Department of Chemistry and Biochemistry and Biomolecular Sciences Institute, Florida International University, Miami, Florida (J.C.H., K.S.S., F.Z.P.); Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia (D.T.S.); and Department of Biological and Medicinal Chemistry, Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland (K.S.S.).
Department of Chemistry and Biochemistry and Biomolecular Sciences Institute, Florida International University, Miami, Florida (J.C.H., K.S.S., F.Z.P.); Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia (D.T.S.); and Department of Biological and Medicinal Chemistry, Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland (K.S.S.)
Mol Pharmacol. 2023 Oct;104(4):154-163. doi: 10.1124/molpharm.123.000698. Epub 2023 Aug 3.
Cytochrome P450 3A4 (CYP3A4) is the dominant P450 involved in human xenobiotic metabolism. Competition for CYP3A4 therefore underlies several adverse drug-drug interactions. Despite its clinical significance, the mechanisms CYP3A4 uses to bind diverse ligands remain poorly understood. Highly monodisperse CYP3A4 embedded in anionic lipoprotein nanodiscs containing an equal mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) were used to determine which of the limiting kinetic schemes that include protein conformational change, conformational selection (CS) or induced fit (IF), best described the binding of four known irreversible inhibitors. Azamulin, retapamulin, pleuromutilin, and mibrefadil binding to CYP3A4 nanodiscs conformed to a single-site binding model. Exponential fits of stopped-flow UV-visible absorption spectroscopy data supported multiple-step binding mechanisms. Trends in the rates of relaxation to equilibrium with increasing ligand concentrations were ambiguous as to whether IF or CS was involved; however, global fitting and consideration of the rate constants favored an IF mechanism. In the case of mibrefadil, a transient complex was observed in the stopped-flow UV-visible experiment, definitively assigning the presence of IF in ligand binding. While these studies only consider a small region of CYP3A4's vast ligand space, they provide kinetic evidence that CYP3A4 can use an IF mechanism. SIGNIFICANCE STATEMENT: CYP3A4 is capable of oxidizing numerous xenobiotics, including many drugs. Such promiscuity could not be achieved without conformational changes to accommodate diverse substrates. It is unknown whether conformational heterogeneity is present before (conformational selection) or after (induced fit) ligand binding. Stopped-flow measurements of suicide inhibitors binding to nanodisc-embedded CYP3A4 combined with sophisticated numerical analyses support that induced fit better describes ligand binding to this important enzyme.
细胞色素 P450 3A4（CYP3A4）是参与人体异生物质代谢的主要 P450。因此，CYP3A4 的竞争是几种不良药物相互作用的基础。尽管其具有临床意义，但 CYP3A4 结合不同配体的机制仍知之甚少。高度单分散的 CYP3A4 嵌入含有 1-棕榈酰-2-油酰基甘油-3-磷酸胆碱和 1-棕榈酰-2-油酰基-sn-甘油-3-磷酸-(1'-rac-甘油）的阴离子脂蛋白纳米盘，用于确定包括蛋白质构象变化、构象选择（CS）或诱导契合（IF）的限制动力学方案中，哪个最好描述了四种已知不可逆抑制剂的结合。阿扎鲁胺、雷他培南、普雷洛姆替林和米布雷弗丁与 CYP3A4 纳米盘的结合符合单一位点结合模型。停止流动紫外可见吸收光谱数据的指数拟合支持多步骤结合机制。随着配体浓度的增加，平衡时的松弛速率趋势对于涉及 IF 或 CS 并不明确；然而，全局拟合和对速率常数的考虑有利于 IF 机制。在米布雷弗丁的情况下，在停止流动紫外可见实验中观察到瞬态复合物，明确确定了 IF 在配体结合中的存在。虽然这些研究仅考虑了 CYP3A4 广阔配体空间的一小部分，但它们提供了动力学证据，表明 CYP3A4 可以使用 IF 机制。意义陈述：CYP3A4 能够氧化许多异生物质，包括许多药物。如果没有构象变化来容纳各种底物，这种混杂性是不可能实现的。在配体结合之前（构象选择）或之后（诱导契合）是否存在构象异质性尚不清楚。自杀抑制剂结合到纳米盘嵌入的 CYP3A4 的停止流动测量结合复杂的数值分析支持，诱导契合更好地描述了这种重要酶的配体结合。
