Genetically engineering a new system for the expression of cytochrome P450 enzymes in insect cells using novel P450 reductases

Human cytochrome P450 enzymes (CYPs) are a superfamily of haem-binding monooxygenase enzymes involved in the metabolism of xenobiotics such as toxins, carcinogens and pharmaceutical drugs as well as in the biosynthesis of cholesterol, vitamins and steroids. In recent years, the area of drug metabolism in the drug discovery process has become crucial for the final clinical success of a drug candidate. Use of CYPs early in the drug discovery process can save significant amount of costs and time required in pre-clinical and clinical studies, thereby greatly facilitating the process. The human CYP superfamily of proteins comprises more than 50 enzymes, each enzyme being able to catalyse multiple reactions. With the exception of some plants, a single NADPH cytochrome P450 reductase (CPR) of a particular eukaryotic species interacts with all CYPs of the same species. For CYP catalytic activity, CPR is absolutely essential, however at the same time CPR is detrimental for the expression of CYPs. Therefore, understanding the process by which the interaction between CYP and CPR occurs is an important biological goal. I have cloned, expressed and studied the interactions of seven CYPs, CYP2D6, CYP3A4, CYP1A1, CYP1B1, CYP1A2, CYP2E1 and CYP2C8 in conjunction with different CPR species (the native human CPR, variants of human CPR and the yeast CPR) using the baculovirus expression system. In my studies I have found that different CPRs have different coupling efficiencies towards the individual CYP 3 isoforms. Use of a high-activity CPR from yeast in this study has allowed us to improve our understanding on CYP-CPR interactions. I have found that the ability of a CPR to reduce an artificial substrate like MTT is not directly proportional to its ability to reduce the physiological substrate, CYP. In other words, the strength of the reductase does not determine CYP activity but it is the ability of CPR to couple with CYP which is crucial. This study has resulted in the identification of ΔhRDM, a genetically engineered variant of human CPR, which couples with CYPs far better than the human native CPR and also offers advantage of better reaction rates for CYPs. ΔhRDM also offers an improvement in the ratio of spectrally active CYP2D6 to spectrally inactive CYP2D6. Identification of ΔhRDM has allowed us to devise an insect cell expression system that genetically provides an improvement in the levels and activities of the drug metabolising cytochrome P450 enzymes.