Institute of Biochemistry, Department of Chemistry, University of Cologne, Cologne, Germany.
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
Clin Pharmacol Ther. 2022 Oct;112(4):808-816. doi: 10.1002/cpt.2637. Epub 2022 May 31.
Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/3A) but normal levels in compound heterozygous carriers (TPMT3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates.
2021 年，美国食品和药物管理局（FDA）批准了钼辅因子（Moco）缺乏症的治疗方法。虽然 Moco 的尿代谢产物尿硫酮被用作 Moco 缺乏症的诊断生物标志物，但它的代谢途径尚不清楚。在这里，我们通过阴离子交换/大小排阻色谱和肽质量指纹图谱，使用小鼠肝组织鉴定了尿硫酮合成甲基转移酶。我们表明，代谢 Moco 的 S-甲基化酶对应于硫嘌呤 S-甲基转移酶（TPMT），这是一种高度多态性的药物代谢酶，与药物相关的血液毒性有关，但未知其生理作用。我们使用重组表达的人 TPMT 蛋白、Tpmt 野生型和敲除（Tpmt ）小鼠的肝裂解物以及人肝胞质体在体外研究了尿硫酮的合成。通过液相色谱串联质谱法在小鼠的肾脏和尿液中定量了尿硫酮的水平。在人类样本中研究了 TPMT 基因型/表型和尿硫酮的排泄水平，并在一项独立的基于人群的研究中进行了验证。由于 Moco 为 TPMT 提供了生理底物（硫代喋呤），因此在小鼠和人类中，与 TPMT 药物底物（6-巯基嘌呤）一起测定的硫代喋呤甲基化活性与 TPMT 活性相关。Tpmt 小鼠的肾脏和尿液中尿硫酮浓度极低。TPMT 缺陷患者的尿硫酮浓度取决于常见的 TPMT 多态性，纯合变异型携带者（TPMT*3A/3A）的水平极低，但复合杂合型携带者（TPMT3A/*3C）的水平正常，这在基于人群的研究中得到了验证。我们的工作新鉴定了 TPMT 的内源性底物，并显示了 Moco 代谢和药物代谢之间前所未有的联系。此外，TPMT 的例子表明，遗传多态性的表型后果可能在药物和内源性底物之间存在差异。
