HTS Assay to Profile Glucuronidation of Small Molecules

Trubetskoy, Vladimir

Abstract

Glucuronidation is an important detoxification pathway that can affect pharmacokinetic and pharmacodynamic properties of therapeutic agents and produce reactive metabolites and potential drug-drug interactions. No suitable HTS assay for assessing drug glucuronidation and related drug-drug interactions exists. The need to study the complexity of UGT-related adverse drug reactions (ADRs) and toxicity in all population groups, including the young, the elderly, and carriers of polymorphic variants, demands development ofefficient HTS assays for small molecule glucuronidation. Attempts to develop robust HTS UGT assays have failed, due to the challenges of working with dilute membrane enzyme preparations [low enzyme stability, significant light scattering, low signal-to-noise ratios, and low assay variability (Z'-factor)]. QBI proposes to develop a reliable, fluorescence-based HTS assay using UGT1A1 as a model enzyme. The assay will use a fluorescent UGT1A1 substrate and a polymeric reagent that acts as a solubilizer, stabilizer, and activator of UGT1A1. This polymer consists of a PEG-based amphiphile that forms stable polymeric micelles that can incorporate UGT1A1 into their core. The research plan includes the following aims. 1. Design and optimize an amphiphilic polymer-based solubilization / stabilization system for UGT1A1 that will maintain enzyme activity. 2. Optimize the solubilization/stabilization system developed in Aim 1 to develop a feasible method for assessing small molecule glucuronidation under HTS conditions. 3. Validate the HTS assay developed in Aim 2 by screening the small molecule library at the Small Molecule Screening Facility at the University of Wisconsin-Madison. The proposed project is important to the public health for 2 reasons. First, the potential for toxicity and adverse drug reactions from UGT activity in all segments of the population, but especially in infants and children, the elderly, and individuals with UGT polymorphisms. Due to the large number of compounds and drug candidates being synthesized, there is a vital need to develop highly robust HTS assays for discovering potentially problematic UGT-related drug-drug interactions.