Metabolism of phenylahistin enantiomers by cytochromes P450: a possible explanation for their different cytotoxicity.

Phenylahistin is a fungal diketopiperazine derived from isoprenylated (Phe-DeltaHis) cyclodipeptide. The (-)-enantiomer is a cell cycle inhibitor, which can be potentially used as an antitumor agent. By contrast, the (+)-enantiomer exhibits no antimicrotubule activity. To better understand the differences that could arise from a difference of bioavailability, we investigated the interaction and metabolism of both enantiomers with mammalian cytochromes P450 (P450s). We found that both enantiomers were metabolized by various isoforms of mammal P450 with a noticeable activity for the (+)-enantiomer. P450 3A isoforms were mainly responsible for this metabolism, the bioactive (-)-enantiomer being 1.5 to 8 times less metabolized than the (+)-enantiomer. Spectral analysis of the interaction with P450s revealed that (-)-phenylahistin led to a hydrophobic type I signature, whereas the (+)-isomer yielded a Fe-N type II one. Structural analysis of metabolites by liquid chromatography-tandem mass spectrometry allowed us to characterize two major metabolites (P1 and P3) for both enantiomers. In human liver microsomal preparations, P1 was predominant in the (-)-phenylahistin metabolic profile. In contrast, (+)-phenylahistin mainly produced P3 in human microsomes and CYP3A human expressed P450s. (-)-Phenylahistin proved to be less toxic on P450-rich hepatocytes than on P450-deprived KB lines. The slower metabolism of this enantiomer could account for its higher toxicity. This is strengthened by the fact that isolated metabolites of (-)-phenylahistin showed no toxic effects toward KB lines. Finally, differences of metabolism and interaction mode between both phenylahistin enantiomers and CYP3A4 were supported by in silico molecular docking calculations.