A Family of Water Immiscible, Dipolar Aprotic, Diamide Solvents from Succinic Acid

Three dipolar aprotic solvents were designed to possess high dipolarity and low toxicity: N , N , N ', N '-tetrabutylsuccindiamide (TBSA), N , N '-diethyl- N , N '-dibutylsuccindiamide (EBSA), N , N '-dimethyl- N , N '-dibutylsuccindiamide (MBSA). They were synthesized catalytically using a K60 silica catalyst in a solventless system. Their water-immiscibility stands out as an unusual and useful property for dipolar aprotic solvents. They were tested in a model Heck reaction, metal-organic framework syntheses, and a selection of polymer solubility experiments where their performances were found to be comparable to traditional solvents. Furthermore, MBSA was found to be suitable for the production of an industrially-relevant membrane from polyethersulphone. An integrated approach involving in silico analysis based on available experimental information, prediction model outcomes and read across data, as well as a panel of in vitro reporter gene assays covering a broad range of toxicological endpoints was used to assess toxicity. These in silico and in vitro tests suggested no alarming indications of toxicity in the new solvents

Free energies of binding of R- and S-propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations

Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches to calculate the free energy differences have been investigated and were compared to the experimental binding data. From the differences between calculations based on forward and backward processes and the closure of thermodynamic cycles, it was clear that not all simulations converged sufficiently. The approach that calculates the free energies of exchanging R-propranolol with S-propranolol in the F483A mutant relative to the exchange free energy in WT CYP2D6 accurately reproduced the experimental binding data. Careful inspection of the end-points of the MD simulations involved in this approach, allowed for a molecular interpretation of the observed differences

Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action

This read-across case study characterises thirteen, structurally similar carboxylic acids demonstrating the application of in vitro and in silico human-based new approach methods, to determine biological similarity. Based on data from in vivo animal studies, the read-across hypothesis is that all analogues are steatotic and so should be considered hazardous. Transcriptomic analysis to determine differentially expressed genes (DEGs) in hepatocytes served as first tier testing to confirm a common mode-of-action and identify differences in the potency of the analogues. An adverse outcome pathway (AOP) network for hepatic steatosis, informed the design of an in vitro testing battery, targeting AOP relevant MIEs and KEs, and Dempster-Shafer decision theory was used to systematically quantify uncertainty and to define the minimal testing scope. The case study shows that the read-across hypothesis is the critical core to designing a robust, NAM-based testing strategy. By summarising the current mechanistic understanding, an AOP enables the selection of NAMs covering MIEs, early KEs, and late KEs. Experimental coverage of the AOP in this way is vital since MIEs and early KEs alone are not confirmatory of progression to the AO. This strategy exemplifies the workflow previously published by the EUTOXRISK project driving a paradigm shift towards NAM-based NGRA.Toxicolog

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.Toxicolog

Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine that are known substrates of human P450s. Interestingly, the metabolism of 3,4-methylenedioxymethylamphetamine and acetaminophen could be stimulated up to 70-fold by the addition of caffeine, a known activator of rat P450 3A2. With testosterone metabolism, homotropic cooperativity was observed. This shows that heterotropic and homotropic cooperativity, known to occur in the P450 3A family, can also take place in BM3. BM3 therefore can be used as a model system to study atypical kinetics in mammalian P450s. Second, this study shows that BM3 can be engineered to a drug-metabolising enzyme, making it a promising candidate to use as biocatalyst in drug discovery and synthesis. © 2006 Elsevier Inc. All rights reserved

Biochemical Similarities and Differences between the Catalytic [4Fe-4S] Cluster Containing Fumarases FumA and FumB from Escherichia coli

Background: The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions. Methodology/Principal Findings: To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S]2+ Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately 2290 mV for both FumA and FumB. Conclusions/Significance: This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.BT/BiotechnologyApplied Science

Metabolism of N-substituted 7-methoxy-4-(aminomethyl) -coumarins by cytochrome P450 2D6 mutants and the indication of additional substrate interaction points.

Previous studies have shown the critical roles residues F120 and F483 play in the oxidative metabolism of 7-methoxy-4-(aminomethyl)-coumarin (MAMC) by cytochrome P450 2D6 (CYP2D6). In the present study, a series of N-alkyl-7-methoxy-4-(aminomethyl)-coumarins (MAMC analogues) were used as substrates for the F120A and F483A mutants in order to probe the CYP2D6 active site. The F120A and F483A mutants of CYP2D6 displayed significant activity towards the MAMC analogues. Automated docking studies of the MAMC analogues in a CYP2D6 homology model suggested a distal hydrophobic active site binding cleft for the substrate N-alkyl chains, consisting of the residues L213 and V308. © 2006 Taylor & Francis

Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates. © 2007 Elsevier Ireland Ltd. All rights reserved