Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55126, USA.
Drug Metab Dispos. 2010 Jun;38(6):1003-9. doi: 10.1124/dmd.109.030155. Epub 2010 Mar 9.
Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b(5) (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K(m) values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.
细胞色素 P450（P450s）与氧化还原转移蛋白相互作用，包括 P450 还原酶（CPR）和细胞色素 b(5)（b5），所有这些都是膜结合的。在多种体外系统中，也观察到了 P450-P450 相互作用，导致酶活性的改变。本研究在重组系统中研究了 CYP2C9 和 CYP3A4 之间相互作用的影响和机制。虽然 S-萘普生和 S-氟比洛芬的 CYP2C9 介导的代谢 K(m) 值没有改变，但 CYP3A4 的共孵育抑制了高达 80%的 CYP2C9 介导的代谢。增加 CYP3A4 浓度增加了抑制程度，而增加 CPR 浓度则导致抑制减少。添加 b5 仅略微影响抑制的幅度。相比之下，CYP2C9 不会改变 CYP3A4 介导的睾酮代谢。通过用全长 CYP3A4 孵育截断的 CYP2C9 并反过来进行孵育，评估了疏水性 N 端在这些相互作用中的潜在作用。在这两种情况下，抑制作用均被完全消除，表明疏水力在 CYP2C9-CYP3A4 相互作用中起重要作用。最后，通过共免疫沉淀技术分离出 CYP2C9/CYP3A4 杂种复合物，证实了蛋白质的物理相互作用。这些结果表明，CYP2C9 和 CYP3A4 的 N 端膜结合结构域参与杂种复合物的形成，至少会导致 CYP2C9 活性降低。
