CYTOCHROMES P450 AS THERAPEUTIC TARGETS AND COUNTER-TARGETS FOR THE PREVENTION OF LUNG CANCER AND TREATMENT OF STEROIDOGENIC DISEASES

Abstract

Cytochrome P450 (CYP) is a superfamily of heme-containing monooxygenase enzymes that metabolize a variety of endogenous and exogenous substrates. These transformations can be advantageous in the role of homeostasis or clearance of foreign compounds. However, aberrant CYP activity or biotransformations of procarcinogens can be detrimental to human health. Thus cytochrome P450 enzymes can be both therapeutic targets and counter-targets. In the process of drug discovery, in vitro evaluation of both the efficacy and selectivity of drug candidates is necessary before in vivo studies can be pursued. In the case of the xenobiotic-metabolizing cytochrome P450 2A13 (CYP2A13), in vitro analysis was used to identify and evaluate selective inhibitors for reducing the risk of lung cancer in tobacco users. Additionally, in vitro biochemical analysis of the steroidogenic cytochromes P450 21A2 (CYP21A2) and 11B1 (CYP11B1) is being pursued for counter-target evaluation in the development of selective CYP17A1 inhibitors for the treatment of prostate cancer and the rational design of selective CYP11B1 inhibitors for the treatment of cortisol-dependent diseases. Lung cancer is the leading cause of all cancer related deaths and results in 6 million annual deaths worldwide. Since 80% of all lung cancer incidence is attributed to tobacco use but tobacco cessation methods are unsuccessful in 95% of users, an increased emphasis has been placed on lung cancer chemoprevention. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most prevalent procarcinogens compounds in tobacco and is selectively activated by CYP2A13 metabolism in the respiratory tract. The resulting diazonium ions are able to form DNA adducts and initiate lung cancer. Therefore, the selective inhibition of CYP2A13 offers a novel therapeutic strategy in the chemoprevention of lung cancer. High throughput screening identified the benzylmorpholine scaffold, and a small library was evaluated for both binding (Kd) and inhibition (Ki) of CYP2A13 versus the 94% identical hepatic cytochrome P450 CYP2A6 (CYP2A6), which does not efficiently metabolize NNK. These investigations identified the structural features of benzylmorpholine analogs responsible for selective binding and inhibition of CYP2A13 versus CYP2A6, leading to the determination of structure-activity relationships for the benzylmorpholine scaffold. Docking and X-ray crystallography studies were further employed to identify the atomic-level interactions between benzylmorpholine analogs and CYP2A13 but were hampered by apparent binding in multiple orientations. Nevertheless, these results could be used to design additional selective and potent CYP2A13 inhibitors for reducing the risk of lung cancer in tobacco users who are unable, unwilling, or in the process of ceasing tobacco use. In a similar pursuit to identify inhibitors of CYP17A1 for the treatment of prostate cancer, it became important to evaluate the selectivity of potential drug candidates against obvious counter-targets. CYP21A2 is involved in the biosynthesis of glucocorticoids and mineralocorticoids and has overlapping substrates with CYP17A1. CYP11B1 follows CYP21A2 in the steroid biosynthetic pathway and is also a counter-target for the development of CYP17A1 inhibitors. Additionally, CYP11B1's crucial role in cortisol production also presents this enzyme as an independent therapeutic target for the treatment of Cushing's disease resulting from cortisol overproduction. However, biochemical studies for both human CYP21A2 and CYP11B1 have been limited by protein availability. Human CYP21A2 was successfully cloned, expressed, purified, and crystallized for the first time, which allows for structural and functional studies of the human enzyme. CYP11B1 was also successfully cloned and expressed, but more optimization is necessary for consistent large-scale expression and purification. This work provides the necessary groundwork for a biochemical and biophysical understanding of both CYP21A2 and CYP11B1 for the evaluation of these enzymes as counter-targets. In addition these studies could lead to the rational design of CYP11B1 inhibitors for the treatment of cortisol dependent diseases.