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 nonsteroidal 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 drugmetabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells.Prostate cancer (PCa) is the second most common type of cancer in men and the fifth leading cause of death worldwide 1 . Several treatments have been developed against PCa, but drug resistance occurs rapidly, leading to a disease state known as castration-resistant prostate cancer (CRPC) 2,3 . In CRPC, androgens produced by the tumour and/or the adrenal gland drive disease progression. Thus, reduction or suppression of hormone levels in the cancer cells remains a key point in advanced stages of the disease.Cytochrome P450 17A1 (CYP17A1) is a monooxygenase involved in the synthesis of steroidal hormones. CYP17A1 converts pregnenolone to dehydroepiandrosterone and progesterone to androstenedione by two subsequent reactions, the 17α -hydroxylase and 17,20-lyase reactions (cf. Fig. 1). The hydroxylase reaction generates intermediates in the biosynthesis of glucocorticoids, while both hydroxylase and lyase reactions are required for biosynthesis of androgens and oestrogens 4 . CYP17A1 is therefore a pivotal target in the treatment of hormone-dependent tumours such as prostate cancer [5][6][7] .Several CYP17A1 inhibitors have been developed over the years, but only abiraterone (cf. Fig. 2) has been approved by the FDA for treating CRPC. Abiraterone consists of a steroidal scaffold with a pyridin-3-yl moiety in position 17 that inhibits CYP17A1 through coordination to the haem iron 8 . Oxygen binding to the haem iron is necessary for all CYP17A1 catalysis, so abiraterone binding is inhibitory. Together, the steroidal scaffold and the aromatic nitrogen-containing ring give abiraterone a promiscuous profile with affinity toward steroid receptors and other CYP enzymes, which likely contribute to the undesirable side effects observed in patients receiving abiraterone treatment 9 . Combinatorial synthesis programmes have been started by pharmaceutical companies to identify non-steroidal inhibitors and two such compounds, orteronel 10 and VT-464 11 , have been evaluate...
Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17-C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have characterized the reaction path for both the hydroxylase and lyase reactions using density functional theory (DFT) calculations and the enzyme-substrate interactions by molecular dynamics (MD) simulations. Activation barriers for positions subject to hydroxylase reaction have values close to each other and span from 54 to 60 kJ·mol with a small preference for 17α hydroxylation, in agreement with experimental observations. For the lyase reaction, two different types of mechanisms, concerted and stepwise, with identical activation energies (87 kJ·mol) were identified. Embedding the DFT-optimized transition states (TSs) for the two reactions into the active site of CYP17A1 showed that the TS for the C17 hydroxylation needs to be distorted by 13 kJ·mol, whereas the TS for the 17,20 lyase reaction easily can be accommodated in the protein. Finally, differences in the hydrogen-bond pattern of the substrates were detected both in the CYP17A1-Cpd I and CYP17A1-POA complexes, with the former found to be more pivotal for the hydroxylation site than the latter, suggesting a possible explanation for the slower conversion of CYP17A1 for 17α-hydroxyprogesterone over 17α-hydroxypregnenolone. The results support the concept that the selectivity of the steroidogenic CYPs is ruled by direct interactions with the enzyme, in contrast to the selectivity of drug-metabolizing CYPs, where the reactivity of the substrates dominates.
Aflatoxin B1 (AFB1) is a chemically intriguing compound because it has several potential sites of metabolism (SOMs), although only some of them are observed experimentally. Cytochrome P450 (CYP) 3A4 and 1A2 are the major isoforms involved in its metabolism. Here, we systematically investigate reactivity and accessibility of all possible SOMs in these two CYPs to elucidate AFB1 metabolism. DFT calculations were used to determine activation energies for each possible reaction. Aliphatic hydroxylation on position 9A and 3α are energetically favored, whereas position 9 is the preferred site for epoxidation. Docking studies, molecular dynamics (MD) simulations, and free energy (MM/GBSA) calculations were applied to elucidate the accessibility of each SOM. The most stable binding modes in CYP3A4 favor the formation of the 3α-hydroxylated and 8,9-exo-epoxide metabolites. Conversion of the methoxy group is also sterically possible, but not observed experimentally due to its low reactivity. In the CYP1A2 active site, AFB1 cannot orient position 3 towards the catalytic center, whereas the 8,9-exo-epoxide and 9A-hydroxylated metabolites are formed from the most stable and the 8,9-endo-epoxide from a less stable binding mode, respectively. The results agree with experimental data and suggest that both reactivity and the shape of the enzyme active site need to be considered to understand the distribution of SOMs and to improve current SOM prediction methods.
Dihydroorotate dehydrogenase (DHODH) is an important drug target due to its prominent role in pyrimidine biosynthesis. Leflunomide and brequinar are two well-known DHODH inhibitors, which bind to the enzyme in the same pocket with different binding modes. We have recently realized a series of new inhibitors based on the 4-hydroxy-1,2,5-oxadiazole ring, whose activity profile was found to be closely dependent on the degree of fluorine substitution at the phenyl ring adjacent to the oxadiazole moiety; a positive influence of fluorine on the DHODH inhibitory potency was observed previously [Baumgartner et al. (2006) J Med Chem 49:1239-1247]. Potential energy surface scans showed that fluorine plays an important role in stabilizing the bioactive conformations; additionally, fluorine influences the balance between leflunomide-like and brequinar-like binding modes. These findings may serve as a guide to design more potent DHODH inhibitors.
The potential endocrine disrupting effects of the commonly prescribed anti-epileptic drug lamotrigine (LAM) were investigated using the H295R steroidogenic in vitro assay and computational chemistry methods. The H295R cells were exposed to different concentrations of LAM, and a multi-steroid LC-MS/MS method was applied to quantify the amount of secreted steroid hormones. LAM affected several steroid hormones in the steroidogenesis at therapeutic concentrations. All progestagens as well as 11-deoxycorticosterone and corticosterone increased 100-200% with increasing concentrations of LAM suggesting a selective inhibitory effect of LAM on CYP17A1, in particular on the lyase reaction. Recombinant CYP17A1 assay confirmed the competitive inhibition of LAM toward the enzyme with IC50 values of 619 and 764 μM for the lyase and the hydroxylase reaction, respectively. Levels of androstenedione and testosterone decreased at LAM concentrations above the therapeutic concentration range. The ability of LAM to bind to CYP17A1, CYP19A1, and CYP21A2 was investigated using docking and molecular dynamics simulations. This in silico study showed that LAM was able to bind directly to the heme iron in the active site of CYP17A1, but not CYP21A2, thus supporting the results of the in vitro studies. The molecular dynamics simulations also suggested binding of LAM to the heme iron in the CYP19A1 active site. No inhibition of the aromatase enzyme was, however, observed in the H295R assay. This could be due to a sequential effect within the steroidogenesis caused by the inhibition of CYP17A1, which reduced the amounts of androgens available for CYP19A1.
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