Enzymology of a carbonyl reduction clearance pathway for the HIV integrase inhibitor, S-1360: role of human liver cytosolic aldo-keto reductases.
S-1360, a 1,3-diketone derivative, was the first HIV integrase inhibitor to enter human trials. Clinical data suggested involvement of non-cytochrome P450 clearance pathways, including reduction and glucuronidation. Reduction of S-1360 generates a key metabolite in humans, designated HP1, and constitutes a major clearance pathway. For characterization of subcellular location and cofactor dependence of HP1 formation, [(14)C]-S-1360 was incubated with commercially available pooled human liver fractions, including microsomes, cytosol, and mitochondria, followed by HPLC analysis with radiochemical detection. Incubations were performed in the presence and absence of the cofactors NADH or NADPH. Results showed that the enzyme system responsible for generation of HP1 in vitro is cytosolic and NADPH-dependent, implicating aldo-keto reductases (AKRs) and/or short-chain dehydrogenases/reductases (SDRs). A validated LC/MS/MS method was developed for investigating the reduction of S-1360 in detail. The reduction reaction exhibited sigmoidal kinetics with a K(m,app) of 2 microM and a Hill coefficient of 2. The ratio of V(max)/K(m) was approximately 1 ml/(min mg cytosolic protein). The S-1360 kinetic data were consistent with positive cooperativity and a single enzyme system. The relative contributions of AKRs and SDRs were examined through the use of chemical inhibitors. For these experiments, non-radiolabeled S-1360 was incubated with pooled human liver cytosol and NADPH in the presence of inhibitors, followed by quantitation of HP1 by LC/MS/MS. Quercetin and menadione produced approximately 30% inhibition at a concentration of 100 microM. Enzymes sensitive to these inhibitors include the carbonyl reductases (CRs), a subset of the SDR enzyme family predominantly located in the cytosol. Flufenamic acid and phenolphthalein were the most potent inhibitors, with > 67% inhibition at a concentration of 20 microM, implicating the AKR enzyme family. The cofactor dependence, subcellular location, and chemical inhibitor results implicated the aldo-keto reductase family of enzymes as the most likely pathway for generation of the major metabolite HP1 from S-1360.
Rosemond
M Jane Cox
MJ
Worldwide Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, NC 27709, USA. jane.c.rosemond@gsk.com
St  John-Williams
Lisa
L
Yamaguchi
Toshiro
T
Fujishita
Toshio
T
Walsh
John S
JS
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D019380:Anti-HIV Agents / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003600:Cytosol / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005663:Furans; D019428:HIV Integrase Inhibitors / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D014230:Triazoles
D019380:Anti-HIV Agents; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D005663:Furans; D019428:HIV Integrase Inhibitors; C451733:S 1360; D014230:Triazoles; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase