A high throughput assay for the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin by recombinant human UDP-glucuronosyltransferases and liver microsomes

Abstract

1. UDP-glucuronosyltransferases (UGTs) are versatile and important conjugation enzymes in the metabolism of drugs and other xenobiotics. 2. We have developed a convenient quantitative multi-well plate assay to measure the glucuronidation rate of 7-hydroxy-4-trifluoromethylcoumarin (HFC) for several UGTs. 3. We have used this method to screen 11 recombinant human UGTs for HFC glucuronidation activity and studied the reaction kinetics with the most active enzymes. We have also examined the HFC glucuronidation activity of liver microsomes from human, pig, rabbit and rat. 4. At a substrate concentration of 20 µM, the most active HFC glucuronidation catalysts were UGT1A10 followed by UGT1A6 >UGT1A7 >UGT2A1, whereas at 300 µM UGT1A6 was about 10 times better catalyst than the other recombinant UGTs. The activities of UGTs 1A3, 1A8, 1A9, 2B4 and 2B7 were low, whereas UGT1A1 and UGT2B17 exhibited no HFC glucuronidation activity. UGT1A6 exhibited a significantly higher Vmax and Km values toward both HFC and UDP-glucuronic acid than the other UGTs. 5. Human, pig and rabbit, but not rat liver microsomes, catalyzed HFC glucuronidation at high rates. 6. This new method is particularly suitable for fast activity screenings of UGTs 1A6, 1A7, 1A10 and 2A1 and HFC glucuronidation activity determination from various samples.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10

“… 44 In addition, 7-hydroxycoumarin and 7-hydroxy-4-(trifluoromethyl)coumarin have both been shown to be glucuronidated by several different UGTs. 25 The hydroxyl group of 7-hydroxycoumarin was oriented toward the UDPGA in the active site of the UGT1A10 model, indicating that there is space in the active site for an additional six or five ring substituent at position C3 of the 7-hydroxycoumarin scaffold ( Figure 2 ). Thus, we synthesized six new UGT1A10 substrates, all of which were 7-hydroxycoumarin derivatives with various substituents at position C3 of the coumarin scaffold.…”

“…The hydroxyl group at C7 on the coumarin scaffold renders the compound fluorescent and glucuronidation of this hydroxyl abolishes the fluorescence ( Figure 1 ). 25 Substituents at positions such as C3 or C4 of the coumarin do not quench the 7-hydroxycoumarin derivatives’ fluorescence but modify its intensity, depending on the substituent’s chemical nature. The 7-hydroxyl group on coumarin is also a good functional group for glucuronidation by many UGTs.…”

Chemical Probes for Human UDP‐Glucuronosyltransferases: A Comprehensive Review

“…However, the design and development of fluorescence probes for UGTs is a very difficult task, due to the following three reasons. First, the fluorescence emission of many fluorophores are often “turned‐off” or “blue‐shifted” following UGT1A1‐mediated O‐glucuronidation, which is unbeneficial for sensitive and precise measurement of the corresponding glucuronides in complex biological samples. Second, the crystal structures of the substrate binding domain (N‐terminal domain) of mammalian UGTs have not been reported yet, so it is very difficult to rationally design the candidate substrate, with the help of molecular docking‐based virtual screening from the views of ligand‐enzyme interactions.…”

“…The first generation of fluorescent probes for human UGTs are typical “On‐off” fluorescent probes, which always use a phenolic fluorophore as a substrate and the fluorescence signals will be turned off following glucuronidation. For instance, 7‐hydroxy‐4‐trifluoromethylcoumarin (HFC), a fluorescent compound, can be glucuronidated by multiple UGTs including UGT1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, and 2B7 . Glucuronidation of HFC at C‐7 phenolic abolishes HFC fluorescence, converting the highly fluorescent HFC into a non‐fluorescent glucuronide .…”

“…For instance, 7‐hydroxy‐4‐trifluoromethylcoumarin (HFC), a fluorescent compound, can be glucuronidated by multiple UGTs including UGT1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, and 2B7 . Glucuronidation of HFC at C‐7 phenolic abolishes HFC fluorescence, converting the highly fluorescent HFC into a non‐fluorescent glucuronide . Similarly, 7‐hydroxy‐6‐methoxycoumarin (7‐HMC, scopoletin) is also a “turn‐off” fluorescent probe substrate for UGTs.…”