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UGT engineering for detoxifying anticancer drug SN-38

抗癌药物SN-38解毒的UGT工程

基本信息

批准号：
8879323
负责人：
Xiaoqiang Wang
金额：
$ 45.16万
依托单位：
UNIVERSITY OF NORTH TEXAS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-07 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8879323
关键词：
Adverse effects Affect Amines Antineoplastic Agents Bacteria Biological Assay Colon DNA DNA-Directed DNA Polymerase Docking Drug Metabolic Detoxication Drug usage Engineering Enzymes Escherichia Escherichia coli Estradiol Flavonoids Glucuronosyltransferase Goals Health Human In Vitro Libraries Medicago truncatula Metabolism Methodology Methods Modeling Molecular Mus Mutagenesis Mutation Nitrosamines Oral Pharmaceutical Preparations Phase Plants Process Protein Engineering Proteins SN-38 Series Site Structure System Toxin UGT1A1 gene Uridine Diphosphate Xenobiotics bacterial vector base chemotherapy design drug development engineering design enzyme activity glycosylation glycosyltransferase improved in vivo insertion/deletion mutation mutant overexpression protein expression public health relevance screening small molecule vector wasting

项目摘要

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.

描述（由申请人提供）：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输送到靶部位，其他药物可能成为废物，甚至对人类有毒。尿苷二磷酸（UDP）糖基转移酶（UGT）是小分子（包括毒素和药物）糖基化的核心参与者，也是人体内关键的II相药物代谢酶。人UDP-葡萄糖醛酸基转移酶1A 1负责消除和解毒结构上不同的异生物质组（例如，SN-38、多环胺和亚硝胺）和内源性化合物。我们发现，植物蒺藜苜蓿UGT 71 G1也可以有效地代谢典型的人类UGT 1A 1底物，包括SN-38和雌二醇在体外。然而，植物UGT 71 G1的活性在体内不足以通过将过表达植物UGT 71 G1的细菌口服递送到小鼠中来解毒SN-38。我们建议开发更有活性的UGT和药物解毒细菌（DDB，使用E。大肠杆菌作为初始模型细菌）用于抗癌药物SN-38的代谢。我们将使用两种不同的方法来寻找更具活性的植物UGT 71 G1突变体，包括基于我们确定的植物截形苜蓿UGT 71 G1结构的合理结构UGT设计和工程改造，以及随机突变。我们将表达和纯化UGT突变体，并进行酶试验，以筛选对SN具有显著改善（至少增加10倍）催化活性的突变体。 38.我们还将筛选不同的E。大肠杆菌表达系统（载体）和细菌菌株。不同的E.大肠杆菌表达系统和细菌菌株可能在小鼠中表现不同并影响酶活性。我们将找到一个合适的E。大肠杆菌表达系统和细菌菌株中表达的抗SN-38活性最高的植物突变体UGT 71 G1与小鼠体内的突变体UGT 71 G1相似。然后，我们将最具活性的植物UGT 71 G1突变体过表达细菌导入小鼠体内，以确定其体内代谢SN- 38的能力和活性，并开发用于SN-38解毒的药物解毒细菌。