UGT1A1/UGT1A3 Inhibition Assay
Uridine 5’-diphospho‐glucuronosyltransferases (UGTs) are a superfamily of enzymes which catalyse the glucuronidation reaction, a major phase II metabolism pathway. Approximately 15% of approved drugs have glucuronide metabolites1, highlighting the importance of UGT enzymes. UGT inhibition is less often observed than CYP inhibition, but is a clinically significant form of drug-drug interaction (DDI), and can lead to toxicity.
DDIs can alter the concentration and duration profile of a co-administered drug, leading to clinical ineffectiveness or adverse effects. The FDA and EMA recommend that all new drugs should be investigated to understand their potential for DDIs. UGT inhibition should be explored when glucuronidation is a predominant metabolic elimination pathway, when the glucuronidation is catalysed by a single enzyme, and when the therapeutic concentrations of the inhibitor are close to the Ki of the target UGT2. It is recommended to study the inhibitory effect of both parent molecule and major metabolites.
The inhibition of UGT1A1 and UGT1A3 can have important implications for drug-induced liver injury. UGT1A1 is the only enzyme responsible for the glucuronidation of bilirubin, which makes the compound less toxic and more readily excreted. The inhibition of this enzyme can therefore lead to inadequate clearance of bilirubin from the body, resulting in a condition known as hyperbilirubinemia4. Glucuronidation is also an important metabolic pathway for hepatic bile acids. UGT1A3 is the enzyme responsible for the glucuronidation of chenodeoxycholic acid (CDCA)3, a major bile acid. Due to its detergent properties, CDCA is toxic. CDCA-glucuronide is less active, and more polar than the parent molecule. Inhibition of UGT1A3 can lead to accumulation of CDCA, and subsequent hepatotoxicity.
Sygnature’s UGT inhibition assay measures the capacity of a compound to inhibit the glucuronidation of a probe substrate. The probe substrate and test compound are incubated at several concentrations at 37°C with recombinant UGT supersomes in the presence of UDPGA. Alamethicin is used to reduce latency, and MgCl2 is added to the assay buffer to increase UGT activity5.
|Compound requirements||10 mM DMSO, 20µL||10 mM DMSO, 20µL|
|Substrate||10 μM β-estradiol||5 μM chenodeoxycholic acid (CDCA)|
|Test Article Concentrations||10-point IC50 in duplicate||10-point IC50 in duplicate|
|Incubation Time||60 min at 37°C||60 min at 37°C|
|Test System||Recombinant UGT1A1 supersomes||Recombinant UGT1A3 supersomes|
Figure 1. Inhibition of UGT1A1 and UGT1A3 by positive control inhibitors Silibinin and Ritonavir
Table 1. Example IC50 values generated in Sygnature’s UGT inhibition assay
|UGT isoform||Probe Substrate||Control Inhibitor||IC50|
|UGT1A1||β-estradiol||Silibinin||1.66 ± 0.41|
|UGT1A3||CDCA||Ritonavir||0.35 ± 0.24|
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- Williams et al., 2004, Drug Metab. Dispos., 32:1201–1208
- Remmel et al., 2007, in Drug Metabolism in Drug Design and Development, (Zhang D et al., eds); pp 37-88, John Wiley & Sons, Inc
- Trottier et al., 2006, Hepatology, 44:1158-1170
- Chang et al., 2013, Mol. Pharm., 10:3067-3075
- Fisher et al., 2000, Drug Metab. Dispos., 28:560-566