Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling

It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial β-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug–glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.越来越多的证据表明，人类肠道微生物组构成的个体间差异会导致药物反应异质性。例如，接受抗癌药物伊立替康（irinotecan）治疗的患者并非均会出现胃肠道（gastrointestinal，GI）毒性，已有研究指出该差异源于肠道细菌β-葡萄糖苷酸酶（β-glucuronidases，GUSs）的种类与水平差异。GUS酶可通过将无活性的药物-葡萄糖醛酸缀合物水解为活性药物，进而损伤胃肠道上皮，促进药物毒性产生。然而，此前尚未能从人类微生物组的复杂环境中，精准鉴定出负责药物再激活的特定GUS酶。本研究依托一套独特的基于活性的蛋白质谱分析（activity-based protein profiling，ABPP）平台，从人类粪便样本中筛选出能够生成伊立替康活性毒性代谢物SN-38的特定细菌GUS酶；我们通过ABPP赋能的蛋白质组学流程对人类粪便中的肠道细菌GUS酶进行鉴定与定量，随后结合离体动力学实验，精准定位出负责SN-38再激活的特定GUS酶。此外，该研究方法还揭示了不同人类粪便样本中肠道细菌GUS抑制作用存在差异的分子机制。综上，本研究构建了一套前所未有的技术与生物信息学流程，可从人类粪便的复杂微生物组中筛选出负责特定生化反应的微生物酶；鉴定此类微生物酶有望助力精准生物标志物的开发与新型药物靶点的发现，从而推动个性化医疗的发展前景。