QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS FOR THE GLUCURONIDATION OF SIMPLE PHENOLS BY EXPRESSED HUMAN UGT 1 A 6 AND UGT 1 A 9

UGT1A6 and UGT1A9 have both been demonstrated to rapidly glucuronidate simple phenolic compounds. A series of simple phenols were selected and screened with both isoforms and then used as model substrates for the generation of Vmax and Km values. UGT1A6 showed a more restricted acceptance of phenolic substrates compared with UGT1A9. However, the affinity of UGT1A6 for these compounds exhibited higher Km values than UGT1A9, although rates of turnover were similar. Molecular surfaceweighted holistic invariant molecular descriptors were generated for each substrate and used to produce the first quantitative structure activity relationship models generated for expressed human UGTs. Models relating log of the Km value to the generated descriptors correlated well with the experimental data r value of 0.996 for UGT1A6 and r value of 0.83 for UGT1A9. Cross validation by a leave-one-out method also showed good predictive capability within the subset with a q value of 0.98 for UGT1A6 and q value of 0.73 for UGT1A9. Empirically, UGT1A6 Vmax decreased as the 4-substituent increased in size, and a trend was observed when UGT1A6 Vmax was plotted against molecular volume. The larger UGT1A6 substrates were typified by low activity and lower Km values than their smaller counterparts. Extrapolating from this, it was demonstrated that phenols with large 4-substituents, which were not UGT1A6 substrates, could inhibit 4-ethylphenol glucuronidation. The Km values for UGT1A9 showed a similar relationship to UGT1A6 but with much lower Km values and greater variability in range of this value. Glucuronidation is an important metabolic process for the clearance of drugs, endobiotics, and xenobiotics in all mammalian species. There are numerous instances where drugs are cleared extensively by direct glucuronidation (3 -azido-2 ,3 -dideoxythymidine, valproic acid, propofol, and morphine; Bertz and Granneman, 1997), although in many other cases, the involvement of glucuronidation is restricted to the conjugation of glucuronic acid to metabolites of phase I oxidative metabolism. Where drugs are significantly glucuronidated independently of phase I metabolism, the functional group to which the glucuronic acid is transferred can be a hydroxy (phenolic or aliphatic), a carboxylic acid or, in some cases, an amino group (primary, secondary, or tertiary) moiety. The UDP-glucuronosyltransferases (UGT) family can be separated into two distinct subfamilies by sequence similarity. The UGT1 family are all derived from a single gene by alternative splicing of four constant exons (exons 2–5) to a variable exon 1. The RNA transcript leads to a transferase protein exhibiting a certain substrate specificity. The UGT2 family of isoforms are known to be encoded by individual genes (Mackenzie et al., 1997). Characterization of cloned and expressed human UGT has demonstrated that many UGT family 1 isoforms are capable of glucuronidating phenols to varying degrees including UGT1A1 (Senafi et al., 1994), UGT1A3 (Green et al., 1998), UGT1A4 (Green and Tephly, 1996), UGT1A8, and UGT1A10 (Cheng et al., 1999). UGT family 2 isoforms also display activity toward phenols, although substrate acceptance and rate of glucuronidation seems to be more restricted (UGT2B15, Green et al., 1994; UGT2B7, Coffman et al., 1998). Two of the earliest UGT isoforms to be characterized were done so on the basis of their ability to glucuronidate phenols. UGT1A6 and UGT1A9 were both classified as phenol UGT isoforms due to the high turnover rates of these substrates (Ebner and Burchell, 1993). UGT1A9 demonstrated greater proficiency in glucuronidating bulky and complex phenols than UGT1A6, which was considered to be only capable of glucuronidating simple or planer phenols (Ebner and Burchell, 1993). The high glucuronidation activity toward simple phenolic substrates has been clearly illustrated to be present in human liver microsomes (Temellini et al., 1991), and UGT1A6 comprises a significant proportion of liver UGT 1-naphthol glucuronidation capacity (Ouzzine et