Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates.

Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.

A Comparative Linear Interaction Energy and MM/PBSA Study on SIRT1-Ligand Binding Free Energy Calculation.

Binding free energy (ΔGbind) computation can play an important role in prioritizing compounds to be evaluated experimentally on their affinity for target proteins, yet fast and accurate ΔGbind calculation remains an elusive task. In this study, we compare the performance of two popular end-point methods, i.e., linear interaction energy (LIE) and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA), with respect to their ability to correlate calculated binding affinities of 27 thieno[3,2-d]pyrimidine-6-carboxamide-derived sirtuin 1 (SIRT1) inhibitors with experimental data. Compared with the standard single-trajectory setup of MM/PBSA, our study elucidates that LIE allows to obtain direct ("absolute") values for SIRT1 binding free energies with lower compute requirements, while the accuracy in calculating relative values for ΔGbind is comparable (Pearson's r = 0.72 and 0.64 for LIE and MM/PBSA, respectively). We also investigate the potential of combining multiple docking poses in iterative LIE models and find that Boltzmann-like weighting of outcomes of simulations starting from different poses can retrieve appropriate binding orientations. In addition, we find that in this particular case study the LIE and MM/PBSA models can be optimized by neglecting the contributions from electrostatic and polar interactions to the ΔGbind calculations.

Characterization of kinetics of human cytochrome P450s involved in bioactivation of flucloxacillin: inhibition of CYP3A-catalysed hydroxylation by sulfaphenazole.

BACKGROUND AND PURPOSE: The aim of this study was to characterize the human cytochrome P450s (CYPs) involved in oxidative bioactivation of flucloxacillin to 5-hydroxymethyl flucloxacillin, a metabolite with high cytotoxicity towards biliary epithelial cells. EXPERIMENTAL APPROACH: The CYPs involved in hydroxylation of flucloxacillin were characterized using recombinant human CYPs, pooled liver microsomes in the presence of CYP-specific inhibitors and by correlation analysis using a panel of liver microsomes from 16 donors. KEY RESULTS: Recombinant CYPs showing the highest specific activity were CYP3A4, CYP3A7 and to lower extent CYP2C9 and CTP2C8. Michaelis-Menten enzyme kinetics were determined for pooled human liver microsomes, recombinant CYP3A4, CYP3A7 and CYP2C9. Surprisingly, sulfaphenazole appeared to be a potent inhibitor of 5'-hydroxylation of flucloxacillin by both recombinant CYP3A4 and CYP3A7. CONCLUSIONS AND IMPLICATIONS: The combined results show that the 5'-hydroxylation of flucloxacillin is primarily catalysed by CYP3A4, CYP3A7 and CYP2C9. The large variability of the hepatic expression of these enzymes could affect the formation of 5'-hydroxymethyl flucloxacillin, which may determine the differences in susceptibility to flucloxacillin-induced liver injury. Additionally, the strong inhibition in CYP3A-catalysed flucloxacillin metabolism by sulfaphenazole suggests that unanticipated drug-drug interactions could occur with coadministered drugs.

Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners.

Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion.

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans.

Glutathione S-Transferase P1 Protects Against Amodiaquine Quinoneimines-Induced Cytotoxicity but Does Not Prevent Activation of Endoplasmic Reticulum Stress in HepG2 Cells.

Formation of the reactive amodiaquine quinoneimine (AQ-QI) and N-desethylamodiaquine quinoneimine (DEAQ-QI) plays an important role in the toxicity of the anti-malaria drug amodiaquine (AQ). Glutathione conjugation protects against AQ-induced toxicity and GSTP1 is able to conjugate its quinoneimine metabolites AQ-QI and DEA-QI with glutathione. In this study, HepG2 cells transiently transfected with the human GSTP1 construct were utilized to investigate the protective effect of GSTP1 in a cellular context. HepG2 cells were exposed to synthesized QIs, which bypasses the need for intracellular bioactivation of AQ or DEAQ. Exposure was accompanied by decreased cell viability, increased caspase 3 activity, and decreased intracellular GSH levels. Using high-content imaging-based BAC-GFP reporters, it was shown that AQ-QI and DEAQ-QI specifically activated the endoplasmic reticulum (ER) stress response. In contrast, oxidative stress, DNA damage, or inflammatory stress responses were not activated. Overexpression of GSTP1 resulted in a two-fold increase in GSH-conjugation of the QIs, attenuated QI-induced cytotoxicity especially under GSH-depletion condition, abolished QIs-induced apoptosis but did not significantly inhibit the activation of the ER stress response. In conclusion, these results indicate a protective role of GSTP1 by increasing enzymatic detoxification of AQ-QI and DEAQ-QI and suggest a second protective mechanism by interfering with ER stress induced apoptosis.

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants.

The 5'-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5'-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5'-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5'-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5'-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity: Application to characterize interindividual variability in human liver fractions.

Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.

Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals.

BACKGROUND: Inter-individual variability in hepatic drug metabolizing enzyme (DME) activity is a major contributor to heterogeneity in drug clearance and safety. Accurate data on expression levels and activities of DMEs is an important prerequisite for in vitro-in vivo extrapolation and in silico based predictions. Characterization and assessment of inter-correlations of the major DMEs cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) have been extensively documented, but simultaneous quantification including other major DMEs has been lacking. OBJECTIVE: Assessment of inter-donor variability and inter-correlations of CYPs, UGTs, sulfotransferases (SULTs), glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) in a set of 20 individual liver homogenates. METHOD: The main drug metabolizing isoforms of CYP and UGT have been reaction phenotype in individual liver microsomes and NQO1, NQO2, GSTT1 and GSTT2 in corresponding cytosol. In addition, we assessed overall SULT activity in liver cytosol using acetaminophen and 7-hydroxycoumarin as non-selective substrates and cytosolic GST activity using the non-selective substrate 1-chloro-2,4-dinitrobenzene (CDNB). Expression of GST isoforms was also assessed. RESULTS AND CONCLUSION: While hepatic NQO1 activity was highly variable, NQO2 activity was more conserved. In addition, we found that of the hepatic GST isoforms, the variation in GSTM3 levels, which is poorly studied, was highest. The majority of significant correlations were found amongst CYP and UGT enzyme activities. The dataset presented provides the absolute quantification of the largest number of hepatic DME activities so far and constitute an essential resource for in silico toxicokinetic and metabolic modelling studies.

Binding free energy predictions of farnesoid X receptor (FXR) agonists using a linear interaction energy (LIE) approach with reliability estimation: application to the D3R Grand Challenge 2.

Computational protein binding affinity prediction can play an important role in drug research but performing efficient and accurate binding free energy calculations is still challenging. In the context of phase 2 of the Drug Design Data Resource (D3R) Grand Challenge 2 we used our automated eTOX ALLIES approach to apply the (iterative) linear interaction energy (LIE) method and we evaluated its performance in predicting binding affinities for farnesoid X receptor (FXR) agonists. Efficiency was obtained by our pre-calibrated LIE models and molecular dynamics (MD) simulations at the nanosecond scale, while predictive accuracy was obtained for a small subset of compounds. Using our recently introduced reliability estimation metrics, we could classify predictions with higher confidence by featuring an applicability domain (AD) analysis in combination with protein-ligand interaction profiling. The outcomes of and agreement between our AD and interaction-profile analyses to distinguish and rationalize the performance of our predictions highlighted the relevance of sufficiently exploring protein-ligand interactions during training and it demonstrated the possibility to quantitatively and efficiently evaluate if this is achieved by using simulation data only.

Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium.

CYP130 belongs to the subset of cytochrome P450s from Mycobacterium tuberculosis (Mtb) that have been structurally characterized. Despite several efforts for its functional characterization, CYP130 is still considered an orphan enzyme for which no endogenous or exogenous substrate has been identified. In addition, functional redox-partners for CYP130 have not been clearly established yet, hampering the elucidation of its physiological role. In the present study, a catalytically active fusion protein involving CYP130 and the NADPH reductase-domain of CYP102A1 from Bacillus megaterium was created. By screening a panel of known substrates of human P450s, dextromethorphan N-demethylation was identified as a reaction catalyzed by CYP130. The fusion enzyme showed higher catalytic activity, when compared to CYP130 reconstituted with a selection of non-native redox-partners. Molecular dynamics simulation studies based on the crystal structure of CYP130 revealed two primary docking poses of dextromethorphan within the active site consistent with the experimentally observed N-demethylation reaction during the entire molecular dynamics simulation. The dextromethorphan N-demethylation reaction was strongly inhibited by azole-drugs and maybe applied to identify mechanism-based inhibitors of CYP130. Furthermore, the present active CYP130-fusion protein may facilitate the identification of endogenous substrates from Mtb.

Drug toxicity profiling of a Saccharomyces cerevisiae deubiquitinase deletion panel shows that acetaminophen mimics tyrosine.

Post-translational protein modification by addition or removal of the small polypeptide ubiquitin is involved in a range of critical cellular processes, like proteasomal protein degradation, DNA repair, gene expression, internalization of membrane proteins, and drug sensitivity. We recently identified genes important for acetaminophen (APAP) toxicity in a comprehensive screen and our findings suggested that a small set of yeast strains carrying deletions of ubiquitin-related genes can be informative for drug toxicity profiling. In yeast, approximately 20 different deubiquitinating enzymes (DUBs) have been identified, of which only one is essential for viability. We investigated whether the toxicity profile of DUB deletion yeast strains would be informative about the toxicological mode of action of APAP. A set of DUB deletion strains was tested for sensitivity and resistance to a diverse series of compounds, including APAP, quinine, ibuprofen, rapamycin, cycloheximide, cadmium, peroxide and amino acids and a cluster analysis was performed. Most DUB deletion strains showed an altered growth pattern when exposed to these compounds by being either more sensitive or more resistant than WT. Toxicity profiling of the DUB strains revealed a remarkable overlap between the amino acid tyrosine and acetaminophen (APAP), but not its stereoisomer AMAP. Furthermore, co-exposure of cells to both APAP and tyrosine showed an enhancement of the cellular growth inhibition, suggesting that APAP and tyrosine have a similar mode of action.

Reduction and Scavenging of Chemically Reactive Drug Metabolites by NAD(P)H:Quinone Oxidoreductase 1 and NRH:Quinone Oxidoreductase 2 and Variability in Hepatic Concentrations.

Detoxicating enzymes NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinone-like compounds. The protective role of the polymorphic NQO1 and NQO2 enzymes is especially of interest in the liver as the major site of drug bioactivation to chemically reactive drug metabolites. In the current study, we quantified the concentrations of NQO1 and NQO2 in 20 human liver donors and NQO1 and NQO2 activities with quinone-like drug metabolites. Hepatic NQO1 concentrations ranged from 8 to 213 nM. Using recombinant NQO1, we showed that low nM concentrations of NQO1 are sufficient to reduce synthetic amodiaquine and carbamazepine quinone-like metabolites in vitro. Hepatic NQO2 concentrations ranged from 2 to 31 µM. NQO2 catalyzed the reduction of quinone-like metabolites derived from acetaminophen, clozapine, 4'-hydroxydiclofenac, mefenamic acid, amodiaquine, and carbamazepine. The reduction of the clozapine nitrenium ion supports association studies showing that NQO2 is a genetic risk factor for clozapine-induced agranulocytosis. The 5-hydroxydiclofenac quinone imine, which was previously shown to be reduced by NQO1, was not reduced by NQO2. Tacrine was identified as a potent NQO2 inhibitor and was applied to further confirm the catalytic activity of NQO2 in these assays. While the in vivo relevance of NQO2-catalyzed reduction of quinone-like metabolites remains to be established by identification of the physiologically relevant co-substrates, our results suggest an additional protective role of the NQO2 protein by non-enzymatic scavenging of quinone-like metabolites. Hepatic NQO1 activity in detoxication of quinone-like metabolites becomes especially important when other detoxication pathways are exhausted and NQO1 levels are induced.

Effect of UGT2B7*2 and CYP2C8*4 polymorphisms on diclofenac metabolism.

The use of diclofenac is associated with rare but severe drug-induced liver injury (DILI) in a very small number of patients. The factors which predispose susceptible patients to hepatotoxicity of diclofenac are still incompletely understood. Formation of protein-reactive metabolites by UDP-glucuronosyl transferases and cytochromes P450 is commonly considered to play an important role, as indicated by the detection of covalent protein adducts and antibodies in the serum of patients suffering from diclofenac-induced liver injury. Since no associations have been found with HLA-alleles, polymorphisms of genes encoding for proteins involved in the disposition of diclofenac may be important. Previous association studies showed that possession of the UGT2B7*2 and CYP2C8*4 alleles is more common in cases of diclofenac-induced DILI. In the present study, the metabolism of diclofenac by UGT2B7*2 and CYP2C8*4 was compared with their corresponding wild-type enzymes. Enzyme kinetic analysis revealed that recombinant UGT2B7*2 showed an almost 6-fold lower intrinsic clearance of diclofenac glucuronidation compared to UGT2B7*1. The mutant CYP2C8*4 showed approximately 35% reduced activity in the 4'-hydroxylation of diclofenac acyl glucuronide. Therefore, a decreased hepatic exposure to diclofenac acyl glucuronide is expected in patients with the UGT2B7*2 genotype. The increased risk for hepatotoxicity, therefore, might be the result from a shift to oxidative bioactivation to cytotoxic quinoneimines.

eTOX ALLIES: an automated pipeLine for linear interaction energy-based simulations.

BACKGROUND: Computational methods to predict binding affinities of small ligands toward relevant biological (off-)targets are helpful in prioritizing the screening and synthesis of new drug candidates, thereby speeding up the drug discovery process. However, use of ligand-based approaches can lead to erroneous predictions when structural and dynamic features of the target substantially affect ligand binding. Free energy methods for affinity computation can include steric and electrostatic protein-ligand interactions, solvent effects, and thermal fluctuations, but often they are computationally demanding and require a high level of supervision. As a result their application is typically limited to the screening of small sets of compounds by experts in molecular modeling. RESULTS: We have developed eTOX ALLIES, an open source framework that allows the automated prediction of ligand-binding free energies requiring the ligand structure as only input. eTOX ALLIES is based on the linear interaction energy approach, an efficient end-point free energy method derived from Free Energy Perturbation theory. Upon submission of a ligand or dataset of compounds, the tool performs the multiple steps required for binding free-energy prediction (docking, ligand topology creation, molecular dynamics simulations, data analysis), making use of external open source software where necessary. Moreover, functionalities are also available to enable and assist the creation and calibration of new models. In addition, a web graphical user interface has been developed to allow use of free-energy based models to users that are not an expert in molecular modeling. CONCLUSIONS: Because of the user-friendliness, efficiency and free-software licensing, eTOX ALLIES represents a novel extension of the toolbox for computational chemists, pharmaceutical scientists and toxicologists, who are interested in fast affinity predictions of small molecules toward biological (off-)targets for which protein flexibility, solvent and binding site interactions directly affect the strength of ligand-protein binding.

Legacy data sharing to improve drug safety assessment: the eTOX project.

The sharing of legacy preclinical safety data among pharmaceutical companies and its integration with other information sources offers unprecedented opportunities to improve the early assessment of drug safety. Here, we discuss the experience of the eTOX project, which was established through the Innovative Medicines Initiative to explore this possibility.

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors.

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

Comprehensive and Automated Linear Interaction Energy Based Binding-Affinity Prediction for Multifarious Cytochrome P450 Aromatase Inhibitors.

Cytochrome P450 aromatase (CYP19A1) plays a key role in the development of estrogen dependent breast cancer, and aromatase inhibitors have been at the front line of treatment for the past three decades. The development of potent, selective and safer inhibitors is ongoing with in silico screening methods playing a more prominent role in the search for promising lead compounds in bioactivity-relevant chemical space. Here we present a set of comprehensive binding affinity prediction models for CYP19A1 using our automated Linear Interaction Energy (LIE) based workflow on a set of 132 putative and structurally diverse aromatase inhibitors obtained from a typical industrial screening study. We extended the workflow with machine learning methods to automatically cluster training and test compounds in order to maximize the number of explained compounds in one or more predictive LIE models. The method uses protein-ligand interaction profiles obtained from Molecular Dynamics (MD) trajectories to help model search and define the applicability domain of the resolved models. Our method was successful in accounting for 86% of the data set in 3 robust models that show high correlation between calculated and observed values for ligand-binding free energies (RMSE < 2.5 kJ mol-1), with good cross-validation statistics.

Human glutathione S-transferases- and NAD(P)H:quinone oxidoreductase 1-catalyzed inactivation of reactive quinoneimines of amodiaquine and N-desethylamodiaquine: Possible implications for susceptibility to amodiaquine-induced liver toxicity.

Amodiaquine (AQ), an antimalarial drug, widely prescribed in endemic areas of Africa and Asia, is used in combination with artesunate as recommended by the WHO. However, due to its idiosyncratic hepatotoxicity and agranulocytosis, the therapeutic use has been discontinued in most countries. Oxidative bioactivation to protein-reactive quinonimines (QIs) by hepatic cytochrome P450s and myeloperoxidase (MPO) have been suggested to be important mechanisms underlying AQ idiosyncratic toxicity. However, the inactivation of the reactive QIs by detoxifying enzymes such as human glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreducatase 1 (NQO1) has not been characterized yet. In the present study, the activities of 15 recombinant human GSTs and NQO1 in the inactivation of reactive QIs of AQ and its pharmacological active metabolite, N-desethylamodiaquine (DEAQ) were investigated. The results showed that GSTP1-1, GSTA4-4, GSTM4-4, GSTM2-2 and GSTA2-2 (activity in decreasing order) were active isoforms in catalyzing GSH conjugation of reactive QIs of AQ and DEAQ. Additionally, NQO1 was shown to inactivate these QIs by reduction. Simulation of the variability of cytosolic GST-activity based on the hepatic GST contents from 22 liver donors, showed a large variation in cytosolic inactivation of QIs by GSH, especially at a reduced GSH-concentration. In conclusion, the present study demonstrates that a low hepatic expression of the active GSTs and NQO1 may increase the susceptibility of patients to AQ idiosyncratic hepatotoxicity.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.