The anti-epileptic drug lamotrigine inhibits the CYP17A1 lyase reaction in vitro.

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.

Dissecting the Cytochrome P450 1A2- and 3A4-Mediated Metabolism of Aflatoxin B1 in Ligand and Protein Contributions.

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.

Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions.

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-1 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-1) 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-1, 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.

Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

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 IC50 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.

Enantioselective endocrine disrupting effects of omeprazole studied in the H295R cell assay and by molecular modeling.

Enantiomers possess different pharmacokinetic and pharmacodynamic properties and this may not only influence the therapeutic effect of a drug but also its toxicological effects. In the present work we investigated the potential enantioselective endocrine disrupting effects of omeprazole (OME) and its two enantiomers on the human steroidogenesis using the H295R cell line. Differences in production of 16 steroid hormones were analyzed using LC-MS/MS. Additionally, to evaluate the differences in binding modes of these enantiomers, docking and molecular dynamics (MD) simulations of S-omeprazole (S-OME) and R-omeprazole (R-OME) in CYP17A1, CYP19A1 and CYP21A2 were carried out. Exposing H295R cells to OME and its enantiomers resulted in an increase of progesterone (PRO) and 17α-hydroxy-progesterone (OH-PRO) levels. At the same time, a decrease in the corticosteroid and androgen synthesis was observed, indicating inhibition of CYP21A2 and CYP17A1. In both cases, the effect of R-OME was smaller compared to that of the S-OME and a certain degree of enantioselectivity of CYP17A1 and CYP21A2 was suggested. Docking indicated that the N-containing rings of OME possibly could interact with the iron atom of the heme for S-OME in CYP17A1 and S- and R-OME in CYP21A2. However, density functional theory calculations suggest that the direct N-Fe interaction is weak. The study demonstrates enantioselective differences in the endocrine disrupting potential of chiral drugs such as omeprazole. These findings may have potential implications for drug safety and drug design.

The role of fluorine in stabilizing the bioactive conformation of dihydroorotate dehydrogenase inhibitors.

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.