27 research outputs found
The molecular basis of dapsone activation of CYP2C9-catalyzed non-steroidal anti-inflammatory drug (NSAID) oxidation
OnlinePublPositive heterotropic cooperativity, or ‘activation’, results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of NSAIDs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4’-hydroxylation and S-naproxen Odemethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MD simulations additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4’-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of NSAIDs with a planar or near planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.Pramod C. Nair, Kushari Burns, Nuy Chau, Ross A. McKinnon, John O. Miner
Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes
Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses N-acetylglucosamine as preferred sugar donor and UGT3A2 uses UDP-glucose. The enzymatic characterization of UGT3A mutants, structural modelling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confer their unique sugar preferences. An asparagine (N391) residue in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-N-acetylglucosamine. Conversely, a phenylalanine (F391) residue in UGT3A2 favors UDP-glucose use. Mutation of N391 to F in UGT3A1 enhances its ability to utilize UDP-glucose and completely inhibits its ability to use UDP-N-acetylglucosamine. An analysis of homology models docked with UDP-sugar donors indicates that N391 in UGT3A1 is able to accommodate the N-acetyl group on C2 of UDP N-acetylglucosamine so that the anomeric carbon atom (C1) is optimally situated for catalysis involving H35. Replacement of N by F at 391 disrupts this catalytically-productive orientation of UDP N-acetylglucosamine but allows a more optimal alignment of UDP-glucose for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing the N391 residue, suggesting that other mammals may not have the capacity to N-acetylglucosaminidate small molecules. In support of this hypothesis, N391-containing UGT3A forms from two non-human primates were found to use UDP-N-acetylglucosamine, while UGT3A isoforms from non-primates could not use this sugar donor. This work gives new insight into the residues that confer sugar specificity to UGT family members and suggests a primate-specific innovation in glycosidation of small molecule
The novel UDP Glycosyltransferase 3A2: cloning, catalytic properties, and tissue distribution
The human UDP glycosyltransferase (UGT) 3A family is one of three families involved in the metabolism of small lipophilic compounds. Members of these families catalyze the addition of sugar residues to chemicals, which enhances their excretion from the body. The UGT1 and UGT2 family members primarily use UDP glucuronic acid to glucuronidate numerous compounds, such as steroids, bile acids, and therapeutic drugs. We showed recently that UGT3A1, the first member of the UGT3 family to be characterized, is unusual in using UDP N-acetylglucosamine as sugar donor, rather than UDP glucuronic acid or other UDP sugar nucleotides (J Biol Chem 283:36205–36210, 2008). Here, we report the cloning, expression, and characterization of UGT3A2, the second member of the UGT3 family. Like UGT3A1, UGT3A2 is inactive with UDP glucuronic acid as sugar donor. However, in contrast to UGT3A1, UGT3A2 uses both UDP glucose and UDP xylose but not UDP N-acetylglucosamine to glycosidate a broad range of substrates including 4-methylumbelliferone, 1-hydroxypyrene, bioflavones, and estrogens. It has low activity toward bile acids and androgens. UGT3A2 transcripts are found in the thymus, testis, and kidney but are barely detectable in the liver and gastrointestinal tract. The low expression of UGT3A2 in the latter, which are the main organs of drug metabolism, suggests that UGT3A2 has a more selective role in protecting the organs in which it is expressed against toxic insult rather than a more generalized role in drug metabolism. The broad substrate and novel UDP sugar specificity of UGT3A2 would be advantageous for such a function
Towards integrated ADME prediction: past, present and future directions for modelling metabolism by UDP-glucoronosyltransferases
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