Research Project
7074044
DESCRIPTION (provided by applicant): Glucuronidation is a major metabolic pathway for the inactivation and detoxification of endogenous and exogenous compounds in the human body. Glucuronides are formed by a super family of uridine diphosphoglucuronosyl transferases (UGTs) that convert a number of lipophilic xenobiotics (drugs, steroids, pollutants and pesticides) into more hydrophilic species by linking them to a polar sugar, which are then more readily eliminated from the body. Glucuronosyl derivatives of xenobiotic compounds are useful as standards in the analysis of urine or blood samples for illegal drugs, or exposure to biocides. They are also important in studies of the metabolism of new drugs, as metabolites might be more toxic or cause undesirable side effects. More recently, glucuronyl derivatives of pharmacologically active compounds have been investigated as potential prodrugs. As a result, efficient and economical methods for glucuronide production have been sought. The two main limitations for the preparative production of glucuronides needed as analytical standards are (1) the high cost of the UDP-glucuronic acid (UDPGA) co-factor and (2) the lack of availability and the relative instability of the human UGT isoforms, themselves. The former problem was resolved in the Phase I sponsored research, in which an efficient method was developed for the regeneration of UDPGA using a coupled enzyme method, thus reducing the cost of glucuronide production significantly. However, the lack of availability of the human UGTs remains to be a problem, as a result of the low yields produced in the baculovirus-infected insect cells currently used. In Phase II, we intend to solve the latter problem, by cloning and expressing 5 different UGT isozymes in E. coll., using our patented method for gene redesign and synthesis. To date, we have used this proprietary method to produce 7 human cytochromes with high activity at quantities not previously commercially available. We intend to use the knowledge gained from this to clone, express and produce human UGTs 1A1, 1A4, 1A6, 1A9 and 2B7 in the amounts necessary for the efficient synthesis of glucuronides at the preparative level.
