FAQs -- Frequently Asked Questions
Fluorescent High Throughput P450 Assays - A High Throughput Method for
Measuring Cytochrome P450 Inhibition
Below are the answers to many frequently asked questions submitted
by our customers. If there are other questions which you would like to
have answered please email them to us at info_gentest@bd.com.
- There are multiple substrates available ... which one
should I use?
- Does organic solvent concentration affect enzyme activity?
- Can I dissolve substrate and positive control (inhibitor)
solutions in organic solvents other than acetonitrile?
- Will my results be affected if I use different plates?
- What is an acceptable range for day to day variability
for IC50 values? Lab to lab variability?
- Does total microsomal protein concentration affect the
IC50 results?
- What can I do if my plate reader does not meet the sensitivity
parameters recommended?
- What Signal/Background is appropriate?
- Do substrates themselves have fluorescence?
- Can I read my plates at different wavelengths than those
recommended?
- What should be my highest test compound concentration?
- How do I calculate an IC50 value if 50% inhibition is
not achieved?
- Can I directly compare IC50 values from this assay to
conventional CYP inhibition methods?
- What is the difference between using NADPH vs. NADP+
with a regeneration system?
Q. There are multiple substrates available ...which
one should I use? Back to TOP
A. We provide multiple substrates because some substrates may be
more operationally suited for your particular needs (e.g. better match
for filters available with your flourometers). Additionally, the availability
of multiple substrates allows users to compare potential substrate-dependent
responses. Substrate dependent responses are particularly common with
CYP3A4. CYP3A4 substrates include DBF, BFC, BzRes and 7-BQ. We provide
multiple CYP3A4 substrates to allow users to get a sense of the magnitude
of qualitative and quantitative responses with their compounds.
For more detailed information, refer to the paper:
Stresser et al. (2000) Substrate-Dependent modulation of CYP3A4 catalytic
activity: analysis of 27 test compounds with four fluorometric substrates.
Drug Metab Dispos. 28: 1440-1448.
Posters:
Substrate Dependent Effects on CYP3A4 Inhibition or Activation by 27 Test
Compounds
poster11.pdf
Fluorometric Cytochromes P450 2C8, 2C9, and 2C19 Inhibition Assays: Testing
the One Substrate Paradigm
post-015.pdf
Q. Does organic solvent concentration affect enzyme
activity? Back to TOP
A. Organic solvents used to dissolve substrates can inhibit
cytochrome P450 enzyme activity [Chauret et al. (1998) Drug Metab. Dispos.
26, 1-4 and Busby et al., (1999) Drug Metab. Dispos. 27, 246-249]. If
solvents other than buffer or water must be used, low concentrations of
water-miscible organic solvents should be used whenever possible. The
use of acetonitrile or methanol is recommended since these solvents have
the lowest P450 inhibition potential of several common organic solvents.
Organic solvent concentration should be controlled and normalized in each
incubation of an experiment. For more detailed information on the effects
of organic solvents on these assays, see …Link to Appendix A Effects of
Solvents on the High Throughput Assay
Q. Can I dissolve substrate and positive control (inhibitor)
solutions in organic solvents other than acetonitrile? Back
to TOP
A. For substrates and positive control inhibitors, we have not
tested solvents other than acetonitrile. Other solvents miscible with
water (e.g methanol or DMSO) are not expected to negatively impact the
assay as long as total solvent concentrations are kept low (e.g. @1% or
less with methanol, @0.2% or less with DMSO). If you decide to dissolve
the compounds in other organic solvents, you may wish to refer to Appendix
A, "Effects of Solvents on the High Throughput Assay". Additionally, some
enzymes are particularly sensitive to organic solvents (e.g CYP2E1).
Q. Will my results be affected if I use different
plates? Back to TOP
A. In general, your choice of plate will not severely affect your
inhibition results as long as adequate sensitivity is achieved. Black
plates are generally preferred over white plates as they reduce light-scattering
resulting in better sensitivity and consequently better reproducibility.
We recommend BD Falcon 3943, 96-well microtiter plates (black, flat bottom)
for fluorometer instruments with top reading capability.
Q. What is an acceptable range for day to day variability
for IC50 values? Lab to lab variability? Back to TOP
A. You can expect a CV (Std dev/mean) of ~ 0.2. Lab to lab variability
would be expected to be higher.
Q. Does total microsomal protein concentration affect
the IC50 results? Back to TOP
A. Increased microsomal protein concentration aids in solubilizing
the test compound and minimizes nonspecific binding to plates. It is recommended
that control protein (cat# 456200 (Old P200)/456201 (Old P201)) be used
to standardize the final protein concentration to 0.25 mg/mL.
Q. What can I do if my plate reader does not meet
the sensitivity parameters recommended? Back to TOP
A. If your plate reader does not meet the sensitivity parameters
outlined in the Instrument Evaluation section then you should ensure that
the wavelengths used are within 10nm or so of those recommended. Alternatively,
you may need to increase the sensitivity of your assay. This can be done
by changing to a higher sensitivity plate (e.g. BD Falcon 3943, 96-well
microtiter plates; black, flat bottom), increasing the pathlength (higher
reaction volume), and/or increasing the amount of metabolite formed by
increasing the enzyme concentration when performing the assay. If the
recommended sensitivity levels are exceeded, the amount of enzyme may
be decreased. Additionally, you may find that a higher level of sensitivity
(i.e. more enzyme) may be needed to allow greater flexibility for the
type or concentration of organic solvent used to dissolve the test compound.
Q. What Signal/Background is appropriate? Back
to TOP
A. It is our recommendation that a minimum signal to background
ratio of 2 be used for these assays. However, a signal to background ratio
of 3 or higher is preferred for optimal reproducibility and robustness
of the assay.
Q. Do substrates themselves have fluorescence? Back
to TOP
A. Yes, the substrates are fluorescent to some extent at the wavelengths
recommended for the assay. This signal is accounted for in the "blank"
samples. The fluorescence contributed by the substrate should be insignificant
in comparison to the metabolite.
Q. Can I read my plates at alternative wavelengths
than those recommended? Back to TOP
A. Yes, you may read the plates at different excitation and emission
wavelengths than recommended. Excitation of a fluorophore at an alternate
wavelength will not change the emission profile, but it may effect the
intensity of the signal. The recommended fluorescent parameters may not
be the excitation and emission maxims for the metabolites, however, GENTEST
has determined the parameters to be optimal for the high throughput assay
using the conditions outlined in the bulletin. For example, interference
from the fluorescence of NADPH (ex = 340, em = 436) is minimized by choosing
a higher excitation filter for the detection of some products and by using
a lower NADPH concentration for some substrates (AMMC). Additionally,
alternate and higher emission wavelengths may be used to overcome interference
exhibited by some test substances (for example: ex = 390, em = 510 or
ex = 390, em = 538 for AMMC).
Q. What should be my highest test compound concentration
? Back to TOP
A. Beyond personal preference, the solubility of the test compound
will often determine the highest test compound concentration. In addition,
the highest test concentration may depend on the tolerance to organic
solvent and therefore the concentration of the stock solution when the
test compound is dissolved in organic solvent. We generally recommend
using a 10 mM stock solution when the test compound is dissolved in acetonitrile,
resulting in a 200 µM upper concentration with a final acetonitrile concentration
of 2%. When the compound must be dissolved in DMSO, we suggest preparing
a stock concentration of at least 50 mM. This would allow an upper concentration
of 100 µM based on the lower tolerance to DMSO in the assay.
Q. How do I calculate an IC50 value if 50% inhibition
is not achieved? Back to TOP
A. If 50% inhibition is not achieved an IC50 value can not be
calculated. The IC50 value is often reported as "greater than X", where
"X" is the highest test concentration, for example, ">200 µM". Testing
a higher concentration may allow you to obtain 50% inhibition or more.
Q. Can I directly compare IC50 values from this assay
to conventional CYP inhibition methods? Back to TOP
A. Yes, you may directly compare IC50 values between this assay
and conventional, low throughput, CYP inhibition methods. Keep in mind
there may be some substrate dependent differences, especially for CYP3A4.
Q. What is the difference between using NADPH vs.
NADP+ with a regeneration system? Back to TOP
A. Either system can be used, however, we recommend using NADP+
with a regenerating system as described in our solutions section. Using
NADP+ with a regeneration system allows a constant concentration of NADPH
in the assay and there is no chance of NADPH depletion.
The NADPH Regenerating System may be purchased from Gentest:
NADPH Regenerating System - Catalog Nos. 451200 (Old B200) and 451220
(Old B220)
Solution A (451220
(Old B220)) Sample Product Insert
Solution B (451200
(Old B200)) Sample Product Insert
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