Automation and Miniaturization of the Bioluminescent UGT-Glo Assay for Screening of UDP-Glucuronosyltransferase Inhibition by Various Compounds

Abstract

The uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17β-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug—drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors.

Screening of six UGT enzyme activities in human liver microsomes using liquid chromatography/triple quadrupole mass spectrometry

“…A high‐throughput UGT enzyme activity assay was developed using HLMs, a generic condition for incubation, and LC/MS/MS quantification. This screening method using a cocktail incubation and LC/MS/MS offers many advantages over previously published methods for evaluating UGT activity and inhibition . It did not require bioluminescence, and interference with or quenching of assays was not been observed.…”

“…This screening method using a cocktail incubation and LC/MS/MS offers many advantages over previously published methods for evaluating UGT activity and inhibition. [1,23] It did not require bioluminescence, and interference with or quenching of assays was not been observed. One major advantage is the increased throughput achieved by performing a cocktail incubation and simultaneous determination of six different metabolites derived from six UGT probe substrates by LC/MS/MS.…”

“…The resultant glucuronide conjugates are more hydrophilic than the parent compound and more favorable to clearance from the body. Due to this function, the UGT enzymes play an important role in the elimination of drugs and other chemicals . Therefore, changes in the glucuronidation rate of a compound can have pharmacological consequences for other drugs that are eliminated primarily via glucuronidation, leading to toxicity and clinically significant drug interactions .…”

“…Successful development of screening methods for P450 enzymes has had an important impact on drug discovery, empowering assessment of P450‐related drug interactions for new chemical entities (NCEs) . To date, only two screening methods for UGT have been developed, luminescence and LC/MS/MS . Luminescence‐based UGT inhibition screening is a high‐throughput method; however, it has limitations, including the cost of substrates, interference from testing chemicals, and use of a recombinant single enzyme due to the insufficient enzyme selectivity of most probe substrates .…”

“…Due to this function, the UGT enzymes play an important role in the elimination of drugs and other chemicals. [1] Therefore, changes in the glucuronidation rate of a compound can have pharmacological consequences for other drugs that are eliminated primarily via glucuronidation, leading to toxicity and clinically significant drug interactions. [2,3] Glucuronide metabolites usually show lower pharmacological activity, but some metabolites can be more active than the parent drug or generate highly reactive metabolites, such as acyl-glucuronide.…”