Abstract

Typical CYP2D6 substrates generally contain a basic nitrogen atom that interacts with Asp³⁰¹ and/or Glu²¹⁶ and an aromatic moiety adjacent to the site of metabolism. Recently, we found novel acidic substrates for CYP2D6, pactimibe, and its indole metabolite, R-125528, that are not protonated but are negatively charged at physiological pH. The K_m value of R-125528 in CYP2D6-expressing microsomes was determined to be 1.74 μM, which was comparable with those of typical basic CYP2D6 substrates (1–10 μM). Pactimibe has lower affinity than R-125528; however, the K_m value was comparable with that of metoprolol. Interestingly, their sites of metabolism, the ω-1 position of the N-octyl indoline/indole group, were relatively distant from the aromatic moiety. A pactimibe analog with an N-decyl chain was similarly labile against CYP2D6; however, analogs with N-hexyl or N-dodecyl chains were stable to CYP2D6. An induced fit docking of the ligands with an X-ray crystal structure of substrate-free CYP2D6 (Protein Data Bank code 2F9Q) indicated the involvement of an electrostatic interaction between the carboxyl group and Arg²²¹ and hydrophobic interaction between the aromatic moiety and Phe⁴⁸³. The docking model correctly positioned the site of metabolism above the heme. The effect of the N-alkyl chain length of pactimibe analogs on their CYP2D6 metabolic stability was plausibly explained by the docking model. In conclusion, we report herein a novel CYP2D6 binding mode for the acidic substrates pactimibe and R-125528. Further investigation, such as a site-directed mutation, will be necessary to directly demonstrate the involvement of Arg²²¹ in CYP2D6 binding.