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On-line electrochemistry-bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38? kinase inhibitors.

ABSTRACT: In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38? 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 p38? kinase is the readout for bioaffinity. Parallel HR-MS(n) 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 p38? 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 p38? bioaffinity assay, which indicates binding to the p38? kinase.

SUBMITTER: Falck D

PROVIDER: S-EPMC3314180 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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

Falck David D de Vlieger Jon S B JS Giera Martin M Honing Maarten M Irth Hubertus H Niessen Wilfried M A WM Kool Jeroen J

Analytical and bioanalytical chemistry 20120108 2

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 t ...[more]

PMID: 22227812

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Project description:IntroductionThe ever-changing market of new psychoactive substances (NPS) poses challenges for laboratories worldwide. Analytical toxicologists are constantly working to keep high-resolution mass spectrometry (HR-MS) screening libraries updated for NPS. This study sought to use the online crowd-sourced HighResNPS database for spectrum comparison screening, thereby broadening its utility to all HR-MS instruments.MethodHighResNPS allows formation of a set of consensus fragment ions for a NPS and prioritises among multiple entries of collision-induced fragment ions. A subset of 42 NPS samples was analysed in data-independent acquisition (DIA) and data-dependent acquisition (DDA) modes on two different instruments. HighResNPS-computed spectra were generated with either Absolute (all fragment ions set to 100%) or Fractional (50% intensity reduction of former fragment ion) intensity. The acquired NPS data were analysed using the consensus library with computed ion intensities and evaluated with vendor-neutral screening software.ResultsOverall, of the 42 samples, 100% were identified, with 88% identified as the top candidate. Three samples had the correct candidate proposed as the second highest ranking NPS. In all three of those samples, the top proposed candidate was a positional isomer or closely related compound. Absolute intensity assignment provided identical scoring between the top two proposed compounds in two samples with DIA. DDA had a slightly higher identification rate in the spectra comparison screening with fractional intensity assignment, but no major differences were observed.ConclusionThe fractional intensity assignment was slightly more advantageous than the absolute assignment. It was selective between proposed candidates, showed a high identification rate and had an overall higher fragmentation scoring. The candidates proposed by the HighResNPS library spectra comparison simplify the determination of NPS for researchers and toxicologists. The database provides free monthly updates of consensus spectra, thereby enabling laboratories to stay at the forefront of NPS screening by LC-HR-MS with spectra screening software.

Dataset Information

On-line electrochemistry-bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38? kinase inhibitors.

Publications

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

Similar Datasets

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