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.
This project is to engineer plant uridine diphosphate (UDP) glycosyltransferases (UGTs) and screen bacterial strains for detoxifying anticancer drug SN-38. We found plant Medicago truncatula UGT71G1 could 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 will develop more active UGTs by rational structure-based protein design and random mutagenesis, screen and identify suitable bacterial expression systems and strains for the development of drug detoxifying bacteria (DDB) for detoxifying anticancer drug SN-38. The successful approach will be also used to develop other plant UGTs for inactivating other substances in the colon that are toxic to humans with broad benefits for human health.
| He, Yu; Zhou, Lei; Gao, Song et al. (2018) Development and validation of a sensitive LC-MS/MS method for simultaneous determination of eight tyrosine kinase inhibitors and its application in mice pharmacokinetic studies. J Pharm Biomed Anal 148:65-72 |
