Department of Chemistry, Washington State University, Pullman, WA, 99164, USA.
Department of Pharmaceutical Sciences, Washington State University, Spokane, WA, 99202, USA.
Arch Biochem Biophys. 2021 Sep 15;708:108937. doi: 10.1016/j.abb.2021.108937. Epub 2021 May 28.
We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450-P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species.
我们研究了药物代谢途径与人类肝微粒体（HLM）中 P450 酶系组成之间的对应关系。我们选择香豆素 152（C152）作为探针，它是一种被多种 P450 物种代谢的荧光底物。通过对 7 种混合 HLM 制剂的底物饱和曲线（SSP）进行研究，我们试图将其与通过靶向蛋白质组学表征的 P450 酶系组成相关联。这项分析，辅以 CYP3A4 和 CYP2C19（主要代谢 C152 的酶）的特异性抑制剂的测定，显示了不同 HLM 样本之间存在显著差异。为了揭示这些差异的来源，我们对具有已知 P450 酶系组成的 HLM 样本的 SSP 系列进行了主成分分析（PCA）。我们的分析表明，C152 代谢的参数主要取决于 CYP2A6、CYP2B6、CYP2C8、CYP2E1 和 CYP3A5 的含量，而只有 CYP2B6 和 CYP3A5 可以代谢 C152。为了验证这一发现，我们研究了用 CYP2A6、CYP2E1 和 CYP3A5 丰富 HLM 的效果。CYP3A5 掺入 HLM 会降低 C152 代谢率，同时增加 CYP2B6 在其周转率中的作用。相反，CYP2A6 和 CYP2E1 的掺入会使 C152 的脱甲基作用转向对底物具有中等亲和力的某些 P450 酶，最有可能是 CYP3A4。我们的结果揭示了个体 P450 特性的明显非加性，并表明 P450-P450 相互作用在确定药物代谢途径方面起着关键作用。这项研究表明，我们基于 PCA 的新方法在揭示不同 P450 物种之间的功能相互关系方面具有很高的潜力。
