Lampe Jed N, Fernandez Cristina, Nath Abhinav, Atkins William M
Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, Washington 98195-7610, USA.
Biochemistry. 2008 Jan 15;47(2):509-16. doi: 10.1021/bi7013807. Epub 2007 Dec 20.
Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.
细胞色素P450 3A4（CYP3A4）在许多药物的代谢过程中起着关键作用。CYP3A4表现出非凡的底物选择性和异常的变构动力学。此外，许多细胞色素P450酶对单一底物催化连续氧化反应，但在大多数情况下，这些过程的机制细节尚未完全明确。因此，基于复杂的体外动力学很难预测许多药物及其代谢产物的体内清除率，需要新的体外探针来了解这些行为。具有强溶剂化显色行为的近红外荧光探针尼罗红被研究作为CYP3A4变构和连续代谢的探针。基于血红素自旋状态对尼罗红浓度的S形依赖性，尼罗红的表观解离常数（Kd）值为0.05和2.3 μM，其中第一个当量的尼罗红使高自旋分数仅增加到饱和时总变化的13%。质谱分析表明，尼罗红被CYP3A4依次代谢为N-单乙基和N-去乙基产物，证实血红素铁的紧邻区域是一个结合位点。在存在CYP3A4的情况下，尼罗红在不同浓度下的稳态荧光发射和激发光谱以及激发态寿命表明存在一个高亲和力位点，该位点调节尼罗红的荧光特性。尼罗红结合位点被伊曲康唑竞争性消除，伊曲康唑是一种已知与血红素铁配位的高亲和力配体。综合这些数据表明，尼罗红以高亲和力（约50 nM）结合到活性位点，在低介电环境中发生去溶剂化。此外，尼罗红的连续氧化速率与其他体外探针相当或更快，这突出了其在体外进一步研究这一重要动力学现象中的实用性。
