Evolution of genome diagnostics in epidermolysis bullosa: Unveiling the power of next-generation sequencing.

BACKGROUND: Genome diagnostics is considered gold standard diagnostics for epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous group of rare disorders characterized by blistering and wounding of mucocutaneous tissues. EB is caused by pathogenic variants in genes encoding proteins of the dermo-epidermal junction. Accurate genetic diagnosis of EB is crucial for prognostication, counselling and precision-medicine. Genome diagnostics for EB started in 1991 with the introduction of Sanger sequencing (SS), analysing one gene at a time. In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel. Several studies have shown a beneficial role for NGS in EB diagnostics, but its true benefit has not been quantified. OBJECTIVES: To determine the benefit of NGS in EB by systematically evaluating the performance of different genome diagnostics used over time based on robust data from the Dutch EB Registry. METHODS: The diagnostic performances of SS and NGS were systematically evaluated in a retrospective observational study including all index cases with a clinical diagnosis of EB in whom genome diagnostics was performed between 01 January 1994 and 01 January 2022 (n = 308), registered at the Dutch EB Expertise Centre. RESULTS: Over time, a genetic diagnosis was made in 289/308 (94%) EB cases. The diagnostic yield increased from 89% (SS) to 95% (NGS). Most importantly, NGS significantly reduced diagnostic turnaround time (39 days vs. 211 days, p < 0.001). The likelihood of detecting variants of uncertain significance and additional findings increased from 5% and 1% (SS) to 22% and 13% (NGS) respectively. CONCLUSIONS: Our study quantifies the benefit of NGS-based methods and demonstrate they have had a major impact on EB diagnostics through an increased diagnostic yield and a dramatically decreased turnaround time (39 days). Although our diagnostic yield is high (95%), further improvement of genome diagnostics is urgently needed to provide a genetic diagnosis in all EB patients.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions.

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric "pentamer" stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450-Catalyzed Substrate Conversion.

The regio- (and stereo-)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (ΔΔGbind ) of the catalytically active substrate-binding poses, and the probability of the substrate to adopt a transition-state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450-catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ΔΔGbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

This review covers the current knowledge of the cytochrome P450 enzymes (CYPs) of the human pathogen Mycobacterium tuberculosis (Mtb) and their endogenous redox partners, focusing on their biological function, expression, regulation, involvement in antibiotic resistance, and suitability for exploitation as antitubercular targets. The Mtb genome encodes twenty  CYPs and nine associated redox partners required for CYP catalytic activity. Transposon insertion mutagenesis studies have established the (conditional) essentiality of several of these enzymes for in vitro growth and host infection. Biochemical characterization of a handful of Mtb CYPs has revealed that they have specific physiological functions in bacterial virulence and persistence in the host. Analysis of the transcriptional response of Mtb CYPs and redox partners to external insults and to first-line antibiotics used to treat tuberculosis showed a diverse expression landscape, suggesting for some enzymes a potential role in drug resistance. Combining the knowledge about the physiological roles and expression profiles indicates that, at least five Mtb CYPs, CYP121A1, CYP125A1, CYP139A1, CYP142A1, and CYP143A1, as well as two ferredoxins, FdxA and FdxC, can be considered promising novel therapeutic targets.

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.

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.

Characterization of human cytochrome P450 mediated bioactivation of amodiaquine and its major metabolite N-desethylamodiaquine.

AIMS: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. METHODS: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. RESULTS: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis-Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km , 11.5 ± 2.0 µmol l-1 , Vmax , 59.2 ± 3.2 pmol min-1  mg-1 and CLint , 5.15 µl min-1  mg-1 . For DEAQ, parameters for bioactivation were Km , 6.1 ± 1.3 µmol l-1 , Vmax , 5.5 ± 0.4 pmol min-1  mg-1 and CLint 0.90 µl min-1  mg-1 . Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. CONCLUSIONS: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations.

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate.

Different Reactive Metabolites of Nevirapine Require Distinct Glutathione S-Transferase Isoforms for Bioinactivation.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase-inhibitor, which is associated with severe idiosyncratic skin rash and hepatotoxicity. These adverse drug reactions are believed to be mediated by the formation of epoxides and/or quinone methide formed by oxidative metabolism by P450s and 12-sulfoxyl-NVP formed by sequential 12-hydroxylation and O-sulfonation. Although different GSH-conjugates and corresponding mercapturic acids have been demonstrated previously in vitro and in vivo, the role of the glutathione S-transferases in the inactivation of the different reactive metabolites has not been studied so far. In the present study the activity of 10 recombinant human glutathione S-transferases (GSTs) in the detoxification of the different reactive metabolites of NVP was studied. The results show that GSTP1-1 is a highly active catalyst of GSH-conjugation of the oxidative metabolites of NVP, even at high GSH-concentration. Experiments with trideuterated NVP suggest involvement of a reactive epoxide rather than quinone methide in the formation of the GSH-conjugate formed after oxidative bioactivation. GSH-conjugation of 12-sulfoxyl-NVP forming NVP-12-GSH was only catalyzed by GSTM1-1, GSTA1-1, and GSTA3-3. Although the exact expression levels of these enzymes in the skin is unknown, the relatively low activity of this catalysis makes it unlikely that GSTs can provide significant protection against this metabolite. However, since NVP-12-GSH is specifically formed via the 12-sulfoxyl-NVP, its corresponding urinary mercapturic acid can be considered as a biomarker for recent internal exposure to this protein-reactive sulfate. However, it has to be taken into account that 12-sulfoxyl-NVP is not completely trapped by GSH and that rates of bioinactivation will differ between patients due to variability in expression of GSTM1, GSTA1, and GSTA3.

Application of a cocktail approach to screen cytochrome P450 BM3 libraries for metabolic activity and diversity.

In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants.

Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).

Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.

Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme.

In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95 % regioselectivity to form 16-ß-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-ß-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Cytochrome P450-mediated bioactivation of mefenamic acid to quinoneimine intermediates and inactivation by human glutathione S-transferases.

Mefenamic acid (MFA) has been associated with rare but severe cases of hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, and hypersensitivity reactions that are believed to result from the formation of reactive metabolites. Although formation of protein-reactive acylating metabolites by phase II metabolism has been well-studied and proposed to be the cause of these toxic side effects, the oxidative bioactivation of MFA has not yet been competely characterized. In the present study, the oxidative bioactivation of MFA was studied using human liver microsomes (HLM) and recombinant human P450 enzymes. In addition to the major metabolite 3'-OH-methyl-MFA, resulting from the benzylic hydroxylation by CYP2C9, 4'-hydroxy-MFA and 5-hydroxy-MFA were identified as metabolites resulting from oxidative metabolism of both aromatic rings of MFA. In the presence of GSH, three GSH conjugates were formed that appeared to result from GSH conjugation of the two quinoneimines formed by further oxidation of 4'-hydroxy-MFA and 5-hydroxy-MFA. The major GSH conjugate was identified as 4'-OH-5'-glutathionyl-MFA and was formed at the highest activity by CYP1A2 and to a lesser extent by CYP2C9 and CYP3A4. Two minor GSH conjugates resulted from secondary oxidation of 5-hydroxy-MFA and were formed at the highest activity by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability of seven human glutathione S-transferases (hGSTs) to catalyze the GSH conjugation of the quinoneimines formed by P450s was also investigated. The highest increase of total GSH conjugation was observed with hGSTP1-1, followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this study show that, next to phase II metabolites, reactive quinoneimines formed by oxidative bioactivation might also contribute to the idiosyncratic toxicity of MFA.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs.

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3'-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4'-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000-6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4' position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies.

Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28 days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.

Characterization of human cytochrome P450s involved in the bioactivation of clozapine.

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and N-oxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.