Affection Of Various Polymorphic Variants Of Cytochrome P450 Oxidoreductase (POR) On Drug Metabolic Activity Of CYP3A4 And CYP2B6

POR (EC 1.6.2.4) is a flavoprotein localized in the endoplasmic reticulum and theouter membrane of the nuclear envelope in eukaryotic cells. It is the only flavoprotein that mediates electron transfer from NADPH to CYP enzymes. Disruption of POR gene in knockout mice results in early embryonic lethality, while liver-specfic knockout of POR results in a profound disruption of hepatic drug metabolism, but these mice are morphologically and reproductively normal. POR has the ability to reduce a host of exogenous electron accptors, includingcytochrome C, potassium ferricyanide, and 2,6-dichloroindophenol (DCIP), as well as the therapeutically important compounds such as mitomycin C and the benzotriazine SR4233, and environmental toxin, paraquat. The gene encoding human POR is quite genetically polymorphic. Since six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y and V608F) have been identified in 2004, a total of 40 have been reported till now. However, those previous work about the POR variants mainly focused on their relation to the P450 oxidoreductase deficiency, this new disorder of steroidogenesis. Seldom study has done about the influence of POR genetic polymorphisms on the metabolic activation of drugs and environmental toxins.The recombinant human metabolism enzyme is a useful tool for the xenobiotics metabolism study in vitro, which can provide interpretation and evidence for metabolism study in vivo, and even predict the situation in vivo to some extent. Using heterologously expressed proteins of variants of a human metabolism enzyme, the variation of the xenobiotic metabolism activities caused by genetic polymorphisms could be valued, the results of which should aid in the designing and interpreting molecular epidemiological studies. In the present study, the wild-type and six polymorphic variants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) with full-length gene were expressed in Sf9 insect cells. First, changes in activities of six human genetic variations POR were determined by measuring the reduction of Cyt c. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, the six mutants of POR were then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters.1. Construction and expression of Human CYP2A13 and its mutantsThere are several expression system used to heterologously express CYP450 protein, including E.coli, yeast, mammalian cell and baculovirus infected insect cells. Bac-to-Bac(?) baculovirus infected Sf9 insect cell expression system were effective in eukaryotic protein expression and CYP3A4. CYP2B6 and PORs were successfully expressed by this system in this experiment. The plasmid with cDNAs encoding wild type POR was use as template to obtain PORs aliens by site-directed mutagenesis. BamH I site and Xho I site were introduced at the 5'terminus and 3'terminus respectively, and a his-tag sequence was added at the 3'-side of PORs sequences. The correct POR alleles were verified by sequencing, and inserted into the pFastBacTM1 vector to generate recombinant pFastBacTM1- POTs. Recombinant pFastBacTM1-PORs was then transformed into MAX Efficiency DH10BacTM competent Escherichia coli to generate recombinant bacmid (Bacmd-PORs) through transposition. After identification of successful transposition by using PCR, the Sf9 cells were transfected with recombinant Bacmid-PORs and generated the recombinant baculovirus, which is the P1 Viral Stock. High-titer baculovirus were obtained by subsequently twice viral stock amplification. The expression conditions were optimized to obtain the highest production, including the multiplicity of infection (MOI). The recombinant viral titers of CYP3A4, CYP2B6 and wild type or mutant PORs were identified using real-time PCR. PORs proteins were expressed with optimized expression conditions and detected using anti-His antibody by western blot and the contents of the CYPs are determined by CO-difference spectrum. POR activity assay was performed by reduction of cytochrome c in the presence of wild type POR and mutants POR respectively. Sf9 cells infected with the virus of POR mutant (e.g., A115V, Y181D, A287P) showed significant desrease in POR activity, about 69%,13% and 21% respectively. G413S variants enhanced the enzyme activity of wide type up to approximately 77%. Regarding the remaining two POR-mutants, namely, S244C and K49N, their activity was similar to the wide-type activity, suggesting that the POR activities can be influenced by mutations in amino acid sequences in POR.2 Influence of various polymorphic variants of POR on drug metabolic activity of CYP3A4In this prat, we choose CYP3A4, the most abundant CYP isoform, as a model to determine the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 in vitro. We also use the testosterone as specific substrat to value the activity variation between CYP3A4-PORWT and mutants. In particular, activity of CYP3A4 was completely inhibited by the co-expression of Y181D and A287P (i.e., CYP3A4-POR (Y181D) and (A287P)) when the concentration of substrate testosterone is at 20μmol/L～400μmol/L. CYP3A4-POR (K49N) and CYP3A4-A115V were exhibited a significant changes in the Km (1.3-folds) and Vmax (1.2-folds) values when compared with CYP3A4-PORWT and increased the catalytic efficiency (CLint) up to 35% of wild-type. In the agreement with 77% increased activity of reduction of Cytochrome C with POR mutant G413S, CLint values of CYP3A4-POR (G413S) were calculated to increase up to 65% of wide-type for metabolism of testosterone. In this part, the results indicated that no single assay based on one kind of metabolism POR directly involved in could predict activities of POR mutations for coupling with all other CYP450 redox partners. Specific effects of many POR variants may vary with different partner proteins and there should be some different mechnisem in the process.3 Influence of various polymorphic variants of POR on drug metabolic activity of CYP2B6In this part, we choose CYP2B6 represents only 2%-10% of total human hepatic complement of cytochrome P450 enzymes, which is identified to participate in the metabolism of many clinically important drugs, as a model to determine the effect of polymorphic variants of POR on drug metabolic activities of CYP2B6 in vitro. We choose bupropion as specific substrat to value the activity variation between CYP3A4-PORWT and mutants. Regarding the impact of six POR genetic mutations on catalytic activities of CYP2B6 (i.e., CYP2B6-POR), we found that influencing of CYP2B6 activities by the six mutants POR were not so strong like that of CYP3A4. On one hand, activity of CYP2B6 was not affected significantly as CYP3A4 by its Y181D and A287P of POR, despite activity of CYP3A4 was completely inhibited by its mutants POR Y181D and A287P. More interestingly, metabolism of bupropion by CYP2B6 with K49N POR (i.e., CYP2B6-POR (K49N) significantly reduced its Vmax to 58% of wide type levels, and also reduced the catalytic efficiencies (CLint) approximately to 74% of CYP2B6-WT. while the metabolism of testosterone by CYP3A4 with mutant K49N (i.e., CYP3A4-POR (K49N)) enhanced its catalytic efficiencies up to 58% of wide-type levels. In the last, CLint values of CYP3A4-POR (Al 15V) and CYP3A4-POR (G413S) were calculated to increase up to 36% and 65% of wide-type for metabolism of testosterone respectively. Moreover, two mutations Al 15V G413S in POR did not influence the activity of CYP2B6. Regarding the previous study, we predict that the extent to which the catalytic activities of CYP450 were altered was not only dependent on the specific POR mutations but also on the different CYP450 redox partners. Therefore, each POR variants will require testing with each CYP superfamily member and other drug metabolizing P450s to assess its effects. To make the CYP-mediated drug metabolism affected by POR mutations more clearly, additional studies covering different other types of CYP450s are required to take either in vitro or in vivo drug clearance tests.Our results suggested that the POR variants may have different effects on supporting different CYPs with different substrates as well. There is no easy way to predict the effect of all the POR variants on all the bioactivation of substrates or the effects on CYP450 metablism. Therefore, each POR variants will require testing with each CYP superfamily member and other drug to assess its effects in order to make the CYP-mediated drug metabolism affected by POR mutations more clearly. So the polymorphic variants of POR should be considered as a new important target gene in pharmacogenetics study and the genetic variations of POR may serve as a biomarker to predict the POR-involved metabolic activity variation of CYP450 and contribute to the development of personalized medicine in the near future. In particular, we proposed that the prescription of medication to the patients with POR mutations identified to cause serious effects on activity of CYP3A4 and CYP2B6 should be monitored carefully. Our work does a good attempt to set up a database about the infuence of POR genetic polymorphisms for the clinical use.