title: Glycosidation of an endothelin ET(A) receptor antagonist and diclofenac in human liver microsomes: aglycone-dependent UDP-sugar selectivity.
abstract: 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 ET(A) 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 UDP-GlcNAc, 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 K(m) values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent K(m) for UDP-GlcA of 96 +/- 17 microM. 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, UDP-Glc and UDP-gal displayed K(i) values of 2054 +/- 108 microM and 1277 +/- 149 microM, >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 aglycone-dependent. 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.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, Sumneytown Pike, P.O. Box 4, WP75-100, West Point, Pennsylvania 19486-0004, USA. cuyue_tang@merck.com
authors.forename: Cuyue
authors.initials: C
authors.lastname: Tang
authors.forename: Bennett
authors.initials: B
authors.lastname: Ma
mesh_terms: D004008:Diclofenac / Q000378:metabolism*; D065130:Endothelin A Receptor Antagonists; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000502:physiology; D006027:Glycosides / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D065130:Endothelin A Receptor Antagonists; D020719:Glucuronides; D006027:Glycosides; D014539:Uridine Diphosphate Sugars; D004008:Diclofenac; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase