Takahashi Ryan H, Forrest William F, Smith Alexander D, Badee Justine, Qiu NaHong, Schmidt Stephan, Collier Abby C, Parrott Neil, Fowler Stephen
Department of Drug Metabolism and Pharmacokinetics (R.H.T.) and Department of OMNI Bioinformatics (W.F.F.), Genentech, Inc., South San Francisco, California; Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B., S.S.); Pharmaceutical Research and Early Development, Roche Innovation Centre Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland (N.Q., N.P., S.F.); Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada (A.D.S., A.C.C.).
Department of Drug Metabolism and Pharmacokinetics (R.H.T.) and Department of OMNI Bioinformatics (W.F.F.), Genentech, Inc., South San Francisco, California; Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B., S.S.); Pharmaceutical Research and Early Development, Roche Innovation Centre Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland (N.Q., N.P., S.F.); Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada (A.D.S., A.C.C.)
Drug Metab Dispos. 2021 Sep;49(9):760-769. doi: 10.1124/dmd.121.000474. Epub 2021 Jun 29.
The expression of ten major drug-metabolizing UDP-glucuronosyltransferase (UGT) enzymes in a panel of 130 human hepatic microsomal samples was measured using a liquid chromatography-tandem mass spectrometry-based approach. Simultaneously, ten cytochromes P450 and P450 reductase were also measured, and activity-expression relationships were assessed for comparison. The resulting data sets demonstrated that, with the exception of UGT2B17, 10th to 90th percentiles of UGT expression spanned 3- to 8-fold ranges. These ranges were small relative to ranges of reported mean UGT enzyme expression across different laboratories. We tested correlation of UGT expression with enzymatic activities using selective probe substrates. A high degree of abundance-activity correlation (Spearman's rank correlation coefficient > 0.6) was observed for UGT1As (1A1, 3, 4, 6) and cytochromes P450. In contrast, protein abundance and activity did not correlate strongly for UGT1A9 and UGT2B enzymes (2B4, 7, 10, 15, and 17). Protein abundance was strongly correlated for UGTs 2B7, 2B10, and 2B15. We suggest a number of factors may contribute to these differences including incomplete selectivity of probe substrates, correlated expression of these UGT2B isoforms, and the impact of splice and polymorphic variants on the peptides used in proteomics analysis, and exemplify this in the case of UGT2B10. Extensive correlation analyses identified important criteria for validating the fidelity of proteomics and enzymatic activity approaches for assessing UGT variability, population differences, and ontogenetic changes. SIGNIFICANCE STATEMENT: Protein expression data allow detailed assessment of interindividual variability and enzyme ontogeny. This study has observed that expression and enzyme activity are well correlated for hepatic UGT1A enzymes and cytochromes P450. However, for the UGT2B family, caution is advised when assuming correlation of expression and activity as is often done in physiologically based pharmacokinetic modeling. This can be due to incomplete probe substrate specificities, but may also be related to presence of inactive UGT protein materials and the effect of splicing variations.
采用基于液相色谱-串联质谱的方法，测定了130份人肝微粒体样品中10种主要药物代谢UDP-葡萄糖醛酸基转移酶（UGT）的表达。同时，还测定了10种细胞色素P450和P450还原酶，并评估了活性-表达关系以作比较。所得数据集表明，除UGT2B17外，UGT表达的第10至90百分位数范围为3至8倍。相对于不同实验室报道的UGT酶平均表达范围，这些范围较小。我们使用选择性探针底物测试了UGT表达与酶活性的相关性。观察到UGT1A（1A1、3、4、6）和细胞色素P450具有高度的丰度-活性相关性（Spearman等级相关系数>0.6）。相比之下，UGT1A9和UGT2B酶（2B4、7、10、15和17）的蛋白质丰度与活性之间没有很强的相关性。UGT 2B7、2B10和2B15的蛋白质丰度高度相关。我们认为多种因素可能导致这些差异，包括探针底物的选择性不完全、这些UGT2B同工型的相关表达，以及剪接和多态性变体对蛋白质组学分析中使用的肽段的影响，并以UGT2B10为例进行了说明。广泛的相关性分析确定了验证蛋白质组学和酶活性方法在评估UGT变异性、群体差异和个体发育变化方面的保真度的重要标准。意义声明：蛋白质表达数据有助于详细评估个体间变异性和酶的个体发育。本研究观察到，肝脏UGT1A酶和细胞色素P450的表达与酶活性密切相关。然而，对于UGT2B家族，在基于生理学的药代动力学建模中像通常那样假设表达与活性的相关性时，建议谨慎行事。这可能是由于探针底物特异性不完全，但也可能与无活性UGT蛋白质材料的存在以及剪接变异的影响有关。
