Eberini Ivano, Fantucci Piercarlo, Rocco Alessandro Guerini, Gianazza Elisabetta, Galluccio Lara, Maggioni Daniela, Ben Ilaria Dal, Galliano Monica, Mazzitello Rosalba, Gaiji Noura, Beringhelli Tiziana
Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, Milano, Italia.
Proteins. 2006 Nov 15;65(3):555-67. doi: 10.1002/prot.21109.
Norfloxacin and levofloxacin, two fluoroquinolones of different bulk, rigidity and hydrophobicity taken as model ligands, were docked to one apo and two holo crystallographic structures of bovine beta-lactoglobulin (BLG) using different computational approaches. BLG is a member of the lipocalin superfamily. Lipocalins show a typical b-barrel structure encompassing an internal cavity where small hydrophobic molecules are usually bound. Our studies allowed the identification of two putative binding sites in addition to the calyx. The rigid docking approximation resulted in strong repulsive forces when the ligands were docked into the calyx of the apo form. On the contrary, hindrance was not experienced in flexible docking protocols whether on the apo or on the holo BLG forms, due to allowance for side chain rearrangement. K(i) between 10(-7) and 10(-6) M were estimated for norfloxacin at pH 7.4, smaller than 10(-5) M for levofloxacin. Spectroscopic and electrophoretic techniques experimentally validated the occurrence of an interaction between norfloxacin and BLG. Changes in chemical shift and dynamic parameters were observed between the (19)F NMR spectra of the complex and of the ligand. A K(i) (ca 10(-7) M) comparable with the docking results was estimated through a NMR relaxation titration. Stabilization against unfolding was demonstrated by denaturant gradient gel electrophoresis on the complex versus apo BLG. NMR experimental evidence points to a very loose interaction for ofloxacin, the racemic mixture containing levofloxacin. Furthermore, we were able to calculate in silico K(i)'s comparable to the published experimental values for the complexes of palmitic and retinoic acid with BLG.
以诺氟沙星和左氧氟沙星这两种具有不同体积、刚性和疏水性的氟喹诺酮类药物作为模型配体,采用不同的计算方法将它们与牛β-乳球蛋白(BLG)的一个脱辅基和两个全蛋白晶体结构进行对接。BLG是脂质运载蛋白超家族的成员。脂质运载蛋白具有典型的β-桶状结构,其内部有一个腔,通常会结合小的疏水分子。我们的研究除了发现萼状结构外,还确定了两个假定的结合位点。当将配体对接至脱辅基形式的萼状结构时,刚性对接近似法产生了强烈的排斥力。相反,无论是在脱辅基还是全蛋白BLG形式上,灵活对接方案都没有遇到阻碍,因为它允许侧链重排。在pH 7.4条件下,诺氟沙星的解离常数(K(i))估计在10⁻⁷至10⁻⁶ M之间,左氧氟沙星的解离常数小于10⁻⁵ M。光谱和电泳技术通过实验验证了诺氟沙星与BLG之间相互作用的发生。在复合物和配体的¹⁹F NMR光谱之间观察到化学位移和动力学参数的变化。通过NMR弛豫滴定法估计出与对接结果相当的K(i)(约10⁻⁷ M)。变性剂梯度凝胶电泳显示复合物相对于脱辅基BLG对解折叠具有稳定性。NMR实验证据表明氧氟沙星(含有左氧氟沙星的外消旋混合物)的相互作用非常松散。此外,我们能够通过计算机模拟计算出与已发表的棕榈酸和视黄酸与BLG复合物实验值相当的K(i)。