Non-steroidal CYP17A1 Inhibitors: Discovery and Assessment.

CYP17A1 is an enzyme that plays a major role in steroidogenesis and is critically involved in the biosynthesis of steroid hormones. Therefore, it remains an attractive target in several serious hormone-dependent cancer diseases, such as prostate cancer and breast cancer. The medicinal chemistry community has been committed to the discovery and development of CYP17A1 inhibitors for many years, particularly for the treatment of castration-resistant prostate cancer. The current Perspective reflects upon the discovery and evaluation of non-steroidal CYP17A1 inhibitors from a medicinal chemistry angle. Emphasis is placed on the structural aspects of the target, key learnings from the presented chemotypes, and design guidelines for future inhibitors

Promising Tools in Prostate Cancer Research:Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC(50) values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells

Synthesis and Structure-Activity Relationships of Novel Non-Steroidal CYP17A1 Inhibitors as Potential Prostate Cancer Agents.

Twenty new compounds, targeting CYP17A1, were synthesized, based on our previous work on a benzimidazole scaffold, and their biological activity evaluated. Inhibition of CYP17A1 is an important modality in the treatment of prostate cancer, which remains the most abundant cancer type in men. The biological assessment included CYP17A1 hydroxylase and lyase inhibition, CYP3A4 and P450 oxidoreductase (POR) inhibition, as well as antiproliferative activity in PC3 prostate cancer cells. The most potent compounds were selected for further analyses including in silico modeling. This combined effort resulted in a compound (comp 2, IC50 1.2 µM, in CYP17A1) with a potency comparable to abiraterone and selectivity towards the other targets tested. In addition, the data provided an understanding of the structure-activity relationship of this novel non-steroidal compound class

Steroidal CYP17 Inhibitors for Prostate Cancer Treatment: From Concept to Clinic

The successful application of therapeutic strategies to block the known growth stimulation property of estrogen in breast cancer, namely the aromatase (CYP19) inhibitors formestane (4-OH) and exemestane (Aromasin) [1], has paved the way for the investigation of inhibitors of other P450 enzymes that might impart the growth of hormone-dependent cancers [2]. Cytochrome P450 17α-hydroxylase,C17,20-lyase (CYP17) is at the crossroads of androgen and corticoid biosynthesis and has become a valuable target in prostate cancer (PC) treatment [3-8]. Androgens, which are produced in steroidogenic tissues, bind to the androgen receptor (AR) and initiate transcription which in turn results in the synthesis of prostate-specific proteins, as well as in cell proliferation. Systemic ablation of androgen by castration, either surgical or chemical, is highly effective in treating PC when the disease is hormone-dependent

Discovery of Novel Non-Steroidal Cytochrome P450 17A1 Inhibitors as Potential Prostate Cancer Agents

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines. The binding mode of these compounds is also discussed

Structural and Functional Evaluation of Steroidogenic Cytochrome P450 Enzymes

Cytochromes P450 (CYP450) are heme-containing monooxygenase enzymes that perform a variety of functions in humans, including xenobiotic metabolism and the production of endogenous signaling molecules. Six isoforms of cytochrome P450 including cytochrome P450 17A1 (CYP17A1) and cytochrome P450 21A2 (CYP21A2), are responsible for the generation of steroid hormones, making these enzymes crucial for development and homeostasis. However in certain pathological conditions, inhibition of steroidogenic cytochrome P450 enzymes can be therapeutically useful. For prostate cancers dependent on androgens such as dihydrotestosterone for tumor growth and proliferation, inhibition of CYP17A1, a required enzyme in androgen biosynthesis, is a promising treatment strategy. Although inhibition of CYP17A1 has been successful clinically, its additional roles in the biosynthesis of other steroid hormones and its similarity to other steroidogenic P450 enzymes can bring about off-target effects. An understanding of the structure and function of CYP17A1, as well as other steroidogenic countertarget enzymes such as CYP21A2, could therefore be fundamental to developing improved inhibitors of this enzyme for the treatment of prostate cancer. CYP17A1 performs two different reactions in the same active site to generate androgens, a hydroxylation reaction followed by a carbon-carbon bond cleavage (or lyase) reaction. While the second, carbon-carbon bond cleavage reaction commits a steroid to the androgen pathway, the initial hydroxylation reaction is also necessary for the production of glucocorticoids. Human CYP17A1 performs the hydroxylation reaction on two structurally similar substrates, Δ4-progesterone and Δ5-pregnenolone, which only exhibit differences on the A ring on the opposite end of the steroid from the carbon which is hydroxylated by CYP17A1. Following hydroxylation, Δ5-17α-hydroxypregnenolone will undergo the second lyase reaction, but the Δ4-17α-hydroxyprogesterone does not. To elucidate a structural basis for preferential lyase turnover of Δ5 steroids by human CYP17A1, the crystal structure of the enzyme containing a background mutation was determined in the presence of both hydroxylase substrates and both lyase substrates. These structures reveal some similarities in binding among all four substrates but also differences in positions relative to the heme iron between hydroxylase substrates, the poor lyase substrate 17α-hydroxyprogesterone, and the efficient lyase substrate 17α-hydroxypregnenolone. Observed differences in distances between 17α-hydroxyprogesterone or 17α-hydroxypregnenolone and the heme iron may reflect differential stabilization of the proposed intermediate for the 17,20-lyase reaction. In addition to substrates, steroidal inhibitors of CYP17A1 can have different configurations of the A ring. X-ray crystal structures of a series of inhibitors with such modifications were also determined with CYP17A1 to compare active site interactions among A-ring modified steroidal inhibitors. Modifications to the A ring of steroidal inhibitor abiraterone did not alter direct contacts with CYP17A1, but did change indirect contacts with the enzyme through active site water networks. The structure of CYP17A1 with one A-ring modified inhibitor was resolved to 2.0 Å, making it the highest resolution crystal structure of CYP17A1 to date, and revealed an additional steroid binding site in the periphery of enzyme that had not been fully appreciated in previous CYP17A1 structures. CYP17A1 and redox partner proteins were recombinantly expressed in E. coli and purified, providing a well-defined and well-controlled system for evaluation of CYP17A1 function and inhibition. This strategy was employed to compare hydroxylase and lyase deficiencies among clinically-reported mutants of the enzyme, as well as steroidal and non-steroidal clinical inhibitors for selective inhibition of the lyase reaction compared to the hydroxylase reaction. Most clinical inhibitors of CYP17A1 demonstrated 1- to 3- fold selectivity for 17,20-lyase inhibition over 17α-hydroxylase inhibition. Only one of the four inhibitors to reach clinical trials, S-orteronel, demonstrated 3- to 5- fold selectivity for 17,20-lyase inhibition compared to inhibition of progesterone and pregnenolone 17α-hydroxylase reactions. However, its enantiomer, R-orteronel demonstrated 8- to 11-fold selectivity. X-ray crystal structures of CYP17A1 with non-steroidal inhibitors reported to selectively inhibit the lyase reaction were also determined to investigate the structural basis for lyase selectivity. Finally, some CYP17A1 inhibitors have also been shown to interact with another P450 responsible for steroid biosynthesis, CYP21A2. The physiological consequences of off-target CYP21A2 inhibition by compounds developed to target CYP17A1 are complex. The crystal structure of human CYP21A2 was determined to provide a structural comparison to CYP17A1 and potentially aid in the design of inhibitors more selective for CYP17A1 over CYP21A2. Some structural features of the CYP17A1 active site, including a hydrophobic pocket over the I helix, are not conserved in CYP21A2. Exploitation of this pocket is a potential strategy for the development of inhibitors with reduced affinity for CYP21A2. In aggregate, the studies described herein use structural information coupled with functional analysis to better understand steroidogenic cytochromes P450. These enzymes act as targets and countertargets in the treatment of hormone dependent diseases including prostate cancer. More detailed knowledge of how these enzymes interact with both substrates and inhibitors could inform the development of better prostate cancer therapeutics

Functional Characterization of Cytochrome P450 17A1 (CYP17A1) Gene Variants for their Steroidogenic Enzymatic Activities.

Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids to drive cellular proliferation. Cytochrome P450 17A1 (CYP17A1), an essential enzyme for sex steroid synthesis, represents a clinically established drug target. Inhibiting CYP17A1 decreases androgen and estrogen biosynthesis and thereby blocks the growth of hormone-dependent cancers. The first part of this dissertation characterizes the previously unreported interaction between the FDA approved CYP17A1 inhibitor, abiraterone, and the estrogen receptor (ER). We show for the first time that abiraterone is a weak ER agonist in preclinical models of ER-positive breast cancer cells. Abiraterone induces cellular growth and expression of the ER response gene, GREB1, by binding to ER, and these effects are inhibited with the ER antagonist fulvestrant (ICI 182,780). To further investigate the impact of CYP17A1 expression in breast cancer, we engineered ER-positive MCF-7 cells to express CYP17A1 (MCF-7/CYP17A1). Progesterone treatment induces cell growth and GREB1 expression in these cells but not in the parental MCF-7 cells, which do not express CYP17A1. Tandem mass spectrometry (LC-MS/MS) analysis confirmed that following progesterone treatment, MCF-7/CYP17A1 cells synthesize downstream steroid products that require CYP17A1 activity including 17OH-progesterone, androstenedione, and testosterone. Treatment of these cells with either abiraterone or a novel CYP17A1 inhibitor decreases progesterone metabolite-induced GREB1 expression in a dose-dependent manner. In addition to studies of CYP17A1 in breast cancer, we further hypothesized that characterization of CYP17A1 genetic variants may lead to insights on enzyme structure and function. We therefore utilized a HEK-293T cell-based expression system to characterize the enzymatic properties of two CYP17A1 gene variants, D216H (rs200063521) and G162R (rs141821705). Our results show that the D216H variant selectively alters 16OH-progesterone production, while no effect on 17OH-progesterone synthesis was observed. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to wild-type. Proteasome inhibition with MG132 indicated that this variant is preferentially ubiquitinated and degraded prematurely. Overall, these studies have broadened our understanding of CYP17A1 enzymatic activity in breast cancer, as well as led to new insights into how CYP17A1 structure relates to enzyme function and stability.PhDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120831/1/ccapper_1.pd

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

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

Bioactivity of Curcumin on the Cytochrome P450 Enzymes of the Steroidogenic Pathway.

Turmeric, a popular ingredient in the cuisine of many Asian countries, comes from the roots of the Curcuma longa and is known for its use in Chinese and Ayurvedic medicine. Turmeric is rich in curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Curcuminoids have potent wound healing, anti-inflammatory, and anti-carcinogenic activities. While curcuminoids have been studied for many years, not much is known about their effects on steroid metabolism. Since many anti-cancer drugs target enzymes from the steroidogenic pathway, we tested the effect of curcuminoids on cytochrome P450 CYP17A1, CYP21A2, and CYP19A1 enzyme activities. When using 10 µg/ml of curcuminoids, both the 17α-hydroxylase as well as 17,20 lyase activities of CYP17A1 were reduced significantly. On the other hand, only a mild reduction in CYP21A2 activity was observed. Furthermore, CYP19A1 activity was also reduced up to ~20% of control when using 1-100 µg/ml of curcuminoids in a dose-dependent manner. Molecular docking studies confirmed that curcumin could dock onto the active sites of CYP17A1, CYP19A1, as well as CYP21A2. In CYP17A1 and CYP19A1, curcumin docked within 2.5 Å of central heme while in CYP21A2 the distance from heme was 3.4 Å, which is still in the same range or lower than distances of bound steroid substrates. These studies suggest that curcuminoids may cause inhibition of steroid metabolism, especially at higher dosages. Also, the recent popularity of turmeric powder as a dilatory supplement needs further evaluation for the effect of curcuminoids on steroid metabolism. The molecular structure of curcuminoids could be modified to generate better lead compounds with inhibitory effects on CYP17A1 and CYP19A1 for potential drugs against prostate cancer and breast cancer

Structural and Functional Evaluation of Clinically Relevant Inhibitors of Steroidogenic Cytochrome P450 17A1

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. (S)-seviteronel binds similarly to both observed CYP17A1 conformations. However, (S)-orteronel and (R)-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the (S)-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent (R)-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site