UGT engineering for detoxifying anticancer drug SN-38

 描述(申请人提供)：化疗中使用的抗癌药物通常有严重的副作用。通常只有一小部分药物被运送到目标部位，其他药物可能会成为废物，甚至对人类有害。尿苷二磷酸(UDP)糖基转移酶(UGT)是包括毒素和药物在内的小分子糖基化的核心分子，是人体内关键的II相药物代谢酶。人UDP-葡萄糖醛酸基转移酶1A1负责消除和解毒结构上不同的外来物质(如SN-38、多环胺和亚硝胺)和内源化合物。我们发现，植物紫花苜蓿UGT71G1在体外也能有效地代谢包括SN-38和雌二醇在内的典型的人类UGT1A1底物。然而，植物UGT71G1的体内活性不足以通过口服高表达植物UGT71G1的细菌进入小鼠体内来解毒SN-38。我们建议开发更具活性的UGT和药物解毒菌(DDB，以大肠杆菌为初始模式菌)来代谢抗癌药物SN-38。我们将使用两种不同的方法来寻找更活跃的植物UGT71G1突变体，包括基于合理结构的UGT设计和基于我们确定的植物UGT71G1结构的工程，以及随机诱变。我们将表达和纯化UGT突变体，并进行酶分析以筛选对SN的催化活性显著提高(至少增加10倍)的突变体 38.我们还将筛选不同的大肠杆菌表达系统(载体)和细菌菌株。不同的大肠杆菌表达系统和菌株在小鼠体内的表现可能不同，从而影响酶的活性。我们将根据其与小鼠共生菌株的相似性，寻找一种合适的大肠杆菌表达系统和细菌菌株，以表达抗SN-38活性最高的植物突变体(S)。然后，我们将最具活性的植物突变体(S)高表达细菌导入小鼠体内，测定其体内代谢SN-38的能力和活性，并开发出用于SN-38解毒的药物解毒菌。

 DESCRIPTION (provided by applicant): There are often serious side effects for anti-cancer drugs used in chemotherapy. Usually only small portion of drug is delivered to the target sites, and others may become waste or even toxic for humans. Uridine diphosphate (UDP) glycosyltransferases (UGTs) are the central players in the glycosylation of small molecules including toxins and drugs, and are key phase II drug metabolizing enzymes in human. Human UDP-glucuronosyltransferase 1A1 is responsible for elimination and detoxification of structurally diverse groups of xenobiotics (e.g., SN-38, polycyclic amines and nitrosamines) and endogenous compounds. We found that plant Medicago truncatula UGT71G1 could also efficiently metabolize typical human UGT1A1 substrates including SN-38 and estradiol in vitro. However, the activity of plant UGT71G1 is not high enough in vivo for detoxifying SN-38 by oral delivery of the plant UGT71G1 overexpressing bacteria into mouse. We propose to develop more active UGTs and drug detoxifying bacteria (DDB, using E. coli as the initial model bacteria) for the metabolism of anticancer drug SN-38. We will use two different approaches for searching more active plant UGT71G1 mutants, including rational structure-based UGT design and engineering based on plant Medicago truncatula UGT71G1 structures we determined, and random mutagenesis. We will express and purify UGT mutants and perform enzyme assays to screen for mutants with significantly improved (at least 10-fold increase) catalytic activity on SN 38. We will also screen different E. coli expression systems (vectors) and bacterial strains. The different E. coli expression systems and bacterial strains may behave differently and affect the enzyme activity in mouse. We will identify a suitable E. coli expression system and bacterial strain with respect to its similarity to commensal one in mouse to express the most active plant UGT71G1 mutant(s) against SN-38. Then we will deliver the most active plant UGT71G1 mutant(s) overexpressing bacteria into mouse to determine its ability and activity for metabolizing SN- 38 in vivo and develop drug detoxifying bacteria for the detoxification of SN-38.