CYP2D6 Human CYP450 Enzymatic LeadHunter Assay - TW

Procedure Summary:

Human recombinant CYP450 2D6 expressed in insect BTI-TN-5B1-4 cells is used. Test compound and/or vehicle is preincubated with 0.05 μM enzyme‡ in phosphate buffer pH 7.4 for 15 minutes at 37°C. The reaction is initiated by addition of 50 μM 3-cyano-7-ethoxycoumarin and cofactor solution (1.3 mM NADP, 3.5 mM glucose-6-phosphate, 3.3 mM MgCl2 and 0.4 U/ml G6P dehydrogenase) for another 45 minute incubation period and terminated by further addition of 80% acetonitrile. Determination of the amount of 3-cyano-7-hydroxycoumarin formed is read spectrofluorimetrically. Compounds are screened at 10 μM.

Substrate:

50 µM 3-Cyano-7-ethoxycoumarin

Incubation:

45 min at 37°C

Control Inhibitor:

Quinidine sulfate

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2