| Description |
3-Substituted indoles like 3-methylindole, zafirlukast and MK-0524 are dehydrogenated by cytochrome P450s to form reactive 3-methyleneindolenine electrophiles, which covalently conjugate with protein and/or DNA nucleophilic residues to cause toxicities. Likewise, the dehydrogenation of indolines to form indoles is a potentially important transformation. However, the mechanisms that govern the dehydrogenation reaction by selected P450s have not been fully established. The goal of this dissertation was to evaluate the ability of P450s to catalyze the dehydrogenation of indole and indoline compounds. In the present study, the in vitro metabolism of another 3-substituted indole, SPD-304, a newly discovered small-molecule TNF antagonist, together with indoline and indoline-containing drugs including indapamide, DW2282 (anti-cancer), SB-206553 (5-HT 2C/2B antagonist), SB-224289 (5-HT 1B inverse agonist) and pyroquilon, and several synthetic indoline derivatives, were investigated. We found SPD-304 was bioactivated through a dehydrogenation mechanism identical to 3-methylindole, to produce an electrophilic 3-methyleneindolenine, which was trapped by the nucleophile glutathione. This potentially important new drug caused the selective mechanism-based inactivation of CYP3A4. In addition, we also found that all the indolines were aromatized to indoles through a novel dehydrogenation pathway, which appeared to be initiated by C-H bond breakage instead of nitrogen oxidation. The metabolic profiles of the substrates were also assessed by in silico molecular docking. Dehydrogenation of SPD-304 appeared to be a major metabolic pathway, and the arginine 212 residue in the active site of CYP3A4 played an important role in hydrogen bonding with all indoline nitrogens by positioning the C-2 and C-3 carbons of indolines close to the heme iron for dehydrogenation. Using this in silico docking strategy, in combination with site-directed mutagenesis and homology modeling techniques, pulmonary cytochrome P450 2F3 and mutants were constructed and docked with substrate 3-methylindole. Substrate orientation favoring dehydrogenation was identified and verified by enzyme kinetic studies, with additional critical residues in the active site of CYP2F3 identified that could direct the selective dehydrogenation reaction. This research has significantly broadened our current understanding about the mechanisms of cytochrome P450-mediated dehydrogenation of xenobiotic indole and indoline compounds. |
