Cypex CYP3A4 Plate Reader Assay

Activity:  7-benzyloxyquinoline (7BQ) O-debenzylase

General description
Incubations are set up in a black 96-well plate, and consist of substrate (7BQ) and CYP3A4 Bactosomes in phosphate buffer containing magnesium chloride. Reactions are initiated by adding 40 µl of 5x NADPH generating system [this is added to all wells, including blanks and standards]. Metabolite (7HQ) formation is measured fluorometrically, using detection wavelengths chosen to minimise interference from NADPH and 7BQ.

The O-debenzylation of 7BQ by CYP3A4 Bactosomes does not follow Michaelis-Menten kinetics. In addition, there is marked substrate inhibition at 7BQ concentrations >70 µM (see data below). The activity determined at 70 µM 7BQ is therefore taken to represent the maximum measurable activity, and this is used to estimate a value for S₅₀ (substrate concentration at which the activity is 50% of the maximum measurable activity) from the experimental data.  Time and P450 linearity tests are then run at a 7BQ concentration ≤ S₅₀.

The substrate, 7BQ, and metabolite, 7HQ, are available from Cypex.

Incubation conditions
Plate type: black, 96-well, flat-bottomed
Assay buffer: 100 mM potassium phosphate pH 7.4, 5 mM MgCl₂
Solvent used to dissolve 7BQ: methanol
Final 7BQ concentration: 13 µM (or experimentally determined S₅₀)
Final CYP3A4 conc.: 0.5 pmol/200 µl (CYP3A4R/CYP3A4BR); 2.5 pmol/200 µl (CYP3A4LR/CYP3A4BLR)
Incubation time: 5 min
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: 7HQ, 50 ng/200 µl (final concentration)
Stop reagent: none (continuous monitoring)

Plate reader parameters
Detection mode: fluorescence, top reading
Excitation wavelength: 430 nm (bandwidth 9 nm)
Emission wavelength: 530 nm (bandwidth 20 nm)
Gain: calculated from 7HQ standard
Sampling frequency: 15 s (time linearity) or 20 s (P450 linearity or kinetics)

Time linearity test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ at a final CYP concentration of 0.5 pmol/200 µl.
The fluorescence was measured every 15 s.

The reaction is linear for approximately 5 min with CYP3A4R Bactosomes.

Time linearity test on CYP3A4BR Bactosomes with 7BQ as substrate:
This test was run at 16 µM 7BQ at a final CYP concentration of 0.5 pmol/200 µl.
The fluorescence was measured every 20 s.

The reaction is linear for approximately 8 - 10 min with CYP3A4BR Bactosomes.

Time linearity test on CYP3A4LR Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ at a final CYP concentration of 2.5 pmol/200 µl.
The fluorescence was measured every 15 s.

The reaction is linear for approximately 10 min with CYP3A4LR Bactosomes.

Time linearity test on CYP3A4BLR Bactosomes with 7BQ as substrate:
This test was run at 15 µM 7BQ at a final CYP concentration of 2.5 pmol/200 µl.
The fluorescence was measured every 20 s.

The reaction is linear for approximately 20 min with CYP3A4BLR Bactosomes.

P450 linearity test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ using an incubation time of 5 min.
The fluorescence was measured every 20 s.

The reaction is linear up to at least 2 pmol/200 µl with CYP3A4R Bactosomes. A similar plot is observed with CYP3A4BR Bactosomes (data not shown).

P450 linearity test on CYP3A4LR Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ using an incubation time of 5 min.
The fluorescence was measured every 20 s.

The reaction is linear up to at least 10 pmol/200 µl with CYP3A4LR Bactosomes. A similar plot is observed with CYP3A4BLR Bactosomes (data not shown).

Kinetics test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 0.5 pmol CYP/200 µl using an incubation time of 5 min.
The fluorescence was measured every 20 s.

The reaction is characterised by non-Michaelis-Menten kinetics in CYP3A4R Bactosomes.
7BQ concentrations >70 µM give rise to substrate inhibition. Similar plots are observed with CYP3A4BR, CYP3A4LR and CYP3A4BLR Bactosomes (data not shown).

Typical kinetic parameters for different enzyme preparations:
The activity measured at 70 µM 7BQ (80 µM for Supersomes) is used as the maximum velocity. The substrate concentration at which the activity is 50% of the maximum (S₅₀) is estimated from the experimental data.

The maximum velocity given for Supersomes has been corrected for CYP content as measured by Cypex, using spectral analysis.

Made under licence from BTG International Ltd (AU730155, EP0914446, US6566108 and other patents pending).

United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No. 2100245 and other patents pending.