Characterization of the Inhibition of Allelic Variants of Cytochrome P450 2A6 with trans ‐Cinnamic Aldehyde

Cigarette smoking and nicotine addiction continues to be a leading cause of preventable death. The enzyme cytochrome P450 2A6 (CYP2A6), which is highly polymorphic with over forty known alleles, is the primary enzyme that catalyzes the initial oxidation of nicotine in humans. Studies have shown that trans ‐cinnamic aldehyde (tCA) is a mechanism‐based inactivator of this enzyme and is therefore a potential smoking cessation aid. However, the inhibitory effectiveness of tCA with the gene products of the different alleles is unknown. The goal of the work presented was to characterize inhibition of the CYP2A6 variant resulting from the *23 allele by tCA. This allele results in a point mutation (R203C). Site‐directed mutagenesis was used to engineer CYP2A6 *23 from a truncated form of the human CYP2A6 *1 gene. The recombinant protein was expressed in E. coli and purified. Using ligand‐binding titrations, we found that the K d for tCA binding to CYP2A6*23 was 7.1 μM, whereas the K d value for CYP2A6*1 was 10.6 μM. Consistent with previous reports, CYP2A6*23 had lower activity than that of CYP2A6*1; however the relative inhibition of both CYP2A6*23 and CYP2A6*1 by 80 μM tCA was similar. Therefore, while tCA has a slightly higher affinity for CYP2A6*23, the change to a cysteine at position 203 does not significantly affect the effectiveness of tCA as an inhibitor. So tCA could still be a potential smoking cessation aid for individuals with the CYP2A6*23 allele. Support or Funding Information National Institute on Drug Abuse Award Number 15DA042341; Pacific University Faculty Development Grant