ABSTRACT

Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial β-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.

摘要

吗替麦考酚酯 (MMF) 是一种重要的免疫抑制剂前药，用于预防器官移植排斥和治疗自身免疫性疾病。然而，MMF 的使用受到严重胃肠道毒性的限制，大约 45% 的 MMF 接受者观察到这种毒性。该药物的活性形式霉酚酸 (MPA) 通过细菌 β-葡萄糖醛酸酶 (GUS) 进行广泛的肠肝再循环，从而在胃肠道内重新激活霉酚酸葡萄糖醛酸 (MPAG) 的 MPA。GUS 酶根据其结构特征表现出不同的底物偏好，并且尚未鉴定出重新激活 MPA 的肠道微生物 GUS 酶。在这里，我们使用鸟枪法宏基因组测序将接受 MMF 的移植受者的粪便微生物组与健康个体进行比较。我们发现微生物组成和 GUS 基因特定结构类别的存在都不足以解释两个队列粪便样本之间测量的 MPA 再激活的差异。接下来，我们采用基于 GUS 特异性活性的化学探针和靶向宏蛋白质组学来识别和量化人类粪便样本中存在的 GUS 蛋白。通过使用从粪便样本中收集的宏基因组数据，改进了特定 GUS 酶的鉴定。我们发现结合黄素单核苷酸 (FMN) 的 GUS 酶的存在与有效的 MPA 重新激活显着相关。此外，结构分析还确定了这些 FMN 结合 GUS 酶特有的基序，为它们处理这种药物葡萄糖醛酸苷的能力提供了分子支持。这些结果表明，FMN 结合 GUS 酶可能是 MPA 重新激活的原因，并且可能是 MPA 诱导的胃肠道毒性的驱动力。