Jessica L. Anderson1, John Schultz1, Wenhui Zhou2, Hui Wang2, Dieter Klaubert2, Terry Riss1 and Bob Bulleit1
1Promega Corporation, Madison, WI
2Promega Biosciences, San Luis Obispo, CA
The UDP glucuronosyltransferase (UGT) family of enzymes are involved in the metabolism of various compounds in the body. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and suitable for excretion. The UGTs act on various endogenous substrates, such as bilirubin, β-estradiol, and testosterone, as well as xenobiotics and drugs, such as diclofenac, morphine and valproic acid. The function of these enzymes is essential for the clearance of drugs and other toxins from the body and alteration of UGT activity could potentially cause drug-drug interactions in vivo. There is an increasing interest in the activity of these enzymes and their involvement in drug metabolism. The current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to higher throughput screening applications for UGT inhibitors or activators. Here, we present a new bioluminescent assay system for measuring UGT enzyme activity in vitro. Our assay does not involve any protein precipitation or chromatographic steps and is easily performed in a multi-well plate format. We have shown the ability of our assay to measure activity of many recombinant UGT enzymes (including UGTs 1A1, 1A4, 1A6, 1A8, 1A9, 1A10, 2B7, and 2B15) as well as assessing endogenous UGT activities from animal tissue microsomes (including human liver, renal, and intestinal microsomes, as well as mouse and rat liver microsomes). We were able to detect inhibition by compounds known to inhibit numerous isozymes (diclofenac) and we verified published data showing that some isozymes are inhibited by the HIV protease inhibitors liponavir and ritonavir while other isozymes are relatively unaffected. Assay variability, as measured by Z′ values, have been calculated for UGT 1A1 [Z′ = 0.83] and UGT 2B7 [Z′ = 0.67]. Our new assay format could greatly increase the throughput for assessing UGT activity and enable efficient screening of UGT isozymes against compound libraries.