Structure-based virtual screening of CYP1A1 inhibitors: towards rapid tier-one assessment of potential developmental toxicants.

Cytochrome P450 1A1 (CYP1A1) metabolizes estrogens, melatonin, and other key endogenous signaling molecules critical for embryonic/fetal development. The enzyme has increasing expression during pregnancy, and its inhibition or knockout increases embryonic/fetal lethality and/or developmental problems. Here, we present a virtual screening model for CYP1A1 inhibitors based on the orthosteric and predicted allosteric sites of the enzyme. Using 1001 reference compounds with CYP1A1 activity data, we optimized the decision thresholds of our model and classified the training compounds with 68.3% balanced accuracy (91.0% sensitivity and 45.7% specificity). We applied our final model to 11 known CYP1A1 orthosteric binders and related compounds, and found that our ranking of the known orthosteric binders generally agrees with the relative activity of CYP1A1 in metabolizing these compounds. We also applied the model to 22 new test compounds with unknown/unclear CYP1A1 inhibitory activity, and predicted 16 of them are CYP1A1 inhibitors. The CYP1A1 potency and modes of inhibition of these 22 compounds were experimentally determined. We confirmed that most predicted inhibitors, including drugs contraindicated during pregnancy (amiodarone, bicalutamide, cyproterone acetate, ketoconazole, and tamoxifen) and environmental agents suspected to be endocrine disruptors (bisphenol A, diethyl and dibutyl phthalates, and zearalenone), are indeed potent inhibitors of CYP1A1. Our results suggest that virtual screening may be used as a rapid tier-one method to screen for potential CYP1A1 inhibitors, and flag them out for further experimental evaluations.

Assessment of the inactivation potential of desethylamiodarone on human CYP1A1.

Desethylamiodarone was reported to inactivate human CYP1A1. To assess this, two protocols were implemented employing dilution and non-dilution of the preincubation mixture. Inactivation studies performed with diluted preincubation mixtures showed no inactivation of CYP1A1 by desethylamiodarone. However there was evidence for a mixed competitive inhibition (competitive and the uncompetitive inhibition constants of 2.1 microM and 9.6 microM, respectively) of CYP1A1 by desethylamiodarone. NADPH addition and/or replenishment were found to be important factors in determining the control activity in inactivation studies.

Further assessment of 17alpha-ethinyl estradiol as an inhibitor of different human cytochrome P450 forms in vitro.

17alpha-Ethinyl estradiol (EE) was systematically evaluated as a reversible and time-dependent inhibitor of 11 human drug-metabolizing cytochromes P450 (P450s) (CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5) in vitro. When ranked, the lowest IC(50) (concentration of inhibitor required to decrease activity by 50%) values were obtained with recombinant CYP1A1 (rCYP1A1) [IC(50(total)) = IC(50(free)) = 2.7 microM] and CYP2C19 activity in human liver microsomes (HLM) [IC(50(total)) = 4.4 microM; IC(50(free)) = 2.8 microM]. For rCYP1A1, formal inhibition studies revealed that EE was a competitive inhibitor [K(i(free)) = 1.4 microM]. All the other IC(50) values were greater than 8.0 microM, and the weakest inhibition was observed with CYP1A2 activity in HLM (IC(50(free)) > 39 microM). In agreement, the IC(50) characterizing the inhibition of melatonin (MEL) 6-hydroxylation in human intestine microsomes (CYP1A1-catalyzed) was lower than that of HLM (0.91 versus >40 microM). Because EE is known to affect the pharmacokinetics of CYP2C19 probe drugs, this result raises the possibility that the concentration of EE during first pass may exceed 1000 nM, sufficient to affect CYP1A1 and CYP2C19, with less impact on CYP3A4 and other P450s. The results implicate intestinal CYP1A1, and possibly CYP2C19, as the loci of EE drug interactions with highly extracted drugs like MEL. Overall, it is very difficult to rationalize drug interactions involving EE based on direct inhibition of CYP2B6 (e.g., selegiline) and hepatic CYP1A2 (e.g., MEL, tizanidine, caffeine, and theophylline).

In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1.

Previously, inhibitors of CYP1A1 were rated as candidate chemopreventive agents against cancer mainly according to their effects on the 7-ethoxyresorufin O-deethylation (EROD) of diagnostic probe substrates. Surprisingly, several polyphenols including resveratrol, formerly identified as potent inhibitors by the EROD assay, exhibited no or weak inhibition of procarcinogen activation. We compared the effects of 11 representative natural polyphenols, which normally occur in food, on different activities of CYP1A1, namely epoxidation of 7,8-dihydrodiol-benzo[a]pyrene, the terminal step in the activation leading to the ultimate carcinogenic diolepoxides, hydroxylation of benzo[a]pyrene, and EROD. For the first time, a reconstituted system was used for the determination of IC(50) values, consisting of purified enzymes (human CYP1A1 and human NADPH-cytochrome P450 reductase) and dilaurylphosphatidylcholine. The results demonstrate that the inhibitory effects of dietary polyphenols on CYP1A1 activity depend on both the structure of the inhibitor and the type of the reaction and substrate used in the assay. Consequently, a potent EROD inhibition alone is insufficient to count a substance among the chemoprotective agents.