Mechanisms for the Time-dependent Inhibition of the Human Nicotine-metabolizing Enzyme CYP2A6 by Cinnamaldehyde: Evidence for Formyl Hydrogen Abstraction and Heme Degradation

trans-Cinnamaldehyde (CA) was previously shown to selectively inhibit cytochrome P450 2A6-mediated metabolism through heme destruction without modification of the apoprotein. The goal of this study was to further investigate factors that influence the mechanism and potency of inhibition. Hydrogen atom abstraction was evaluated by measuring how the CYP2A6 inactivation rate (kinact) changed when protium atoms (1H) were selectively replaced with deuterium atoms (2H) at three different carbons (carbonyl, alpha, and beta) in CA. The structure-inhibitor relationship for CA analogs was advanced by evaluating how substituents at the ortho position influenced CYP2A6 inactivation rates, in a recombinant system using coumarin hydroxylase as a reporter of 2A6 activity. Metabolite identification was conducted since it is an indirect indicator of substrate orientation in the enzyme active site, and can provide clues about reaction routes that lead to inactivation. Heme analysis and metabolite identification studies used a reconstituted system with purified human 2A6 and rat reductase, and a QTOF mass spectrometer incorporating a mass defect filter for detection. 2H incorporation at the formyl position demonstrably slowed the rate of inactivation (kinact = 0.043 min−1 +/− 0.001; p < 0.00002; remaining activity = 17.1 +/− 0.6 and 10.5 +/− 0.4 pmol/min/pmol for 2H-CA and 1H-CA, respectively). The kinact for the alpha- and beta-2H-labeled CA did not differ from 1H-CA (0.058 +/− 0.004, 0.060 +/− 0.006, and 0.061 +/− 0.002 min−1, respectively). Also, kinact decreased as a function of the 2H-CA/1H-CA ratio. Mass defect filtering (range = 0.01–0.06 Da) revealed that the primary product from CYP2A6-mediated metabolism of 1H-CA and 2H-CA is cinnamic acid. Metabolites from epoxidation and aromatic hydroxylation were not observed. The relative peak area of cinnamic acid decreased 29% (n=5; p=0.025) for 2H-CA compared to 1H-CA. Electron withdrawing groups substantially increased kinact: CA (0.039 min−1), nitro-CA (0.223 min−1), trifluoromethyl-CA (0.142 min−1) and difluoro-CA (0.244 min−1). Substitution with an electron donating group (methyl) resulted in complete disappearance of time dependent inhibition. Thus, both resonance and inductive effects have a significant role in inactivation. No heme adducts were detected using mass defect filtering, suggesting adduct instability compared to positive controls (aminobenzotriazole). Together the results support the hypothesis that the formyl proton is abstracted during inactivation and the potency of CA analogs can be substantially enhanced by structural changes that stabilize the radical. Support or Funding Information Research reported in this presentation was supported by the National Institute On Drug Abuse of the National Institutes of Health under Award Number 15DA042341. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.