Metabolic activation of a novel 3-substituted indole-containing TNF-alpha inhibitor: dehydrogenation and inactivation of CYP3A4.
SPD-304 is a recently discovered small-molecule TNF-alpha antagonist. However, SPD-304 contains a potentially toxic 3-alkylindole moiety. Previous studies on 3-methylindole and the 3-alkylindole-containing drugs zafirlukast and MK-0524 structural analogues found that they were bioactivated by cytochrome P450s through a dehydrogenation process to form 3-methyleneindolenine intermediates that are electrophilic alpha,beta-unsaturated iminium species. These electrophiles could react with protein and/or DNA nucleophilic residues to cause toxicities. In the present study, we found that SPD-304 was bioactivated through a similar dehydrogenation mechanism to produce a similar electrophilic 3-methyleneindolenine intermediate. The electrophile was trapped with nucleophilic glutathione and identified by LC/MS/MS. The iminium or another reactive intermediate also was a mechanism-based inactivator of CYP3A4. The inactivation parameters were K I = 29 microM and k inact = 0.047 min (-1). In addition, SPD-304 was metabolized through hydroxylation, N-dealkylation, and epoxidation pathways, and several metabolites and glutathione adducts were characterized by tandem mass spectrometry. The metabolism profile was also evaluated by in silico molecular docking of SPD-304 into the active site of CYP3A4, which predicted that the dehydrogenation reaction was initiated by 3-methylene C-H atom abstraction at the trifluoromethylphenyl-1 H-indol-3-ylmethyl portion of SPD-304. Hydroxylation of the 6'-methyl of the dimethylchromone portion of SPD-304 was the other major predicted metabolic pathway. The molecular models correlated precisely with experimental metabolic results. In summary, dehydrogenation of SPD-304 may cause toxicities through the formation of electrophilic intermediates and cause drug-drug interactions through CYP3A4 inactivation.
Sun
Hao
H
Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Room 201, Salt Lake City, Utah 84112-5820, USA.
Yost
Garold S
GS
D000478:Alkylation; D002839:Chromans / Q000737:chemistry / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000493:pharmacokinetics*; D004852:Epoxy Compounds; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006865:Hydrogenation; D007211:Indoles / Q000737:chemistry / Q000493:pharmacokinetics*; D008862:Microsomes, Liver / Q000378:metabolism; D008961:Models, Structural; D012862:Skatole / Q000031:analogs & derivatives*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D002839:Chromans; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D007211:Indoles; C527814:SPD-304; D014409:Tumor Necrosis Factor-alpha; D012862:Skatole; D051544:Cytochrome P-450 CYP3A; D005978:Glutathione