Pearson Josh T, Hill John J, Swank Jennifer, Isoherranen Nina, Kunze Kent L, Atkins William M
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
Biochemistry. 2006 May 23;45(20):6341-53. doi: 10.1021/bi0600042.
The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is approximately 25 A from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits "equilibrium overshoot". Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low-spin spectra and K(D) values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.
含血红素的细胞色素P450（CYPs）是药物清除和药物-药物相互作用的主要酶学决定因素。CYP3A4同工型受到抗真菌咪唑类或三唑类的抑制，它们通过形成氮-铁离子配位键形成低自旋血红素铁配合物。然而，CYP3A4也会在距三唑氮原子约25 Å的位点缓慢氧化抗真菌药物伊曲康唑（ITZ），这表明大型抗真菌唑类在CYP3A4活性位点内可以采取多种取向。在此，我们报告了一种表面等离子体共振（SPR）分析，该分析以动力学分辨率研究了ITZ和相关药物酮康唑（KTZ）两种结合模式。SPR揭示了一种结合取向的解离速率非常缓慢。观察到多相结合动力学，并且通过曲线拟合解析的两个结合组分之一表现出“平衡超调”。用血红素配体咪唑预加载CYP3A4消除了抗真菌唑类结合轨迹的这一组分，并消除了明显缓慢的解离速率。通过改变药物暴露脉冲的持续时间，可以控制与不同药物取向相对应的CYP3A4复合物的分数群体。紫外-可见差示吸光度滴定产生与SPR解析的高亲和力复合物一致的低自旋光谱和K(D)值。这些结果表明，ITZ和KTZ以多种取向结合，包括催化活性模式和缓慢解离的抑制模式。最重要的是，它们提供了基于SPR的方法用于动力学表征药物与任何人类CYP结合的首个实例，包括其他方法无法获得的机制见解。
