Glycosidation of an endothelin ETA receptor antagonist and diclofenac in human liver microsomes:: Aglycone-dependent UDP-sugar selectivity

Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ETA receptor antagonist, Compound A [(+)-(5S, 6R, 7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno[1,2-b] pyridine 6-carboxylic acid], to form an acyl glucuronide and a glucoside ( Tang et al., 2003), two additional nucleotide sugars [UDP- galactose (UDP- gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP- gal, but not UDPGlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP- sugars showed pH-dependent kinetics and appeared to compete with each other, with IC50 values parallel to their respective apparent Km values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent Km for UDP-GlcA of 96 +/- 17 mu M. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDPGlc and UDP-gal displayed K-i values of 2054 +/- 108 mu M and 1277 +/- 149 mu M, > 10-fold greater than the K-m for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglyconedependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.