CYP Inhibition Studies Using Recombinant P450 Isoenzymes

Usually, the NCE is pipetted together with the en-zyme/substrate complex and the reaction is started with addition of the cofactor solution. Incubation times vary between 15 and 45 min at 37 °C. Afterwards, the reaction is stopped by addition a TRIS/Acetonitile solution and applied to fluorescence read out. 

The assays are usually performed in parallel to solvent control and a well-known inhibitor of the P450 isoenzyme investigated. 

CRITICAL ASSESSMENT OF THE METHOD For over-expressing P450 isoenzymes, several heterologous expression systems have been established, that 

CYP Substrate Substrate Cone. (/uM) Enzyme (nM) NADP+ 0 M) G6P (mM) MgCl2 (mM) G6P-DH (U/ml) Incubation Time (min) 

2D6 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7 -methoxy-4-methyl-coumarin (AMMC) 1.5 7.5 8.1 0.4 0.4 0.2 30 

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II.K.2 CYP Inhibition Studies Using Recombinant P450 Isoenzymes. 552... 

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