Miniaturization and automation of cytochrome P450 inhibition assays
Bryan D. Marks and Brad R. Larson

Invitrogen Discovery Sciences • 501 Charmany Drive • Madison, Wisconsin 53719 • USA

P450 metabolism

2C9
BOMF

2C9
OOMR

2C19
EOMCC

2D6
EOMCC

3A4
BOMCC

3A4
DBOMF

120

4000

amitriptyline

>100

>100

60

9.9

2.5

23

32

100

3000

astemizole

>100

13

7.7

7.5

5.8

1.2

2.5

80

RFU

2C19 EOM CC

2000

signal1000

Vivid®
substrates
arealkoxymethyl
andbenzyloxymethyl ethers of fluorescent dyes.
Cytochrome P450 metabolism cleaves the quenchedsubstrate, resulting in a highly fluorescent metabolite.
P450 inhibitors are identified by their ability toprevent formation of fluorescent signal in the Vivid®
assay. The structures of the five substrates used inthis study are indicated below.

No metabolism

2000

backgroundbufuralol

>100

>100

>100

>100

3.8

>100

>100

bupropion

ND

>100

>100

11

2.0

>100

>100

clotrimazole

10

0.44

0.20

0.97

9.1

0.013

0.011

dextromethorphan

ND

450

>1000

180

0.66

120

54

15000

diclofenac

>1000

4.9

3.0

190

630

>1000

750

10000

fluoxetine

41

23

12

0.46

0.92

11

21

signalbackground

1000

0

0

0

10

20
30
reaction time (min)

40

0

10

20
30
reaction time (min)

40

15000
20000

2C9 BOM F

2C9 OOM R

RFU

12000
9000

RFU

Vivid® Metabolite
Highly Fluorescent

RFU

Vivid® Substrate
Low Fluorescence

Low Fluorescence

1A2
EOMCC

5000

3000

6000
signal

EOMCC

3000

signal

5000

backgroundfurafylline

2.6

>300

>300

120

160

110

60

imipramine

>100

>100

98

23

0.69

21

49

ketoconazole

65

7.6

3.3

5.1

7.2

0.13

0.016

mifepristone

>100

3.4

3.1

6.5

11

1.2

2.7

miconazole

2.8

1.1

2.2

0.069

0.43

0.64

0.44

nifedipine

7.9

1.7

1.0

1.3

>100

0.72

0.29

background

α-naphthoflavone

0.029

4.2

2.4

6.8

ND

24

9.6

40

omeprazole

250

5.9

2.9

1.7

120

24

31

background

BOMF
0

0
0

BOMCC

10

20
30
reaction time (min)40

0

3A4 BOM CC

5000

OOMR

RFU

RFU

3000
signalbackground

1000

2500
0

0

Assay

[Substrate](µM)

Excitation/Emission(nm)

1A2 EOMCC

53

415/460

0

10

20
30
reaction time (min)

0

40

10

20
30
reaction time (min)4000

2C9 BOMF
2C9 OOMR

Materials – P450 Baculosomes® (microsomes prepared from insect cells
containing a single human P450 and P450 reductase), control Baculosomes®
(microsomes prepared from insect cells infected with a control virus),
Regeneration System (glucose-6-phosphate dehydrogenase and glucose-6phosphate), NADP+, Vivid® Substrates, and assay buffer were from Invitrogen
Corp. (Madison, WI). Assay plates were 384-well low volume black polystyrene
plates (cat. no. 3677) from Corning (Acton, MA).
Automation Equipment – A Biomek FX Laboratory Automation Workstation(Beckman Coulter, Fullerton, CA) was used for test compound/DMSO control
stamping and to dispense all assay reagents. In order to increase throughput, aCytomat Microplate Hotel (Beckman-Coulter, part no. 394597) was partnered with
the Biomek FX for extra storage of assay plates, reagents, and tip boxes.

Z’-factor is a parameter used to measure the robustness of an assay, taking into
account both assay window and variability. Values >0.5 are generally considered
suitable for high-throughput screening, while a value of 1 indicates a theoretical
ideal assay with no variability (Zhang et al, 1999).

550/590510415/460

2D6 EOMCC

20

10415/460

3A4 BOMCC

2.5

10415/460

3A4 DBOMF

2.5

2490/520

signal1000

background

0

10

20
30
reaction time (min)40

Figure 3 depicts the generation of fluorescent metabolite in each assay over time at room
temperature. “Signal” indicates the fluorescent intensity measured in the presence of P450
Baculosomes®, while “background” represents the fluorescent intensity of wells containing
control Baculosomes®. Error bars represent the standard deviation of 3 replicate assay wells.
A 25-minute incubation time was used for screening test compounds in the Vivid® P450
inhibition assays.

Figure 4 –Signal Stability After Addition of Stop
Solution

1A2 EOMCC
2C19 EOMCC

A

2D6 EOMCC

0

1

2

3

4

2C9 OOMR

0

3A4 BOMCCC

3A4 DBOMF

0

1

2

3

4

reagent storage time prior to assay (hrs)2

3

120 min180 min
240 min10 -4 10 -3 10 -2 10 -1 10 0
[α -naphthoflavone] (µM )

4

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

1A2 EOMCC

2D6 EOMCC

2C9 OOMR

3A4 BOMCC

2C9 BOMF

3A4 DBOMF

2C19 EOMCC

0

1

2

3

D
4

10 1

110
100
90
80
70
60
50
40
30
20
10
0
-10
10 -6

20
0
-20
10 -4

560

590

390

ND

470

150

>100

>100

>100

1.1

>100

>100

quinidine

ND

>100

>100

>100

0.008

20

29

saquinavir

ND

9.2

5.8

15

6.2

2.3

3.2

sulfaphenazole

ND

0.31

0.13

>300

ND

190

>300

terfenadine

78

15

12

10

0.55

0.34

0.86

testosterone

ND

88

45

69

>100

A

45

0

troglitazone

36

1.0

0.64

3.1

32

2.1

3.9

-20

troleandomycin

ND

ND

ND

ND

>100

1.6

5.0

verapamil

ND

>100

51

>100

88

3.2

9.2

warfarin

ND

11

9.0

>200

>200

ND

>200

180 min
240 min

10 -5

10 -4 10 -3 10 -2 10 -1[ketoconazole] (µM )

10 0

10 1

110
100
90
80
70
60
50
40
30
20
10
0
-10
10 -4

5 min
15 min60 min
120 min180 min
240 min

10 -3

10 -2 10 -1
10 0
10 1
[sulfaphenazole] (µM )

10 2

Assay

Signal-tobackgroundratio(avg ± std dev)

% std dev
(signal, background)Z’-Factor(avg ± std dev)

1A2 EOMCC

63±46.2, 2.80.81±0.03

2C9 BOMF

10.8±1.1

3.0, 1.7

0.90±0.02

2C9 OOMR

56±142.8, 6.4

0.91±0.02

2C19 EOMCC

43±34.7, 3.0

0.85±0.02

2D6 EOMCC

10.2±0.7

4.7, 2.4

0.84±0.04

3A4 BOMCC

160±204.7, 4.8

0.86±0.04

3A4 DBOMF

33±9

3.0, 3.3

0.90±0.03

reagent storage time prior to assay (hrs)

Each 3X Baculosomes®/Vivid® Substrate reagent was prepared in assay buffer andincubated at room temperature for varying time periods prior to measurement ofactivity (Figures 2A, 2B, 2C). Note that while not all P450/substrate reagents retain full
activity at later timepoints, the quality of the data remains robust even when stored for
4 hours as indicated by Z’-factor values >0.7 (Figure 2D). The 3X Regeneration
System/NADP+ reagent was found to be stable for at least 4 hours when stored atroom temperature (data not shown).

Because the assays were performed using a single plate read in endpoint mode, a basic tris
solution (pH 10.5) was used to stop the reaction at the end of the desired incubation time.
After addition, the pH value was increased from 7.4 to approximately 9.5. Acetonitrile, avolatile solvent commonly used as a stop solution component in other methods, was notneeded for the Vivid® assays. The pH increase was sufficient to stop P450 activity, andfollowing addition, the signal was found to be stable for several hours in all assays. Example
data illustrating the stability of IC50 measurements is shown for assays utilizing
cyanocoumarin (EOMCC), fluorescein (DBOMF), and resorufin-based (OOMR) substrates.

10 -3

10 -2 10 -1 10 0
10 1
[quinidine] (µM )

10 2

10 3

120
100

1A2 EOMCC
2C9 BOMF
2C19 EOMCC
2D6 EOMCC
3A4 BOMCC
3A4 DBOMF

80
60
40
20

10 -3

10 -2 10 -1 10 0
10 1
10 2
[sulfaphenazole] (µM )10 3

Test compounds were tested against the panel of P450 inhibition assays using an
11-point titration (n=2) and 3-fold serial dilution scheme. The example data
above illustrates screening results using the potent CYP3A4 inhibitor
ketoconazole, selective CYP2D6 inhibitor quinidine, and selective CYP2C9
inhibitor sulfaphenazole.

Results and Conclusions

Table 3 – Vivid® Assay Robustness
120 min

1A2 EOMCC
2C9 BOMF
2C19 EOMCC
2D6 EOMCC
3A4 BOMCC
3A4 DBOMF

40

1.1

60 min

10 3

60

9.1

5 min
15 min2C9 OOM R

reagent storage time prior to assay (hrs)Z'-factor% activity

120
110
100
90
80
70
60
50
40
30
20
10
0

1

60 min%activity

reagent storage time prior to assay (hrs)

5 min
15 min10 -5

B

2C9 BOMF

3A4 DBOM F%activity

120
110
100
90
80
70
60
50
40
30
20
10
0

%activity% activity% activity

120
110
100
90
80
70
60
50
40
30
20
10
0

110
100
90
80
70
60
50
40
30
20
10
0
-10

10 2

80

phenacetin

ND indicates inhibition not detected, A indicates apparent activation of P450-mediated probe substrate metabolism by test
compound, “>” is used to denote cases where inhibition was observed but ≥50% inhibition was not achieved at the highest
concentration of test compound used. None of the compounds above exhibited fluorescent interference in any of the Vivid®
assays.

10 -2 10 -1 10 0
10 1
[ketoconazole] (µM )100propranolol

0IC50 Determination
Percent activity of sample wells was plotted versus the log value of each test
compound concentration to generate an 11-point dose-response curve. Nonlinear
regression was performed with GraphPad Prism software (San Diego, CA) using
an equation for sigmoidal dose-response (variable Hill slope) to determine the IC50
value.

2000

1A2 EOM CC% activity = [(RFUsample – RFUbackground)/(RFUsignal – RFUbackground)] x 100%

Z’ = 1- [(3xStd devsignal RFU+ 3xStd devbackground RFU)/(meansignal RFU – meanbackground RFU)]

1

Figure 2 – Room Temp Reagent Stability

Percent Activity
where RFU = “relative fluorescence units”, fluorescent intensity

20

490/520

2C19 EOMCC

Data Analysis

Z’-Factor

1

Each assay also contained 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6phosphate dehydrogenase, and 30 µM NADP+ to supply a constant amount of thecofactor NADPH. The buffer was 100 mM potassium phosphate pH 7.4, except for the
CYP2C9 assays, which contained 60 mM potassium phosphate pH 7.4. All assays also
contained 1% DMSO, introduced as the test compound vehicle solvent or included incontrol assay wells. A 12 nm bandwidth was used for both excitation and detection oflight emission, centered on the wavelengths listed above.

Compound Preparation – Serial dilutions of test compounds were performed in
100% DMSO at 100X the final assay concentration. Compounds were then diluted
to 3X concentration using assay buffer. Assay plates were then stamped with 5 µL
of 3X test compound to sample wells or 5 µL of 3% DMSO to control wells.
Vivid P450 Inhibition Assay – All steps were conducted at room temperature.
5 µL of 3X P450 Baculosomes®/Vivid® Substrate or 3X control
Baculosomes®/Vivid® Substrate in assay buffer (0.2X assay buffer for CYP2C9
assays) were dispensed to the assay plate and shaken on an orbital shaker for 30
seconds. Each test compound concentration was tested in duplicate in thepresence of P450 Baculosomes® to assess inhibition as well as in the presence ofcontrol Baculosomes® to detect autofluorescent compounds. 1% DMSO in thecontrol wells was used in the presence of P450 Baculosomes® (n=16) to represent
100% activity (“signal”) or in the presence of control Baculosomes® (n=16) torepresent 0% activity (“background”). Plates were incubated for 10 min prior tostarting the reactions with 5 µL of 3X Regeneration System/NADP+ to all wells.
Plates were shaken for 30 sec as above and incubated for 25 min. The reaction
was stopped by addition of 5 µL of 0.5 M tris base (pH 10.5) to all wells andshaken for 30 sec as above. Plates were then read on a Safire (Tecan, Durham,NC) plate reader using the fluorescent excitation and emission settings indicatedin Table 1.

20

RFU

Methods

10 -3

120

2D6 EOM CC
3000

40

0

signal

5000

60

-20
10 -4

10000
7500

1A2 EOMCC
2C9 BOMF
2C19 EOMCC
2D6 EOMCC
3A4 BOMCC
3A4 DBOMF

20

40

12500

2000

[P450](nM)

20
30
reaction time (min)

3A4 DBOMF

15000

4000

Table 1 – Vivid® Assay Conditions

10

17500

6000

DBOMF

Figure 5 – Example Inhibition Curves

Compound

4000
1A2 EOM CC

P450 inhibition

Table 2 – Vivid® Assay Example IC50 Values (µM)% activity

Members of the cytochrome P450 (CYP) family of metabolic enzymes
are recognized as a major factor in adverse drug interactions, andscreening new chemical entities (NCEs) for P450 inhibition potential isa critical step in the drug discovery process. Hence, there is a need for
rapid, reliable assessment of the P450 inhibition potential of NCEs. To
this end, cytochrome P450 inhibition assays were developed to detectdrug-drug interactions (DDI) using microsomes containing cDNAexpressed human CYP1A2, 2C9, 2C19, 2D6, or 3A4 with cytochrome
P450 reductase (P450 Baculosomes® Reagents) and fluorogenic
Vivid® substrates in a 384-well plate format using a reaction volumeof 15 µL. Several factors were determined to be especiallyadvantageous in developing a streamlined assay protocol compatible
with automation. The assay reagents were shown to be stable atroom temperature, enabling automated reagent dispensing over thecourse of several hours. The rate of metabolite formation in eachassay was observed to be linear for at least 30 minutes at room
temperature, allowing use of a uniform reaction time. Each assay wascompatible with the presence of 1% DMSO, allowing standardized
compound handling. Use of a universal stop solution, which lacks avolatile organic solvent, enabled the assays to be performed in
endpoint mode. After stopping the reaction, the fluorescent assay
signal was found to be stable for several hours. Multiple structurally
unique substrates were used for CYP3A4 and 2C9 to detect potential
probe substrate-dependent inhibition, heteroactivation, or othercomplex DDI. The use of multiple substrates with distinct opticalproperties, as well as rigorous intra-plate controls, were also used to
identify and account for possible test compound autofluorescence.
The miniaturized P450 inhibition assays are robust, with average Z’factor values >0.8 and inhibition results in agreement with those of
known inhibitors for each P450. The assays described here are offeredand used in a custom P450 profiling service.

Figure 3 – Assay Linearity% activity

Figure 1 – Vivid® Assay Principle% activity

Abstract

Table 3 lists performance parameters for each assay, calculated using the control wells on asingle assay plate taken from five independent experiments. Signal (“100% activity”) was
calculated from the fluorescence intensity of wells containing P450 Baculosomes® (n=16), andbackground (“0% activity) was determined from the fluorescence intensity of wells containing
control Baculosomes® (n=16), both in the presence of 1% DMSO. All of the assays were very
robust, with Z’-factor values ≥0.81 and low % standard deviation values.

•

A robust automated high-throughput screening method was developed toassess inhibition of the five major cytochrome P450 enzymes involved in
drug metabolism using P450 Baculosomes® and Vivid® fluorogenic
substrates.

•

Reagent stability, assay linearity, and fluorescent signal stability were key
factors that enabled development of a robust automation method. Assay
compatibility with 1% DMSO enabled use of a standardized compoundstorage and handling system.

•

The Vivid® assays have proven to be robust, with low variability andexcellent Z’-factor values (>0.8).

•

The suite of Vivid® P450 inhibition assays has been validated with a panelof compounds with known effects on P450-mediated metabolism. Results
were in agreement with reported P450 interactions of these drugs.

•

Using this method, a P450 inhibition profile can be generated for any
novel test compound both quickly and with a high degree of confidence.
This service is available from Invitrogen on a custom basis with a rapid
project turnaround time.