On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSn experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase

Métabolites de l’ecstasy et phénomènes de cytotoxicité

International audienc

Further Studies on the Role of Metabolites in (±)-3,4-Methylenedioxymethamphetamine-Induced Serotonergic Neurotoxicity

The mechanism by which the recreational drug (±)-3,4-methylenedioxymethamphetamine (MDMA) destroys brain serotonin (5-HT) axon terminals is not understood. Recent studies have implicated MDMA metabolites, but their precise role remains unclear. To further evaluate the relative importance of metabolites versus the parent compound in neurotoxicity, we explored the relationship between pharmacokinetic parameters of MDMA, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA) and indexes of serotonergic neurotoxicity in the same animals. We also further evaluated the neurotoxic potential of 5-(N-acetylcystein-S-yl)-HHMA (5-NAC-HHMA), an MDMA metabolite recently implicated in 5-HT neurotoxicity. Lasting serotonergic deficits correlated strongly with pharmacokinetic parameters of MDMA (Cmax and area under the concentration-time curve), more weakly with those of MDA, and not at all with those of HHMA or HMMA (total amounts of the free analytes obtained after conjugate cleavage). HHMA and HMMA could not be detected in the brains of animals with high brain MDMA concentrations and high plasma HHMA and HMMA concentrations, suggesting that HHMA and HMMA do not readily penetrate the blood-brain barrier (either in their free form or as sulfate or glucuronic conjugates) and that little or no MDMA is metabolized to HHMA or HMMA in the brain. Repeated intraparenchymal administration of 5-NAC-HHMA did not produce significant lasting serotonergic deficits in the rat brain. Taken together, these results indicate that MDMA and, possibly, MDA are more important determinants of brain 5-HT neurotoxicity in the rat than HHMA and HMMA and bring into question the role of metabolites (including 5-NAC-HHMA) in MDMA neurotoxicity

Separation of 248Cm from a 252Cf Neutron Source for Production of Cm Targets.

Abstract not availableJRC.E-Institute for Transuranium Elements (Karlsruhe