Human Cytochrome P450 2D6 Mechanism-based Inhibition by Schering 66712: Investigation of Site of Adduction
Butler, Brendan F.
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The inactivation of human liver cytochrome P450 2D6 (P450 2D6) by the mechanismbased inhibitor Schering 66712 (SCH66712) is not well understood. The irreversible, timedependent inhibition of P450 2D6 by SCH66712 was demonstrated by Palamanda et al and first published in 2001 [Palamanda, et al (2001) Drug Met. Dispos. 29, 863-7]. They also showed that the inactivation was not protected by superoxide dismutase, glutathione, or mannitol and that P450 2D6 could be partially protected by the competitively binding compound, quinidine [Palamanda, et al. (2001) Drug Met. Dispos. 29, 863-7]. The site of adduction on P450 2D6 has not yet been shown. Information about which amino acid residues are involved in this inhibition, however, may give insight to both the structure and function of P450 2D6. In this investigation, we attempted to determine if metabolites of SCH66712 bound to P450 apoprotein, heme prosthetic group, or both. A comparison of loss of P450 heme-CO spectrum after incubation with SCH66712 (~39% loss after 40 min) and loss of heme content after incubation with SCH66712 (~30% loss after 40 min) did not conclusively demonstrate adduction to heme. Autoradiography was used to visualize adduction to P450 apoprotein after incubation with 14C-SCH66712. The results suggested that radiolabeled metabolites bound to the apoprotein, but radioactive bands in the gel were very faint. Finally, ESI-LC-MS was used to detect adduction to either heme or apoprotein. No heme adduction was detected. A species with mass 57,109.0 amu, slightly larger than the sum of the P450 2D6 apoprotein (56,729.0 amu) and SCH66712 (~338 amu) was detected, suggesting that a metabolite bound to the P450 apoprotein. These results, however, were partially obscured by lipids in the reconstituted mixture.