Trubetskoy
ARTICLE
10.1177/1087057104269731
HighlyMiniaturized
et al.
Formats for P450 Assays

Highly Miniaturized Formats for In Vitro Drug
Metabolism Assays Using Vivid® Fluorescent Substrates
and Recombinant Human Cytochrome P450 Enzymes
1

2

2

OLGA V. TRUBETSKOY, JASMIN R. GIBSON, and BRYAN D. MARKS

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate®
format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic
substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of®
the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus
on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The
screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selectedassay modifiers, including isozyme-specific substrates and inhibitors. IC50 values obtained for the modifiers in 96- and 1536well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z′ factor, demonstrated that these assays are arobust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput
screening format. (Journal of Biomolecular Screening 2005:56-66)
Key words: cytochrome P450, CYP1A2, CYP2C9, CYP2D6, CYP3A4, CYP2C19, fluorescent substrate, drug metabolism,
P450 assay, P450 inhibition, high-throughput screening (HTS), ultra-high-throughput screening (uHTS)
INTRODUCTIONA

LARGE NUMBER OF POTENTIAL DRUG CANDIDATES fail in thelater stages of drug development due to lack of efficacy andunpredicted effects of human metabolism, such as toxicity and unfavorable pharmacokinetic properties.1,2 At the present time, development of high-throughput screening (HTS) and automation
technologies, together with substantial advances in combinatorial
chemistry, predictive modeling, and bioinformatics, has significantly increased the number of new chemical entities entering drug
discovery programs. In addition, the recent completion of the Human Genome Project is expected to raise the number of moleculartargets from 500 to as many as 3000 to 4000,3 further increasing
the flow of compounds into pharmaceutical screens. The current
situation is changing the role of in vitro drug metabolism and toxicology studies, which have traditionally been performed with alimited set of compounds in the latest stages of drug development,
1

Quintessence Biosciences, Madison, WI, olgat@quintbio.com.

2

Invitrogen Drug Discovery Solutions, Madison, WI.

Received Mar 26, 2004, and in revised form Jun 16, 2004. Accepted for publication Jun 21, 2004.
Journal of Biomolecular Screening 10(1); 2005
DOI: 10.1177/1087057104269731

56 www.sbsonline.org

and is shifting it toward integrating absorption, disposition, metabolism, and excretion (ADME), and toxicology tests at the earliest
possible stages in the drug discovery process.4 Cytochrome P450s,
a superfamily of critical enzymes involved in human drug metabolism, are implicated in many clinical cases of adverse drug reactions and toxicity stemming from mechanism-based enzyme inhibition and drug-drug interactions.5 Performing HTS for P450
inhibition or induction earlier in the process helps to eliminate
molecules with unwanted metabolic properties and guides medicinal chemists to produce better clinical candidates based on predictive cytochrome P450 structure-activity relationship models.6 The
need to screen for ADME properties at the stage of compound libraries is driven by the necessity not just to accelerate the development of the new chemical entities but to decrease rates of drug failure later in the drug development process,2,7 creating a demand fora new generation of assays with higher throughput capability
alongside higher sensitivity and reproducibility.8 A number of approaches to screen for metabolism and inhibition of the major human cytochrome P450 enzymes have been developed. These techniques include radiometric assays based on the isolation ofradiolabeled metabolites,9-12 the use of colorimetric or fluorescentreagents for quantitation of formaldehyde formation during P450dependent demethylation reactions,13,14 and high-performance liquid chromatography (HPLC) methods, including rapid liquid© 2005 The Society for Biomolecular Screening

Highly Miniaturized Formats for P450 Assays
chromatography/mass spectrometry (LC/MS) approaches for metabolite analysis.15,16 However, most of these assays include relatively cumbersome postreaction separation steps that limit theirusefulness for HTS. The approach that shows the most promise forhigh-throughput inhibitor screens is the use of fluorescent or luminescent17 assays, which can be done in a homogeneous format (i.e.,they require no postreaction separation steps). Fluorescent HTS
methods employ fluorescent P450 substrates that are efficiently
metabolized by specific P450 isozymes to yield a product with altered fluorescent properties, usually increased fluorescent intensity.18,19 Metabolism assessment with fluorogenic substrates isbased on the ability of an unknown drug to inhibit or compete withthe fluorogenic compound in the P450 reaction and is dependent
on the kinetic parameters of such an interaction.20,21 The hits fromthese competitive inhibition screens must be further evaluated todetermine whether they are inhibitors or substrates for the indicated isozyme. Several of the generic fluorescent cytochrome
P450 substrates have been applied for the development of 96-wellcompatible fluorometric assays employing different cytochrome
P450 isozymes.20-22 In addition, fluorometric assays in a 384-wellformat for the CYP3A4 and CYP2D6 isozymes have also been described.23 However, many undesirable features of the generic (unmodified) fluorescent molecules, such as poor aqueous solubility,
high background fluorescence (e.g., the fluorescence ofunmetabolized substrate), and low signal-to-background ratios,
limit their applications for low-volume ultra-high-throughput formats. Recently, highly miniaturized formats for primary screening
assays involving several important pharmaceutical targets weredeveloped.24,25 The ability to perform primary screening assays in
microwell plates at 1- to 3-µL volumes has made a significant impact on the early stages of drug discovery by accelerating and reducing the cost of the drug discovery process.8 The ultra-highthroughput screening (uHTS) assays currently available for many
drug discovery targets demand similar speed, precision, and highthroughput capabilities from the ADME assays used to prescreen
compound libraries, particularly for the cytochrome P450 metabolism and inhibition assays.1,2,6,7 Here we describe the development
and characterization of fluorescent assays for the detection of substrates and inhibitors of major human cytochrome P450 isozymes,
enabling rapid and sensitive evaluation of P450 activity in a 1536well plate format. The assays use the Vivid® fluorogenic substrates
that were originally developed to address many issues existingwith fluorescent substrates of the previous generations, such as
poor solubility, high background fluorescence of the substrate, undesirable excitation and emission wavelengths, and insufficient
rates of metabolism.26 Previous applications of Vivid® substrates inthe development of several cytochrome P450 screening assays in a
96-well plate format have resulted in highly robust and reliable
HTS assays,27-29 indicating that these substrates are excellent candidates for further miniaturization and development of low-volume reactions. 30 To estimate the performance of Vivid ®
fluorogenic substrates in a 1536-well plate format, we combined

Journal of Biomolecular Screening 10(1); 2005

these substrates with baculovirus-expressed human P450
isozymes CYP3A4, CYP2D6, CYP2C9, CYP2C19, andCYP1A231 and applied them for the study of isozyme-specificdrug interactions and inhibition. The IC50 values obtained in the1536-well assays demonstrated a high degree of correlation withdata obtained in 96-well assays and conventional HPLC-based assays.32 In addition, the 1536-well assay characteristics, such as stability and reproducibility of the enzyme component, signal-tobackground ratio, Z′ factor in the range of 0.66 to 0.82, and tolerance to organic solvent, were indicative of highly robust and reliable assays. An overall extension of the current assays for the principal human cytochrome P450 enzymes to the uHTS format isviewed as an advanced and cost-effective approach for ADME
screening and presents a valuable new addition to the modern drug
discovery process. The advantages and limitations of the assays forvarious applications are further discussed.
METHODS
Chemicals
The Vivid® substrates, their corresponding fluorescent standards, and NADP+ were obtained from Invitrogen Drug Discovery
Solutions (Madison, WI). Ellipticine was obtained from
Calbiochem (San Diego, CA), and (±)-bufuralol hydrochloride
was purchased from BD Biosciences (San Jose, CA). Amitriptyline, clomipramine, diclofenac, erythromycin, furafylline,
imipramine, ketoconazole, α-naphthoflavone, omeprazole, (±)propranolol, quinidine, sulfaphenazole, tamoxifen, ticlopidine,
tranylcypromine, troleandomycin, (±)-verapamil, and warfarin
were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Allother reagents used were of the highest commercial grade
available.
Enzymes
Microsomes from baculovirus-infected cells coexpressing human CYP1A2, CYP2D6, CYP2C9, CYP2C19, or CYP3A4 andNADPH-cytochrome P450 reductase (P450 BACULOSOMES®),as well as the NADPH regeneration system containing glucose-6phosphate and glucose-6-phosphate dehydrogenase, were obtained from Invitrogen Drug Discovery Solutions (Madison, WI).
Cytochrome P450 content was determined spectrally by themethod of Omura and Sato.33 Protein concentrations were measured using the BCA protein assay kit (Pierce, Rockford, IL).
Particle sizing
The particle-sizing experiment was performed using a Zeta
Plus Particle Analyzer (Brookhaven Instruments Corp., Holtsville,NY) equipped with a solid-state laser operating at the wavelengthof 532 nm. BACULOSOMES® Reagents were diluted in 100 mM
potassium phosphate buffer, pH 8.0, at a concentration of 0.1 mg/

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Trubetskoy et al.
Table 1. General Reaction Conditions Used in 1536-Well Plate Assays
®

P450
®
BACULOSOMES

Vivid
Substrate

CYP450 (nM)

Vivid Substrate ( M)

Acetonitrile from®
Vivid Substrate (%)

Buffer (mM)

1A2
2C9
2C9
2C19
2D6
3A4
3A4

1A2 Blue
2C9 Green
2C9 Red
2C19 Blue
2D6 Blue
3A4 Green
3A4 Red

5
10
10
5
10
5
5

3
2
2
10
10
2
3

0.15
0.1
0.1
0.5
0.5
0.1
0.15

100
50
50
50
100
100
100

®

Reaction
Time (min)
20
60
60
30
60
10
20

+

All assays were performed at room temperature with 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 100 µM NADP in varying amounts of potassium phosphate at pH 8.0.

mL of protein. The scattering signal was acquired for 2 min at 25°C. The particle size was assessed using Multimodal Size Distribution (MSD) software.
96-Well assay incubation conditions
The 100-µM stock solutions of the Vivid® assay standards wereprepared in DMSO, and 2-mM stock solutions of Vivid® substrates
were prepared in acetonitrile according to the manufacturer’s instructions. Costar 96-well black, nontreated, flat-bottom assay
plates (#3915, Corning Costar, Acton, MA) were used for the fluorescent assays with a volume of 100 µL per well. All incubations
were performed at room temperature using buffer conditions andenzyme concentrations as described in assay protocol (www.
invitrogen.com/content/sfs/panvera/L0504.pdf). Fluorescentreadings were obtained for 96-well plates kinetically over thecourse of the reaction using a Gemini XS instrument (Molecular
Devices Corp., Sunnyvale, CA). The excitation and emission
wavelengths used for each Vivid® substrate were as follows:
Vivid® Red substrates (excitation 530 nm, emission 585 nm, andcutoff 570 nm), Vivid® Green substrates (excitation 485 nm, emission 530 nm, and cutoff 515 nm), and Vivid® Blue substrates (excitation 409 nm, emission 460 nm, and cutoff 455 nm). The same excitation and emission wavelength sets were used for both theVivid® substrates and corresponding standards. Product formation
was determined using a calibration curve of the corresponding fluorescent standard prepared by dilution into the appropriate buffer.
1536-Well assay incubation conditions
For the ultra-high-throughput assays, Greiner 1536-well blackplates (#782076, Greiner Bio-One, Inc., Longwood, FL) wereused with a total reaction volume of 8 µL using conditions described in Table 1. Reagents were dispensed using the Flying Reagent Dispenser I (FRD I) from Aurora Biosciences (San Diego,
CA). Assays were conducted in end-point mode by dispensing 1µL of inhibitor to stop the reaction at the end of the incubation period for a final concentration of 10 µM ketoconazole (CYP3A4),
10 µM α-naphthoflavone (CYP1A2), 1 µM quinidine (CYP2D6),58 www.sbsonline.org

10 µM sulfaphenazole (CYP2C9), or 100 µM tranylcypromine(CYP2C19). The plates were read on an Ultra plate reader (Tecan,
Inc., Durham, NC) using the following filter sets: Vivid® Red substrates (excitation 535 ± 25 nm, emission 590 ± 20 nm), Vivid®
Green substrates (excitation 485 ± 20 nm, emission 535 ± 25 nm),and Vivid® Blue substrates (excitation 405 ± 20 nm, emission 465± 35 nm).
96-Well inhibition studies
Vivid® assays conducted in 96-well plates were used to determine IC50 values of a selected panel of drugs known to be metabolized by specific P450 isozymes, as indicated in Table 2. Available
information about each drug’s solubility was used to prepare stock
solutions and serial dilutions of the drugs employed in a current
study. The 10-mM stock solutions of amitriptyline, (±)-bufuralol,
clomipramine, diclofenac, imipramine, (±)-propranolol,
ticlopidine, tranylcypromine, and (±)-verapamil were preparedand serially diluted in water to generate 20 concentrations in duplicate of each inhibitor. Ellipticine, erythromycin, furafylline,
ketoconazole, omeprazole, quinidine, sulfaphenazole, tamoxifen,
troleandomycin, and (R,S)-warfarin were prepared in DMSO atconcentrations ranging from 5 to 100 mM and serially diluted into
water containing an appropriate, uniform amount of DMSO; final
DMSO content in most cases ranged from 0.1% to 0.5% and did
not exceed 1%. Each experiment was conducted twice. The inhibition assays were performed as described in the section on 96-wellassay incubation conditions, with final concentrations as describedin Table 1. At twice the final reaction concentration, 50 µL of a
mixture of the BACULOSOMES® Reagent and the NADPH regeneration system in phosphate buffer (pH 8.0) was preincubatedfor 15 min in the presence of varying concentrations of 40 µL inhibitor prior to dispensing 10 µL of a mixture of 1 mM NADP+ andVivid® substrate. Final concentrations of the Vivid® substrates
were 2 µM Vivid® 2C9 Green, 2 µM Vivid® 2C9 Red, 2 µM Vivid®
3A4 Green, 3 µM Vivid® 1A2 Blue, 3 µM Vivid® 3A4 Red, 10 µM
Vivid® 2C19 Blue, or 10 µM Vivid® 2D6 Blue. Fluorescence values were then read in end-point mode after a 10-min (3A4 Green),
20-min (1A2 Blue and 3A4 Red), 30-min (2C19 Blue), or 60-min

Journal of Biomolecular Screening 10(1); 2005

Highly Miniaturized Formats for P450 Assays
Z factor and signal-to-background determination
A full 96- or 1536-well plate was used for each signal-tobackground and Z′ factor determination. Volumes of reagents andincubation times were identical to those used for the inhibition
studies. In 1536-well plates, the experiments were performed in
the absence and presence of 1% DMSO; 3.2 µL of 2.5% DMSO inwater was substituted in place of the inhibitor described in the1536-well inhibition studies. The Z′ factor was determined as described previously by Zhang and coworkers.34
RESULTS®

FIG. 1. Particle size distribution in CYP3A4 BACULOSOMES suspension. Measurements were performed as described in the section on
particle sizing conditions.

(2C9 Green, 2C9 Red, and 2D6 Blue) incubation period. Background fluorescence was established by wells containing all of theassay components, except BACULOSOMES® Reagent, and subtracted before further analysis. Percent inhibition was then calculated for each concentration of inhibitor by comparison of fluorescence values to those wells without inhibitor. Controls were used
to account for inhibition by DMSO where appropriate. IC50 values
were then determined by nonlinear regression using Prism software (version 3.03, GraphPad Software, San Diego, CA).
1536-Well inhibition studies
For inhibition studies in the 1536-well format, serial dilutions
were made in the same manner as described for 96-well inhibition
studies in quadruplicate, and final reaction concentrations were thesame as described for the 1536-well assay incubation conditions.
The volume of reagents was scaled down to 4 µL of a mixture of
the BACULOSOMES® Reagent and the NADPH regeneration
system in phosphate buffer (pH 8.0) at twice the final reaction concentration, 3.2 µL of various concentrations of the inhibitor, and0.8 µL of a 1-mM NADP+ and Vivid® substrate mixture. Final concentrations of the Vivid® substrates were 2 µM Vivid® 2C9 Green,
2 µM Vivid® 2C9 Red, 2 µM Vivid® 3A4 Green, 3 µM Vivid® 1A2
Blue, 3 µM Vivid® 3A4 Red, 10 µM Vivid® 2C19 Blue, or 10 µM
Vivid® 2D6 Blue. Then, 1 µL of isozyme-specific inhibitor wasadded to stop the reaction, as described for the 1536-well assay incubation conditions, after a 10-min (3A4 Green), 20-min (1A2
Blue and 3A4 Red), 30-min (2C19 Blue), or 60-min (2C9 Green,
2C9 Red, and 2D6 Blue) incubation period. Fluorescence values
were measured using the Ultra plate reader. Background fluorescence was established by wells containing all of the assay components, except BACULOSOMES® Reagents, and was subtracted
before analysis. Percent inhibition was calculated for each concentration of the inhibitor by comparison of fluorescence values tothose wells without the inhibitor. Controls were used to account for
inhibition by DMSO where appropriate. IC50 values were determined by nonlinear regression using Prism software.

Journal of Biomolecular Screening 10(1); 2005

Stability and homogeneity assessmentof the P450 enzyme component
P450 BACULOSOMES® Reagents were used as a source of recombinant cytochrome P450 enzymes to perform metabolic reactions with Vivid® substrates in HTS and uHTS formats. Enzyme
stability is a feature of high importance for uHTS technology, inwhich preservation of the enzyme activity for several hours may berequired. To address these issues, we performed a characterizationof BACULOSOMES® Reagents by particle sizing to assess the homogeneity of the preparation and to check for possible aggregate
formation. Such aggregates could cause variability in P450 activity
levels and could increase light scattering, affecting fluorescentassay performance. Particle size was measured in BACULOSOMES®
suspension and followed a peak distribution in a range from 50 to
500 nm, with the majority of BACULOSOMES® present as 250nm diameter particles (Fig. 1). Relatively narrow variation in particle size in membrane preparations is an indicator of system homogeneity, which is an important parameter contributing to the stability and reproducibility of the enzyme component of the assay. To
follow up the changes in particle size after multiple freeze/thaw cycles, we subjected BACULOSOMES® to 5 freeze/thaw cycles andthen assessed homogeneity. No significant shift in particle size distribution was observed following the freeze/thaw cycles, indicating the absence of aggregates due to the procedure (data notshown). In addition, we tested the stability of BACULOSOMES®
Reagents (CYP3A4, 2D6, 1A2, 2C9, and 2C19) by measuring
their activity with Vivid® fluorogenic substrates before and after a
1- to 7-h incubation on ice or at room temperature (Fig. 2). The
tube containing P450 BACULOSOMES® was stored on ice or atroom temperature, and at the indicated time points, aliquots weretaken out and assayed for activity. No significant loss of P450 activity was observed for the nondiluted stock of CYP3A4, 2D6,
2C9, or 2C19 BACULOSOMES® within the indicated time frameand conditions, as shown in Figure 2. At the same time, an increase
in CYP1A2 activity during the first 60 min of storage on ice or atroom temperature with Vivid® CYP1A2 Blue substrate was observed (Fig. 2). The latter effect is probably related to theconformational rearrangements previously reported for thisisozyme.35 After the initial 1-h equilibration stage, CYP1A2
BACULOSOMES® activity remained stable for up to 7 h whenwww.sbsonline.org 59

Trubetskoy et al.

®

FIG. 2. Nondiluted P450 BACULOSOMES stability at different temperatures. Reactions were performed in triplicate in a 96-well plate, essentiallyas described in the section on 96-well assay incubation conditions.

stored on ice or at room temperature (Fig. 2). The overall 1- to 7-hstability studies performed for all BACULOSOMES® Reagentsindicated that 1 thawed tube of P450 enzyme could be stored in
nondiluted format and used in uHTS applications within the several hours of experimental procedures without significant precipitation or loss of enzymatic activity. At the same time, stability ofthe diluted P450 BACULOSOMES® can be affected by the presence of the components of the NADPH regeneration system anddoes not allow multiple reactions with the 1-step diluted enzyme
preparation. The optimal conditions for the storage of the diluted
P450 enzymes without significant loss of their activity are subjectsof further investigations.
Assay linearity
In addition to the enzymatic component, the chemical and fluorescent characteristics of Vivid® fluorogenic substrates contributed
to the successful development of uHTS applications. The suggested reaction time for each assay was established by exploring
the linearity of metabolite formation in the reactions of CYP3A4,
2D6, 1A2, 2C9, and 2C19 BACULOSOMES® with their corresponding Vivid® substrates (Fig. 3). The readings were obtained in
kinetic mode using the assay and instrument settings described in
Methods and were compared to control reactions performed in theabsence of the enzymatic component. Metabolic rates for the
CYP2D6 Blue and CYP2C9 Red and Green reactions were linear
for at least 60 min at room temperature (Fig. 3). The time linearityof the other Vivid® assays was restricted to 10- to 30-min reactions,depending on the P450 isozyme and fluorescent substrate used

60 www.sbsonline.org(Fig. 3). These reaction times were also used as guidelines to conduct all inhibition assays, as described in the Methods section.
P450 inhibition assays in 1536-well format
The characterization and validation studies in the 1536-wellplate format were carried out to establish the usefulness of the assay in predicting isozyme-specific metabolism and inhibition. This
type of analysis included a titration of test compounds and the generation of dose-dependent curves used for calculating apparent
IC50 values as described previously.27-29 Therefore, 20 concentration point curves for 19 different compounds with 7 different
P450/Vivid® fluorogenic substrate combinations using 3 known
substrates and 1 inhibitor per each pair were generated. The inhibition studies were conducted at room temperature under optimized
buffer conditions (Table 1) at Vivid® substrate concentrations approaching the apparent Km, enabling the detection of weak inhibitors.27,36 The inhibition studies in the 1536-well plate format were
performed in end-point mode, after the fluorescent reaction was
stopped by the addition of an isozyme-specific inhibitor. Apparent
IC50 values obtained in the 1536-well reactions were similar to IC50
values obtained for the same drugs in the 96-well plate format (Table 2), therefore allowing an easy cross-format transition of data.
In all of the cases studied, ranking of a drug’s inhibition potency
was similar between fluorescent and conventional assays, thoughapparent IC50 values obtained with either fluorescent or conventional assays were not identical (Table 2). For a majority of thedrugs, apparent IC50 values in fluorescent assays were similar toIC50 values obtained by screening with conventional substrates.33

Journal of Biomolecular Screening 10(1); 2005

Highly Miniaturized Formats for P450 Assays®

®

FIG. 3. Time linearity and representative dose-response inhibition curves for P450 BACULOSOMES and Vivid substrates reactions. Assays forlinearity of the reaction over time were performed in quadruplicate in 96-well plates, as described in the section on 96-well assay incubation conditions.
The plates were read continuously at room temperature for the indicated periods of time. Dose-response curves were obtained in quadruplicate in 96- and1536-well plate formats, as described in the section on 96- and 1536-well inhibition studies.

Journal of Biomolecular Screening 10(1); 2005

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Trubetskoy et al.
Table 2. IC50 Values (µM) Obtained with Vivid® Substrates in 96- and 1536-Well Formats and Conventional Assays
Vivid Assay

®

Drug

96-Well IC50

1536-Well IC50

Conventional Assay IC50

1A2 Blue

Propranolol
Furafylline
Ellipticine
Ticlopidine
Sulfaphenazole
Diclofenac
Tamoxifen
Warfarin
Sulfaphenazole
Diclofenac
Tamoxifen
Warfarin
Amitriptyline
Tranylcypromine
Omeprazole
Ticlopidine
Quinidine
Bufuralol
Imipramine
Clomipramine
Ketoconazole
Erythromycin
Verapamil
Troleandomycin
Ketoconazole
Erythromycin
Verapamil
Troleandomycin

0.3 ± 0.1
1 ± 0.1
0.02 ± 0.01
1.2 ± 0.4
0.3 ± 0.02
3.3 ± 18.6 ± 0.5
18 ± 30.1 ± 0.02
2.1 ± 0.1
5.1 ± 0.4
5.4 ± 0.5
2.0 ± 0.2
2.9 ± 0.1
2.2 ± 0.5
0.2 ± 0.02
0.004 ± 0.002
7±1
1 ± 0.13
0.3 ± 0.03
0.004 ± 0.0004
2.4 ± 0.1
2.7 ± 0.1
0.21 ± 0.01
0.1 ± 0.02
0.4 ± 0.04
0.3 ± 0.1
0.1 ± 0.03

0.6 ± 0.04
1.9 ± 0.1
0.02 ± 0.001
1.4 ± 0.2
0.3 ± 0.02
3.5 ± 0.8
12 ± 1.0
18 ± 0.5
0.1 ± 0.001
1.8 ± 0.15±2
4.5 ± 0.3
2.9 ± 0.1
2.9 ± 0.1
2.4 ± 0.1
0.3 ± 0.02
0.002 ± 0.0001
9.3 ± 0.8
1.4 ± 0.2
0.4 ± 0.03
0.009 ± 0.003
2.0 ± 0.1
3.9 ± 0.3
0.22 ± 0.03
0.06 ± 0.01
0.6 ± 0.1
0.5 ± 0.02
0.2 ± 0.02

3.84 ± 0.63
1.2-2.9
0.11 ± 0.01
6.1 ± 0.45
2.3-2.5>30
10 ± 222 ± 2
2.3-2.5>30
10 ± 222 ± 23.3 ± 1.2
8.2 ± 0.67
2.1 ± 0.36
0.40 ± 0.04
0.005 ± 0.001
6.1 ± 1.6
3.8 ± 0.99
1.6 ± 0.17
0.005-0.046
2.4 ± 0.35
2.0 ± 0.28
1.0 ± 0.95
0.005-0.046
2.4 ± 0.35
2.0 ± 0.28
1.0 ± 0.95

2C9 Green

2C9 Red

2C19 Blue

2D6 Blue

3A4 Green

3A4 Red32

As reported by Cohen et al. All assays were performed in quadruplicate, n = 4.

However, for some of the assay modifiers, such as propranolol(CYP1A2) or sulfaphenazole (CYP2C9), an overprediction of theinhibition potency (lower IC50 values) in fluorescence-based competitive assays versus the conventional assays was observed. Although similar issues have been reported previously by attempts at
direct correlations between conventional and fluorescent assays,33
they do not invalidate the application of fluorescent assays for theassessment of P450 metabolism and inhibition. Although a direct
correlation between the IC50 values obtained in both types of assays is not always advisable, the fluorescent assays are rapid
screening tools for the ranking of compounds’ inhibitory potency
and may be followed by detailed characterization of the identified
inhibitors by lower throughput applications or in vivo assessment.
1536-Well plate assay characteristics
To assess the robustness of the uHTS format for cytochrome
P450 metabolism and inhibition, several characteristics of the assay, such as signal-to-background ratio and Z′ factor, were calculated and compared to those obtained in the 96-well plate format(Table 3). All assays employing the fluorogenic Vivid® substrates
in the 1536-well format demonstrated broad dynamic range (Table

62 www.sbsonline.org

3) and high Z′ factors in a range of 0.66 to 0.82, therefore exhibiting characteristics of highly reliable and reproducible assays.
DISCUSSION
During the past decade of drug discovery, the application of
HTS approaches has succeeded in accelerating the generation of
new lead compounds in the pharmaceutical industry. One of the
new challenges for HTS now lies in the area of early ADME
screening, demanding the development of technologies amenable
to highly miniaturized formats. Here we have described the development of fluorescence-based uHTS assays for inhibitor screening, employing Vivid® fluorogenic substrates and recombinantly
expressed principal human cytochrome P450 isozymes in the1536-well plate format. Drug metabolism assays using humanliver microsomes (HLMs), hepatocytes, and tissue slices arewidely used in preclinical drug metabolism studies and ADME
screening in vitro. Many in vitro studies are done with HLMs, buthigh interindividual variability in the expression levels of particular P450 isoforms, population-dependent polymorphism, and heterogeneity due to the presence of multiple cytochrome P450
isoforms limit their use in HTS applications. Recently, progress in

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Highly Miniaturized Formats for P450 Assays
Table 3. Vivid® Assay Signal-to-Background (S/B) and Z′ Factor
96-Well, No DMSO

1536-Well, No DMSO

1536-Well, 1% DMSO®

Vivid Assay

S/B

Z

S/B

Z

S/B

Z

1A2 Blue
2C9 Green
2C9 Red
2C19 Blue
2D6 Blue
3A4 Green
3A4 Red57 ± 73.0 ± 0.1
52 ± 1053 ± 45.5 ± 0.4
29 ± 280 ± 8

0.89
0.87
0.90
0.80
0.86
0.92
0.88

12 ± 2
10.3 ± 1.0
66 ± 825 ± 23.5 ± 0.2
40 ± 690 ± 11

0.71
0.77
0.75
0.80
0.76
0.72
0.76

10.8 ± 0.9
9.6 ± 0.955 ± 419.6 ± 1.4
2.10 ± 0.12
22 ± 341 ± 4

0.73
0.79
0.82
0.81
0.66
0.72
0.80

recombinant human protein production has allowed a variety of recombinant human P450 enzymes to be expressed in Escherichia
coli, insect, and mammalian cell expression systems.31,37,38 A typical cytochrome P450 system of this type consists of a microsomal
fraction of the baculovirus- or lymphoblast-expressed P450,
coexpressed with P450 oxidoreductase and, in some cases,
cytochrome b 5 . 39 One of the main advantages of theseheterologously expressed individual P450 isozymes is that they
serve as a more reliable and consistent source of P450 enzyme andshow significantly increased rates of metabolism that are suitablefor detection in the uHTS format. Although recombinant P450s do
not have exactly the same properties as HLMs, they have an advantage in that only 1 cytochrome P450 isozyme is present, allowing
one to exclude the contributions from other P450 isozymes andother drug-metabolizing enzymes, such as flavin monooxygenases and UDP-glycosyltransferases. This simplifies the assessment of isozyme involvement in the metabolism of a particulardrug, the conclusions about inhibitor potency, and determining relative affinities and contributions of the involved enzymes without
requiring the use of isozyme-specific inhibitors or inhibitory antibodies. To be used as a tool in HTS or uHTS assays, the recombinant P450 proteins must be reliable in terms of homogeneity, enzyme stability, and reproducibility. In many cases, recombinant
enzymes produced as a microsomal fraction from overexpressing
cell lines have limited stability due to the heterogeneity of the preparation and the precipitates form following a limited storage atroom temperature. Exposing the enzyme to an extended storage atroom temperature is often a necessary step in an HTS assay inwhich more than 1 multiwell plate at a given time is used. The precipitated fractions may reduce enzyme activity and affect the accuracy and predictability of the assay. This is especially important formembrane and lipid-containing fractions, such as microsomes,
in low-volume fluorescent assays in which precipitation may significantly increase the background fluorescence due to a lightscattering effect. In the current study, extended stability (7 h atroom temperature) and homogeneity of the nondiluted
BACULOSOMES® stock were demonstrated. In addition to thestability, the high specific content and activity of these recombinant enzymes made them appropriate for the development of low-

Journal of Biomolecular Screening 10(1); 2005

volume uHTS fluorescent assays with Vivid® substrates, whichhave been previously employed in studies of metabolism and inhibition with several P450 isozymes in the 96-well plate format.27,28
Several distinctive features of the Vivid® substrates employedin the current study made the development of uHTS screening assays possible. Vivid® substrates (http://gcms.invitrogen.com/content. cfm?pageid=10058) were originally developed as benzyloxymethyl and alkoxymethyl ethers of available phenolic dyes,
such as resorufin, fluorescein, 3-cyano-7-hydroxycoumarin, andthe structurally related 7-hydroxy-4-trifluoromethylcoumarin.26,30
The nonmodified phenolic dyes and their commercially available
benzyl derivatives often suffer from high initial fluorescent background, interference from NADPH fluorescence, and low turnoverrates that prevent or complicate their use in uHTS assays, necessitating several additional steps or coupled reactions. For example,
an application of DFB, a fluorescent CYP3A4 substrate, requires
an additional step for the removal of excess NADPH at the end of
incubation to prevent NADPH interference with the fluorescentmetabolite detection. This is accomplished by adding oxidized
glutathione and glutathione reductase to convert NADPH to
NADP+, which is not fluorescent.21 In other cases, a derivatization
step with alkali to enhance the signal-to-background ratio may berequired.40
Vivid® fluorogenic substrates exhibit a very low level of background fluorescence (Fig. 3) and do not require an additional stepor coupled reaction to achieve high signal-to-background ratios inP450 reactions (Table 3). Metabolites of the Vivid® substrates havefavorable fluorescent characteristics, such as excitation in the visible range of the light spectrum, preventing interference from
NADPH and the majority of compounds with an absorbance peakin the UV range. Although recently developed alternative technology employing chemiluminescent P450 substrates presents an additional advantage for screening fluorescent compound libraries
and exhibits high signal-to-background ratios,17 these assays require a coupled reaction with luciferase and are prone to the detection of false positives due to the inhibition of luciferase by reaction
modifiers. In addition, high aqueous solubility of the Vivid® substrates allows lowering the organic solvent concentration used inassays by keeping it at or below 0.5% acetonitrile in all final reac-

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Trubetskoy et al.
tion mixtures (Table 1). This latter feature is extremely importantfor several cytochrome P450 isozymes due to known effects of organic solvents on the course of their metabolism.41
Assay characterization and validation studies were carried out
to establish the usefulness of the 1536-well format for theprediction of isozyme-specific metabolism and inhibition. Doseresponse curves for 19 different compounds were generated with 7
different P450/Vivid® fluorogenic substrate combinations (3isozyme specific substrates and 1 inhibitor per each pair). Here wedemonstrate that Vivid® fluorescent assays with recombinant P450
isozymes performed in the 1536-well format were able to detect
isozyme-specific substrates and inhibitors for CYP1A2, 2C9,
2C19, 2D6, and 3A4. The apparent IC50 values obtained for eachdrug in the 1536-well plate format were in good agreement withthe values obtained in the 96-well plate format, as indicated in Table 2. Application of only 1 fluorescent probe to metabolism andinhibition studies with CYP1A2, 2D6, and 2C19 was sufficient. At
the same time, more complex interactions between multiple substrates at the active site of CYP3A442 and CYP2C943 have been reported, indicating a need to apply multiple probes for these
isoforms’ metabolic assessment. The advantages of applying several structurally unrelated fluorescent probes have been discussed
previously for CYP2C929 and CYP3A4.44 In our studies, we applied both Vivid® Green and Red fluorescent substrates to
CYP3A4 and CYP2C9 metabolism and inhibition. Apparent IC50
values obtained with Vivid® Green substrates better correlatedwith results obtained with conventional substrates for bothisozymes (Table 2). At the same time, Vivid® Red fluorescent substrates may have the advantage of more sensitive detection andmay avoid interference from library compounds with fluorescentbackground in the blue and green ranges of the fluorescent
spectrum.
Typically, assay miniaturization will result in greater data variability and affect several assay parameters such as dynamic range
and Z′ factor. It is important that Vivid® assay miniaturization did
not show any significant negative effects on assay performance.
Vivid® assays carried out in the 1536-well plate format demonstrated only a slight deviation from the assay parameters obtainedin the 96-well format. All Vivid® assays performed in the 1536well plate format have a very high Z′ factor in a range of 0.66
(CYP2D6 Blue) to 0.82 (CYP2C9 Red), making them highly reliable for uHTS applications. One of the most significant effects ofassay miniaturization was reduction in the signal-to-backgroundratio observed for CYP1A2 Blue and CYP2C19 Blue Vivid® reactions performed in the 1536-well plate format, but broad dynamic
range was maintained in both applications (Table 3) and did not affect the detection of assay modifiers. Some of the variations in dynamic range observed between the 96- and 1536-well formats
could be due to the use of filter-based instruments (Ultra plate
reader) versus monochromator-based instruments (Gemini XS
plate reader) for detection. The effects of several organic solventson the performance of assays employing Vivid® fluorogenic substrates and recombinant cytochrome P450 enzymes have been re-

64 www.sbsonline.org

ported previously.27-29 Here we explored assay tolerance to thepresence of DMSO, an organic solvent commonly used to dissolve
compound libraries in HTS applications. The presence of 1%
DMSO did not significantly affect the Z′ factor in any of the 1536well Vivid® assays but did result in the reduction of signal-to-background ratios, which was most dramatic in the CYP3A4 Vivid®
Green and Red assays. However, high signal-to-background ratios
in a range from 22 to 41 were still maintained under these conditions (Table 3), thus confirming the robustness and reliability of theassay performance in the presence of 1% DMSO. As a general
guideline, the recommendation is to apply a proper control to account for the effect of DMSO or any organic solvent used in Vivid®
assays.27-29 Alternatively, to minimize the negative effect of DMSO
on assay performance, test compounds could be delivered as
redissolved dry films, according to recent data demonstratingcomparable activity for compounds delivered in DMSO stocks or
as dried compound films.23 The latter approach may be preferable
for further development of fully automated miniaturized assays
and robotic applications.
Overall, we proved that HTS methods for in vitro cytochrome
P450 metabolism and inhibition studies employing the Vivid®
P450 fluorescent substrates and major human P450 isozymes described here are sensitive, homogeneous, robust, and amenable tominiaturization, allowing for ultra-high-throughput formats. The
strong fluorescent signal, high signal-to-background ratio, and excellent Z′ factor value make these assays valuable tools for screening large compound libraries for cytochrome P450 metabolism
and inhibition in a cost-effective, low-volume screening format.
Furthermore, reduced amounts of enzyme used in uHTS present
the advantage of minimizing enzyme variability by conducting
multiple P450 assays with the same batch of the recombinantcytochrome P450 enzyme. This enables more accurate and consistent data generation during the extended lead optimization process.
One potential drawback of assays based on fluorescent reporter
reactions is that both substrates and inhibitors act as assay modifiers by inhibiting the reporter fluorescent reaction, therefore preventing discrimination between allosteric and mechanism-based
inhibition. However, this is a common issue for all of the known
fluorescent reporter reactions and may be resolved in the future byapplication of more sophisticated kinetic models45 that were not a
subject of the current investigation. In summary, highly miniaturized formats of the Vivid® fluorescent assays for the assessment of
human cytochrome P450 metabolism and inhibition present a
valuable contribution to the area of early ADME toxicology
studies.
ACKNOWLEDGMENTS
The authors would like to acknowledge Steve Duff, James
Farnham, Naveeda Qadir, Hildegard Eliason, and Mohammed
Shekhani for their contributions in the synthesis and purification of
the Vivid® fluorogenic substrates used in the current study and
Gregory Parker for his assistance in the preparation of the figures.

Journal of Biomolecular Screening 10(1); 2005

Highly Miniaturized Formats for P450 Assays
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Address reprint requests to:
Bryan D. Marks
Invitrogen Drug Discovery Solutions
501 Charmany Drive
Madison, WI 53719
E-mail: Bryan.Marks@invitrogen.com

66 www.sbsonline.org

Journal of Biomolecular Screening 10(1); 2005