TY - JOUR T1 - Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.120.265850 SP - jpet.120.265850 AU - Matthew G. McDonald AU - Lindsay M. Henderson AU - Sutapa Ray AU - Catherine K. Yeung AU - Amanda L. Johnson AU - John P. Kowalski AU - Helmut Hanenberg AU - Constanze Wiek AU - Kenneth E. Thummel AU - Allan E. Rettie Y1 - 2020/01/01 UR - http://jpet.aspetjournals.org/content/early/2020/05/18/jpet.120.265850.abstract N2 - CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaskan Native people, ultra-rare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862) and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well-characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in E. coli, towards (S)-warfarin, phenytoin, flurbiprofen and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I and P279T variants but, as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wildtype protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 reductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen and (S)-naproxen to the expected mono-hydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wildtype CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen and (S)-naproxen. We conclude that carriers of the variant N218I and especially the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance.SIGNIFICANCE STATEMENT Novel gene variants of CYP2C9; M1L and N218I, along with P279T (CYP2C9*29), are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency towards several substrates, including the low therapeutic index drugs, (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this under-studied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities. ER -