Centre for Applied Pharmacokinetic Research, Division of Pharmacy and Optometry, University of Manchester, Manchester, United Kingdom (B.A., J.B., A.R.-H.); Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (J.K.F., P.C.S.); Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut (A.D., M.N., J.J.N., T.C.G.); and Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, United Kingdom (A.R.-H.).
Centre for Applied Pharmacokinetic Research, Division of Pharmacy and Optometry, University of Manchester, Manchester, United Kingdom (B.A., J.B., A.R.-H.); Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (J.K.F., P.C.S.); Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut (A.D., M.N., J.J.N., T.C.G.); and Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, United Kingdom (A.R.-H.)
Drug Metab Dispos. 2017 Oct;45(10):1102-1112. doi: 10.1124/dmd.117.076703. Epub 2017 Aug 2.
Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, < 0.0001; = 0.30; = 24]. SIL-based abundance measurements correlated well with enzyme activities, with correlations ranging from moderate for UGTs 1A6, 1A9, and 2B15 (Rs = 0.52-0.59, < 0.0001; = 0.34-0.58; = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79-0.90, < 0.0001; = 0.69-0.79). QconCAT-based data revealed generally poor correlation with activity, whereas moderate correlations were shown for UGTs 1A1, 1A3, and 2B7. Spurious abundance-activity correlations were identified in the cases of UGT1A4/2B4 and UGT2B7/2B15, which could be explained by correlations of protein expression between these enzymes. Consistent correlation of UGT abundance with catalytic activity, demonstrated by the SIL-based dataset, suggests that quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions.
定量表征 UDP-葡糖醛酸基转移酶 (UGT) 对葡醛酸化反应表型分析、基于药代动力学模型外推研究预测代谢清除率和药物相互作用非常有价值。不同的定量蛋白质组学工作流程已被用于各种系统中 UGT 酶的定量，有报道表明表达存在很大的可变性，这不能仅用个体间的可变性来解释。为了评估方法差异对终点 UGT 丰度定量的影响，两个实验室使用稳定同位素标记 (SIL) 肽或定量连接子 (QconCAT) 标准，在 24 个匹配的肝微粒体样本中定量了 8 种 UGT 酶，测量结果与 7 种酶的催化活性进行了比较（=59）。两种方法报告的个体丰度水平之间几乎没有一致性；只有 UGT1A1 表现出较强的相关性[Spearman 等级相关系数 (Rs)=0.73，<0.0001；=0.30；=24]。基于 SIL 的丰度测量与酶活性相关性良好，UGTs1A6、1A9 和 2B15 的相关性为中度（Rs=0.52-0.59，<0.0001；=0.34-0.58；=59），而 UGTs1A1、1A3、1A4 和 2B7 的相关性较强（Rs=0.79-0.90，<0.0001；=0.69-0.79）。基于 QconCAT 的数据与活性的相关性一般较差，而 UGTs1A1、1A3 和 2B7 的相关性为中度。UGT1A4/2B4 和 UGT2B7/2B15 出现了虚假的丰度-活性相关性，这可以用这些酶之间的蛋白表达相关性来解释。SIL 数据集显示 UGT 丰度与催化活性的一致性相关性表明，只要有可能，定量蛋白质组学数据应与催化活性进行验证。此外，代谢反应表型分析应考虑虚假的丰度-活性相关性，以避免得出误导性结论。
