Journal: Scientific Reports

Article Title: A combinatorial approach for the discovery of cytochrome P450 2D6 inhibitors from nature

doi: 10.1038/s41598-017-08404-0

Figure Lengend Snippet: Essential steps of the luminescence-based, high-throughput P450-Glo CYP2D6 inhibition assay. ( A ) Methoxy-luciferin-ethylene glycol ester is a CYP2D6 substrate that is demethylated to luciferin-ethylene glycol ester in the presence of NADPH, which serves as an electron source. ( B ) The read-out of the CYP2D6 reaction is based on the treatment of the reaction mixture with the detection reagent that consists of a detergent, an unspecific esterase and a modified firefly-luciferase. ( C ) The esterase continuously generates luciferin, which is oxidized by the firefly-luciferase and thereby a stable luminogenic signal is produced.

Article Snippet: The CYP2D6 enzyme reaction was initiated by the addition of 25 μl of 2× NADPH regeneration system (2.6 mM NADP + , 6.6 mM glucose-6-phosphate, 6.6 mM MgCl 2 and 0.8 U/ml glucose-6-phosphate dehydrogenase buffered in 0.1 mM sodium citrate at pH 5.5; Promega).

Techniques: High Throughput Screening Assay, Inhibition, Modification, Luciferase, Produced