A New Co Expressed P and UGT Platform for the Simultaneous Preparation of P Metabolites and Their Glucuronides

Toxins pharmaceuticals and physiologically indogenous compounds are metabolized by the liver via several classes of enzymes The uridine diphosphate glucuronosyltransferases UGTs are concentrated in the liver but also present in other tissues These enzymes add UDP glucuronic acid UDPGA to a compound thereby increasing its solubility and ability to be excreted in the bile and urine Oxford Biomedical Research has created a proprietary method for processing a strain of Pichia pastoris optimized for expressing the cytochromes P CYP and oxidoreductase resulting in a growing product line tailored to pharmaceutical research This process transforms the yeast into a stabilized dry powder SDP format which exhibits remarkable organic solvent and thermal tolerance along with extended catalysis when compared to traditional recombinant enzymes The proposed multi catalytic platform capable of simultaneous P oxidation followed by UGT glucuronidation would give the pharmaceutical researcher a valuable tool for preparing glucuronides as well as studying the P UGT synergistic relationship This Phase I proposal will involve producing and optimizing three catalytic systems the first Pichia system will express the dehydrogenase responsible for converting UDP glucose to UDPGA the cofactor required for glucuronide production The second system will include expand on this platform and include the UGT B enzyme to create a catalyst capable of glucuronidating methylumbelliferone The third and final system will add into this expression system the CYP A and P oxidoreductase POR enzymes with development focusing on the optimization of andapos hydroxymidazolam glucuronide production Achieving these goals will give pharmaceutical research a robust easy to use system for performing two metabolically important reactions in one system P oxidation followed by UGT glucuronidation Pharmaceutical discovery involves the preparation and isolation of metabolites formed by the action of liver enzymes on promising drug candidates The cytochromes P and UDP glucuronosyltransferases are two of these enzymes which require mild and specific operating conditions and usually have short operating lifetimes We have created an enzymatic system which greatly improves the stability operating time and cost effectiveness of the cytochromes P and intend to integrate the UDP glucuronosyltransferases into the same system to improve their properties