Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence

Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3-mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents

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

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3 g/L 15β-OH-NET and 0.16 g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes

Application of a fluorescence-based continuous-flow bioassay to screen for diversity of cytochrome P450 BM3 mutant libraries

A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants. © 2011 Society for Laboratory Automation and Screening

Advances in Understanding Xenobiotic Metabolism.

The understanding of how exogenous chemicals (xenobiotics) are metabolized, distributed, and eliminated is critical to determine the impact of the chemical and its metabolites to the (human) organism. This is part of the research and educational discipline ADMET (absorption, distribution, metabolism, elimination, and toxicity). Here, we review the work of Jan Commandeur and colleagues who have not only made a significant impact in understanding of phase I and phase II metabolism of several important compounds but also contributed greatly to the development of experimental techniques for the study of xenobiotic metabolism. Jan Commandeur's work has covered a broad area of research, such as the development of online screening methodologies, the use of a combination of enzyme mutagenesis and molecular modeling for structure-activity relationship (SAR) studies, and the development of novel probe substrates. This work is the bedrock of current activities and brings the field closer to personalized (cohort-based) pharmacology, toxicology, and hazard/risk assessment.publishersversionpublishe

Supplemental Material, DS1_IJT_10.1177_1091581818777998 - Development of a Phototoxicity Testing Strategy for Accurate Photosafety Evaluation of Pharmaceuticals Based on the Assessment of Possible Melanin-Binding Effects

<p>Supplemental Material, DS1_IJT_10.1177_1091581818777998 for Development of a Phototoxicity Testing Strategy for Accurate Photosafety Evaluation of Pharmaceuticals Based on the Assessment of Possible Melanin-Binding Effects by Jelle Reinen, Pieter van Sas, Ton van Huygevoort, Leticia Rubio, Kevin Scase, and Mira Wenker in International Journal of Toxicology</p