In silico prediction of the site of oxidation by cytochrome P450 3A4 that leads to the formation of the toxic metabolites of pyrrolizidine alkaloids.

In humans, the metabolic bioactivation of pyrrolizidine alkaloids (PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation of their necine bases at C3 or C8 of heliotridine- and retronecine-type PAs or at the N atom of the methyl substituent of otonecine-type PAs. However, no attempts have been made to identify which C atom is the most favorable site for hydroxylation in silico. Here, in order to determine the site of hydroxylation that eventually leads to the formation of the toxic metabolites produced from lasiocarpine, retrorsine, and senkirkin, we utilized the ligand-based electrophilic Fukui function f(-)(r) and hydrogen-bond dissociation energies (BDEs) as well as structure-based molecular docking. The ligand-based computations revealed that the C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of senkirkin were chemically the most susceptible locations for electrophilic oxidizing reactions. Similarly, according to the predicted binding orientation in the active site of the crystal structure of human CYP3A4 (PDB code: 4I4G ), the alkaloids were positioned in such a way that the C3 atom of lasiocarpine and retrorsine and the C26 of senkirkin were closest to the catalytic heme Fe. Thus, it is concluded that the C3 atom of lasiocarpine and retrorsine and C26 of senkirkin are the most favored sites of hydroxylation that lead to the production of their toxic metabolites.