑醇 293 0.93×105 0.93×1013 0.952 3 22.08×105 1.56 0.991 2 -94.0 -15.5 -268.23 0.987 7 303 1.16×105 1.16×1013 0.991 1 0.81×105 1.21 0.998 1 -17.3 -253.25 313 1.29×105 1.29×1013 0.989 6 0.08×105 0.95 0.979 5 -12.8 -259.69 BSA与R-烯唑醇 293 0.97×105 0.97×1013 0.946 8 79.78×105 1.66 0.994 9 -74.9 -13.6 -209.03 0.982 9 303 1.32×105 1.32×1013 0.993 6 4.01×105 1.26 0.999 2 -11.1 -210.60 313 1.64×105 1.64×1013 0.991 1 0.53×105 1.12 0.983 4 -12.3 -199.98 BSA与S-烯唑醇 293 0.93×105 0.93×1013 0.950 9 22.96×105 1.56 0.993 3 -74.9 -15.5 -202.72 0.989 6 303 1.17×105 1.17×1013 0.989 7 1.68×105 1.21 0.998 1 -17.4 -189.79 313 1.33×105 1.33×1013 0.988 3 0.25×105 1.07 0.993 6 -13.6 -195.84 
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表 2 HSA-烯唑醇和BSA-烯唑醇复合物的分子对接参数
Table 2 The molecular docking parameters for binding between HSA- and BSA-diniconazole complexes
作用体系 疏水相互作用 氢键 π-阳离子相互作用 对接能量/
(kJ·mol-1)残基 距离/nm 残基 距离/nm 残基 距离/nm HSA与R-烯唑醇 ARG117 0.342 ARG117 0.252 ARG117 0.441 -26.4 ILE142 0.365 ARG117 0.232 TYR161 0.349 LEU182 0.201 — — ARG186 0.322 HSA与S-烯唑醇 ILE142 0.334 TYR161 0.219 — — -23.6 PHE157 0.381 LEU185 0.195 ARG186 0.384 GLY189 0.337 ARG186 0.397 — — BSA与R-烯唑醇 LEU454 0.324 THR190 0.194 ARG458 0.454 -27.6 TYR451 0.326 SER428 0.217 ILE455 0.336 TYR451 0.303 TYR451 0.338 — — GLU186 0.344 — — BSA与S-烯唑醇 LYS431 0.344 GLU186 0.198 — — -23.3 TYR451 0.309 LEU189 0.202 ILE455 0.349 THR190 0.295 ILE455 0.378 — —
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