Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 May 17.
Published in final edited form as: Methods Mol Biol. 2013;987:157–162. doi: 10.1007/978-1-62703-321-3_14

High-throughput fluorescence assay of cytochrome P450 3A4

Qian Cheng, F Peter Guengerich
PMCID: PMC4024206  NIHMSID: NIHMS578518  PMID: 23475676

Summary

Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence.

Keywords: P450, fluorescence, high-throughput assay, enzyme inhibition

1. Introduction

Cytochrome P450 oxygenases (P450s) are the major enzymes involved in the oxidative metabolism of drugs and other xenobiotics (1). Inhibition of P450s catalytic activities is a principal mechanism for in vivo drug-drug interactions (2–4). Various in vitro assays have been developed to measure the activity or inhibition of P450s (5). Historically, the most commonly applied method is to use recombinant P450s with probe substrates, with the products identified by high-performance chromatography (HPLC) coupled with mass spectrometry (MS) (6–8). However, this method is relatively time-consuming and labor intensive and cannot be readily adapted to high-throughput formats (e.g. testing the inhibitory properties of a chemical library). Thus, microtiter plate-based fluorescence assays are more efficient and cost effective than HPLC-MS methods, allowing rapid in vitro testing of many samples in parallel (9, 10), if they provide accurate reports of the function of the enzymes. In a fluorescent assay, a “pro-fluorescent” molecule is oxidized by P450 to a fluorescent product, which can be directly measured using a fluorescence microplate reader. P450 3A4 is, in most individuals, the most abundant P450 in the liver and small intestine and is involved in the metabolism of about one-half of the drugs on the market. Here we describe a high-throughput fluorescence assay for measuring the activity of P450 3A4. A number of pro-fluorescence substrates (mainly benzyl ethers) have been used in P450 3A4 fluorescence assays (11, 12). 7-Benzoyloxy-4-trifluoromethyl coumarin (BFC) is selected in this protocol because BFC metabolism by P450 3A4 is linear with respect to BFC concentration up to 100 μM (13). A typical P450 3A4 inhibitor, ketoconazole, is used to demonstrate the use of microtiter plate-based fluorescence assays to assess the activity of P450 3A4 and characterize the IC50 of this inhibitor.

2. Materials

2.1 P450 assay

  1. Human P450 3A4 ‘bicistronic’ membranes (14) containing both P450 3A4 and NADPH-P450 reductase (concentration of both enzymes is 1 μM). Similar commercial products are available from BD Biosciences (see Note 1).

  2. 10 mM NADP+ stock solution: 382 mg NADP+ in 50 ml Milli-Q water. Store at 4 °C.

  3. 100 mM glucose-6-phosphate: 3.4 g in 100 ml Milli-Q water. Store at 4 °C.

  4. 103 IU/ml yeast glucose-6-phosphate dehydrogenase: 1.0 mg in 1 ml 10 mM Tris-acetate buffer (pH 7.4), containing 20% glycerol (v/v) and 1.0 mM EDTA. Store at 4°C.

  5. 4 mM BFC stock solution: 2.56 mg in 2 ml methanol. Stored in Teflon-sealed amber glass vial at 4 °C.

  6. 1 mM ketoconazole stock solution: 5.31 mg in 10 ml methanol. Stored in Teflon-sealed glass vial at 4°C.

  7. NADPH-generating system: combine 50 parts 10 mM NADP+, 100 parts 100 mM glucose-6-phosphate and 1 part 1 mg/ml yeast glucose 6-phosphate dehydrogenase. Prepare fresh daily and store on ice when not in use (see Note 2).

  8. Stop buffer: 80% acetonitrile and 20% 0.5 M Tris-base (v/v), stored at ambient temperature.

  9. P450-BFC 2× mix: mix P450 3A4 bicistronic membranes and 4 mM BFC stock solution in 100 mM potassium phosphate buffer (pH 7.4) so that the final concentration of P450 is 20 nM and BFC is 40 μM. Prepare the mix fresh daily and keep on ice.

2.2. Equipment

  1. Microplate reader. Polarstar microplate reader is used in this protocol (BMG Labtech)

  2. 96-well black microtiter plate (Corning Costar)

  3. Microchannel pipette (Gilson)

  4. Software to process the data. Prism (Graphpad.com) is used in this protocol (see Note 3).

3. Methods

3.1 Plate setup

The assay is performed in duplicate and the plate setup is summarized in Table 1.

Table 1.

The scheme of plate setup: The assay are performed in duplicated as designated by row I and row II. The concentration of ketoconazole (nM) in each well is listed.

1 2 3 4 5 6 7 8 9 10 11 12
I 5000 2500 1250 625 312 156 78 39.1 19.5 9.76 Blank No inhibitor
II 5000 2500 1250 625 312 156 78 39.1 19.5 9.76 Blank No inhibitor
Open in a new tab

  1. Dispense 60 μl 100 mM potassium phosphate buffer into the wells (column 2–12) in a 96-well microplate using a multichannel pipette.

  2. Dispense 118 μl 100 mM potassium phosphate and 2 μl of 1 mM ketoconazole into the wells (column 1).

  3. Serially dilute 60 μl inhibitor solution from the wells in column 1 to the other wells (2–10). Discard the extra 60 μl solution in the wells in column 10.

  4. Dispense 100 μl P450-BFC 2× mix in all the wells. Add 75 μl stop buffer to the wells in column 11.

  5. Preincubate the plate at 37 °C for 5 minutes ((see Note 4).

  6. Add 40 μl NADPH generating system into each well to initiate the reaction.

  7. Incubate the plate at 37 °C for 20 minutes.

  8. Stop the reaction by adding 75 μl stop buffer to each well (except wells in column 11).

3.2 Data acquirement and processing

  1. Scan the plate using the Polarstar plate reader. Average the replicates of the reading for each column.

  2. Subtract the blank from the mean value of all other columns and calculate the percentage of inactivated enzyme for each ketoconazole concentration (designated as I%). I=(1 − (mean of individual column − mean of column 11)/(mean of column 12 − mean of column 11)) × 100.

  3. Convert the concentration of inhibitor into log format (Designated as X).

  4. Fit the data with the 4-parameter logistic fit: I = b + (a−b)/(1+10^((LogIC50−X) × HillSlope)) using Prism. (Note 5)

  5. The processed data is shown in Table 2 and fitted plot is shown in Figure 1.

  6. Subtract the blank from the mean value of all other columns and calculate the percentage of inactivated enzyme for each ketoconazole concentration (designated as I%). I=(1 − (mean of individual column − mean of column 11)/(mean of column 12 − mean of column 11)) × 100.

  7. The calculated log IC50 is 1.57 to 1.92. Thus the measured IC50 in this experiment is 37–83 nM. (Note 6)

Table 2.

Fluorescence reading of each well and data processing. Data listed in bold font is used for 4-parameter logistic fit: I=b + (a−b)/(1+10^((LogIC50−X) × HillSlope)).

1 2 3 4 5 6 7 8 9 10 11 12
I 7845 8072 8229 8729 9536 11753 15018 18213 22589 23408 7489 24886
II 7571 7647 7850 8861 9827 10940 14914 16373 20459 21789 7223 23760
Mean 7708 7859 8039 8795 9681 11346 14966 17293 21524 22598 7356 24323
Mean-blank 424 503 683 1439 2325 3990 7610 9937 14168 15242 0 16967
I (%) 97.5 97 96 91.5 86.3 76.5 55.1 41.4 13.8 9.1
Inhibitor concentration (nM) 5000 2500 1250 625 312 156 78 39.1 19.5 9.76
X 3.7 3.4 3.1 2.8 2.5 2.2 1.9 1.6 1.3 1
Open in a new tab

Figure 1.

Figure 1

Open in a new tab

Calculated IC50 of ketoconazole with P450 3A4. The response (inhibition percentage) is plotted against the log(10) of ketoconazole concentration.

Footnotes

1

The activities of P450 3A4 ‘bicistronic’ membranes may decrease over the time. Avoid extensive thaw-freeze cycles.

2

Determine that the NADPH-generating system is active by monitoring the increase in absorbance at 340 nm.

3

Other statistical software (than GraphPad Prism) can perform nonlinear regression can be used for this experiment. Web-based curve fitting programs are also available, e.g. www.chanbioscience.com/stat/ec50.html.

4

Preincubation prior to the addition of NADPH to equilibrate temperature is a critical step. Insufficient preincubation can result in inconsistent readings between replicates.

5

Although simple linear regression may be used in certain situation, the 4-parameter logistic fit is more appropriate in most cases. Parameter a is the highest response and b is the lowest response.

6

The raw data used in this protocol was reported previously (15).

References

  • 1.Isin EM, Guengerich FP. Complex reactions catalyzed by cytochrome P450 enzymes. Biochim Biophys Acta. 2007;1770:314–329. doi: 10.1016/j.bbagen.2006.07.003. [DOI] [PubMed] [Google Scholar]
  • 2.Lamb DC, et al. Cytochromes P450 and drug discovery. Curr Opin Biotechnol. 2007;18:504–512. doi: 10.1016/j.copbio.2007.09.010. [DOI] [PubMed] [Google Scholar]
  • 3.Di L, et al. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS. Int J Pharmaceut. 2007;335:1–11. doi: 10.1016/j.ijpharm.2006.10.039. [DOI] [PubMed] [Google Scholar]
  • 4.Wienkers LC, Heath TG. Predicting in vivo drug interactions from in vitro drug discovery data. Nat Rev Drug Discov. 2005;4:825–833. doi: 10.1038/nrd1851. [DOI] [PubMed] [Google Scholar]
  • 5.Fowler S, Zhang H. In vitro evaluation of reversible and irreversible cytochrome P450 inhibition: current status on methodologies and their utility for predicting drug-drug interactions. AAPS J. 2008;10:410–424. doi: 10.1208/s12248-008-9042-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Youdim KA, et al. An automated, high-throughput, 384 well cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point. J Pharm Biomed Anal. 2008;48:92–99. doi: 10.1016/j.jpba.2008.05.011. [DOI] [PubMed] [Google Scholar]
  • 7.Lin T, et al. In vitro assessment of cytochrome P450 inhibition: strategies for increasing LC/MS-based assay throughput using a one-point IC(50) method and multiplexing high-performance liquid chromatography. J Pharm Sci. 2007;96:2485–2493. doi: 10.1002/jps.20884. [DOI] [PubMed] [Google Scholar]
  • 8.Turpeinen M, et al. Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay. Eur J Pharm Sci. 2005;24:123–132. doi: 10.1016/j.ejps.2004.10.006. [DOI] [PubMed] [Google Scholar]
  • 9.Schaeffner I, et al. A microtiterplate-based screening assay to assess diverse effects on cytochrome P450 enzyme activities in primary rat hepatocytes by various compounds. Assay Drug Dev Technol. 2005;3:27–38. doi: 10.1089/adt.2005.3.27. [DOI] [PubMed] [Google Scholar]
  • 10.Yamamoto T, Suzuki A, Kohno Y. Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns. Drug Metab Pharmacokinet. 2002;17:437–448. doi: 10.2133/dmpk.17.437. [DOI] [PubMed] [Google Scholar]
  • 11.Nakamura K, et al. Coumarin substrates for cytochrome P450 2D6 fluorescence assays. Anal Biochem. 2001;292:280–286. doi: 10.1006/abio.2001.5098. [DOI] [PubMed] [Google Scholar]
  • 12.Crespi CL, Miller VP, Penman BW. Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem. 1997;248:188–190. doi: 10.1006/abio.1997.2145. [DOI] [PubMed] [Google Scholar]
  • 13.Stresser DM, et al. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab Dispos. 2000;28:1440–1448. [PubMed] [Google Scholar]
  • 14.Parikh A, Gillam EM, Guengerich FP. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol. 1997;15:784–788. doi: 10.1038/nbt0897-784. [DOI] [PubMed] [Google Scholar]
  • 15.Cheng Q, Sohl CD, Guengerich FP. High-throughput fluorescence assay of cytochrome P450 3A4. Nat Protoc. 2009;4:1258–1261. doi: 10.1038/nprot.2009.123. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES