Download Novel Fluorescent Probes Detect Different Types Of CYP450

Novel Fluorescent Probes Detect Different Types Of CYP450-Drug Interactions In High Throughput Mode
Gregor Zlokarnik, Laurie P. Volak, Michele M. Andrew, Chinh Tran, Thomas Cleveland, Lewis R. Makings; Chemistry Department, Aurora Biosciences Corporation, 11010 Torreyana Road, San Diego, CA 92121
0
5
C
R1
15
0
20
D
VividTM 3A4 Blue
R2
R2
+
H2CO
+
+
20
6
8
10
O
0
O
Metabolism of
VividTM 3A4 Substrates
VividTM 3A4 substrates are benzyloxymethyl ethers
of phenolic fluorescent dyes. Unlike their benzylic
counterparts, they possess two potential sites of
oxidation that can lead to release of a fluorescent
metabolite. Ab initio calculations suggest that the
benzylic position (marked in red) is the more likely
site of metabolism (6) as hydrogen abstraction
creates a more stable radical intermediate.
BFC
v {min }
5
-1
4
O
O
O
3
25
0
% Activity
400
This work was supported in part by
NIH grant GM60114
O
3
2
Red
Green
Blue
Cyan
300
200
50
100
25
0
-8 -7 -6 -5 -4
HLM
100
75
50
25
Red
Green
Blue
Cyan
Red
Green
Blue
Cyan
TM
15
-1
v {min }
O
O
O
O
CN
10
5
1A
2
2A
6
2B
6
2C
8
2C
2C 9
18
2C
19
2D
6
2E
1
3A
4
3A
5
0
15
-1
v {min }
VividTM 3A4 Cyan
O
O
O
O
10
5
0
Vivid
Drug
Licensing opportunities:
Michael J. Dunn
Vice President, Business Development
Aurora Biosciences Corporation
11010 Torreyana Road
San Diego, CA 92121
(858) 404-6769;
[email protected]
VividTM 3A4 substrates were treated with
individually expressed human cytochrome P450
isozymes.
Substrates are predominantly
metabolized by CYP3A isozymes, except for
coumarin-based substrates which are also
metabolized by CYP2B6. (Error bars represent
standard deviation).
RHI
3A4 Green
HLM
RHI
3A4 Blue
HLM
RHI
3A4 Cyan
HLM
RHI
HLM
mean
SD
(log)
mean
SD
(log)
mean
SD
(log)
mean
SD
(log)
mean
SD
(log)
mean
SD
(log)
mean
SD
(log)
mean
0.02
0.2
1.0
0.1
0.03
0.3
0.02
0.1
0.3
0.3
1
0.1
0.4
0.1
2
0.1
Miconazole
0.9 - 1.3
0.03
0.1
0.1
0.1
0.1
0.02
0.1
0.01
0.1
0.1
0.1
0.05
0.4
0.1
0.2
0.05
Nifedipine
10 - 22
0.4
0.1
20
0.1
0.2
0.1
2
0.1
0.8
0.1
10
0.1
1
0.1
70
0.1
Verapamil
24 - 82
0.5
0.05
80
0.01
2
0.1
10
0.2
1
0.1
30
0.04
2
0.5
100
0.04
Troleandomycin
10 - 51
1
0.1
0.2 *
0.3
0.7
0.1
5
0.05
1
0.1
7*
0.1
1
0.03
>>100
n/a
Erythromycin
16 - 194
1
0.2
8*
0.1
3
0.1
100
0.2
7
0.2
50 *
0.3
2
0.1
>>300
n/a
47
1*
0.3
>100
n/a
5
0.04
30
0.04
1
0.2
40 *
0.2
2
0.03
>100
n/a
50 - 75
3
0.1
60
0.2
4
0.1
20
0.02
4
0.01
40 *
0.02
2
0.04
>400
n/a
34
10
0.2
20
0.1
5
0.04
20
0.03
4
0.05
30
0.01
4
0.1
80
0.1
Testosterone
40 - 60
(3)
0.1
>>100
n/a
5*
0.5
20
0.1
60
0.3
>100
n/a
>>80
n/a
>>100
n/a
Progesterone
8 - 45
(4)
0.2
200
0.3
10 *
0.2
20
0.01
50
0.3
100
0.1
>>80
n/a
200
0.1
Diltiazem
RHI : Recombinant Human Isozyme (baculovirus expression, insect cell microsomes);
HLM : pooled Human Liver Microsomes
mean : geometric mean of triplicates; SD (log) : standard deviation of geometric mean in log units;
( ) : activation; * : partial inhibition
Apparent Ki values with Recombinant CYP3A4 and Human Liver Microsomes
75
50
% Activity
CYP2C
CYP3A4
CYP2D6
75
75
Blue
Red
Green
50
25
50
0
-7 -6 -5 -4
[Drug] log {M}
-8
-7 -6 -5 -4
[Drug] log {M}
Blue
Red
Green
200
25
0
0
-8 -7 -6 -5 -4
-8 -7 -6 -5 -4
[Sulfaphenazole]
-8 -7 -6 -5 -4
[Warfarin] log {M}
Dose-Response Curves
VividTM substrates were used to assess interaction of CYP2C9 with inhibitors (A, B),
and an activator (C). Assuming a two-site model for the CYP2C9 active site (or a large
TM
2C9 Blue, a
binding site permitting simultaneous binding of two substrates), Vivid
coumarin-based substrate, is most sensitive to drug-interaction at the activator site (panel
TM
C). Also, lansoprazole and sulfaphenazole inhibit Vivid 2C9 Blue turnover only partially,
while they fully inhibit turnover of the other substrates. The resorufin-based substrate ,
TM
Vivid 2C9 Red is somewhat more sensitive to drug-interactions with CYP2C9 than
TM
Vivid 2C9 Green.
XI
VividTM
SD
(log)
mean
SD
(log)
mean
SD
(log)
0.09 - 1.6
0.3 *
0.1
0.01
0.1
0.6
0.1
5 - 15
3*
0.1
0.3
0.1
6
0.1
Lansoprazole
45 - 52
5*
0.1
2
0.03
10
0.2
Omeprazole
40 - 240
5*
0.1
0.4
0.03
10
0.1
Tolbutamide
96 - 371
100
0.2
> 200
n/a
>>800
n/a
Warfarin
0.25 - 27
(6)
0.1
10
0.1
20
0.2
Sulfaphenazole
Diclofenac
IX
VividTM
2C9 Green
2C9 Blue 2C9 Red 2C9 Green
Lit. K (µM) mean
Drug
mean : geometric mean of triplicates; ( ) : Kactivation; * : partial inhibition
1.5
SD (log) : standard deviation of geometric mean in log units
1.0
Apparent Ki Values with Recombinant CYP2C9
The spreadsheet lists apparent Ki values of drugs determined with VividTM 2C9
substrates and recombinant human CYP2C9 isozyme. Shown for comparison are
values from the recent literature in which drug substrates and human liver
microsomes were used.
2C9 Blue
0.5
2C9 Red
0.0
0
5
Lit K : Ko, J.W. et al. (1997). Drug Metab. Dispos., 25, 853;
Mancy, A. et al. (1996). Biochemistry, 35, 16205
10
[substrate] {µM}
Substrate Kinetics
VividTM 2C9 substrates display Michaelis-Menten type
kinetics.
Line color indicates the color of the fluorescent
metabolite generated.
TM
Km= 20 µM, kcat = 1.5 min-1
Km= 2 µM, kcat = 0.4 min-1
TM
2C9 Green Km= 2 µM, kcat = 3.0 min-1
Vivid
25
-8
C
Blue
Red
Green 400
[Lansoprazole]
Vivid 2C9 Blue
TM
Vivid 2C9 Red
XII
VividTM 2C9
Substrates Detect
Drug Interactions
100
Assays were performed with 10nM human CYP2C9 (insect
cell microsomes containing baculovirus-expressed 2C9 and
NADPH-P450 reductase; Panvera, WI).
60
TM
40
20
Vivid
2C9
Green
Red
0
Blue
>100 Drugs ranked by Potency (Blue)
Shown are the %
inhibition values for >100
drugs in VividTM 2C9
assays at 10µM [drug].
VividTM 2C9 Blue detects
inhibitors ( ) and activators
(off scale, not shown) of
CYP2C9. At the screening
concentration all substrates
detect the potent inhibitors
of CYP2C9.
CYP3A4 Assay in NanoplateTM
Dose responses for Ketoconazole ( ), Verapamil ( )
and Yohimbine ( ) were measured side-by-side using
the VividTM 3A4 Red substrate and recombinant human
CYP3A4 in NanoplateTM (2µl) and 96-well formats
(100µl). Comparable IC50 values were obtained.
SD
(log)
0.015 - 8
Ethinylestradiol
Isozyme Selectivity of
VividTM 3A4 Substrates
Lit. K (µM)
3A4 Red
Ketoconazole
Amiodarone
1A
2
2A
6
2B
6
2C
8
2C
2C 9
18
2C
19
2D
6
2E
1
3A
4
3A
5
CF3
VI
TM
96-Well Plate
100
Exemplary IC50 curves of four drugs known to interact with CYP3A4 are shown. Vivid 3A4 substrates were either used with
recombinant human CYP3A4 (RHI) or with pooled human liver microsomal preparations (HLM). In general, drugs were less potent
in inhibiting VividTM 3A4 probe metabolism in liver microsomes than in recombinant CYP3A4 preparations (A, B, D). Interestingly,
the profound activation of VividTM 3A4 Red metabolism by testosterone in recombinant CYP3A4 occurs to a much lesser degree in
human liver microsomes (C). As in the recombinant system, partial inhibition of probe metabolism (D) is found for certain drugprobe pairs in human liver microsomes, but for a different pair (B). Troleandomycin, a potent inhibitor of recombinant CYP3A4 only
TM
TM
partially inhibits Vivid 3A4 Red metabolism. Vivid 3A4 Green turnover is partially inhibited by steroids, both in recombinant
TM
and liver preparations. The two coumarin-based substrates Vivid 3A4 Blue and Cyan give similar results to each other.
B
100
80
NanoplateTM
[Amiodarone] log {M}
A
100
0
2.0
-8 -7 -6 -5 -4
-9 -8 -7 -6 -5 -4
CYP1A2
VIII
Dose-Response Curves with Recombinant CYP3A4 and Human Liver Microsomes
VividTM 3A4 Blue
Oxprenolol
Nitrofurantoin
Diphenylhydantoin
0
-9 -8 -7 -6 -5 -4
[Testosterone] log {M}
1A
2
2A
6
2B
6
2C
8
2C
2C 9
18
2C
19
2D
6
2E
1
3A
4
3A
5
O
0
-9 -8 -7 -6 -5 -4
1
0
O
-9 -8 -7 -6 -5 -4
RHI
Red
Green
Blue
Cyan
Isoniazid
Retinoic acid
Phenylbutazone
When the assay is performed in competitive
TM
mode Vivid 3A4 Green metabolism by CYP3A4
is inhibited by fewer compounds than any of the
other probe substrates. This permits distinction of
turnover-dependent inhibitors from competitive
inhibitors by a simple change in the assay
protocol. Drugs were either pre-incubated under
conditions permitting turnover (turnover), or added
to the enzyme at the same time as the fluorogenic
substrates (competitive). Drugs whose CYP3A4
inhibitory activity has a substantial turnoverdependent component are highlighted in bold.
Shown is % inhibition at 10µM [drug] relative to
10µM ketoconazole (competitive mode) or 10µM
ellipticine (turnover mode).
Red
Green
Blue
Cyan
Red
Green
Blue
Cyan
D
100
75
HLM
[Troleandomycin] log {M}
HLM
Doxorubicin
Ticlopidine
IX
0
-9 -8 -7 -6 -5
Tamoxifen
100
102
101
96
84
100
98
97
94
94
93
90
89
86
86
83
78
56
45
44
36
35
35
34
21
19
12
10
8
Turnover-dependence
75
[Ketoconazole] log {M}
1A
2
2A
6
2B
6
2C
8
2C
2C 9
18
2C
19
2D
6
2E
1
3A
4
3A
5
-1
v {min }
O
RHI
25
RHI
Poten
cy Red
100
50
C
ked b
y
Shown are the % inhibition values for >100 drugs in
TM
3A4 assays at 10µM [drug] using
Vivid
recombinant human CYP3A4. Drugs were ranked by
TM
potency in the Vivid 3A4 Red assay and the 15
most potently inhibiting drugs are indicated in black.
VividTM 3A4 Red detects inhibitors ( ) and activators
TM
(off scale, not shown) of CYP3A4, Vivid 3A4 Green
implicates a lesser number of competitive inhibitors
(fewer black dots above 50% inhibition potency), while
TM
Vivid 3A4 Blue and Cyan detect additional drugs
that interact with CYP3A4 (blue & cyan dots inside
yellow oval).
B
Red
Green
Blue
Cyan
-9 -8 -7 -6 -5
VividTM 3A4 Green
O
20
50
0
O
15
HLM
Red
Green
Blue
Cyan
1
4
10
BR: Benzylresorufin; DBF: Dibenzylfluorescein; BCC: 7-Benzyloxy-3-cyanocoumarin
BFC: 7-Benzyloxy-4-trifluoromethylcoumarin
2
N
5
[substrate] {µM}
Assays were performed with 5nM human CYP3A4 (insect cell microsomes
containing baculovirus-expressed 3A4 and NADPH-P450 reductase; Panvera, WI).
75
Green
s ran
VividTM 3A4 Substrates Detect
Drug-Substrate Interactions
Kinetic properties of VividTM substrates (solid lines) were compared sideby-side with the structurally related substrates (dashed) and with ( , )
or without ( , ) cytochrome b5. Line color indicates the color of the
fluorescent metabolite generated. Red fluorogenic substrates display
Michaelis-Menten type kinetics while all other substrates show sigmoidal
velocity curves with Hill coefficients ~2.
% Activity
VividTM 3A4 Red
Drug
10
0
RHI
0
>100
CYP3A4 Substrate Kinetics
100
Ellipticine
Mifepristone
Asarinin
Papaverine
Verapamil
Dicyclomine
Erythromycin
Clemastine
Amiodarone
Thioridazine
Vinblastine
Nortryptyline
Clomipramine
Disulfiram
Omeprazole
20
20
40
A
Dihydroergotamine
Blue
[substrate] {µM}
V
II
20
40
-20
0
0
60
Cyan
VividTM 3A4 Cyan
BCC
H
O
4
-1
40
H
O
O
2
30
60
-1
HO
10
v {min }
R1
v {min }
HO
0
0
R2
Nicardipine
Clotrimazole
Bromoergocryptine
98
98
95
93
71
40
22
-7
48
36
30
30
32
1
6
13
-3
0
-11
4
8
3
-9
-9
-1
-13
1
-9
21
X
CYP2E1
% Inhibition
-1
BR
R1
DBF
5
80
turnover
-1
O
ABSTRACT
2
10
competitive
v {min }
H
4
Ketoconazole
% Activity
Kcal/mol
H
15
4
O
Kcal/mol
79.5
MODE
DRUG
100
id TM
3A
H
H
VII
IV
VividTM 3A4 Green
20
v {min }
-1
v {min }
53.9
Here we present two diverse sets of
fluorogenic substrates for CYP3A4
and 2C9. Vivid™ substrates are
highly sensitive probes for drug
interactions with these isozymes and
permit assay miniaturization to < 2µl
volumes and enable automated high
throughput screening. This panel of
sensitive CYP450 probes also
permits the distinction of different
modes of drug-CYP450 interactions.
Competitive, noncompetitive, and
activator interactions are readily
distinguished. The large amount of
information generated in high
throughput assays is likely to speed
the drug-lead optimization process
and to give guidance to chemists in
the development of leads that are
less likely to fail because of drug
interactions.
VividTM 3A4 Red
6
TM
Inhibitory interactions of drugs with
metabolizing enzymes are a major
cause for adversial drug-drug
interactions. Testing compounds
early in drug discovery for these
unwanted interactions will permit
elimination of unsuitable compounds
and chemical series from further
drug development. With the everexpanding pool of candidate drugs
and increasing number of hits
emerging from primary screening
this task is becoming increasingly
more laborious. To complicate
matters, CYP3A4 and CYP2C9
display complex kinetic behaviors
and inhibitory profiles that are
dependent on the probe substrate
used. This is due to their large active
sites that may bind two substrates
simultaneously. To obtain meaningful SAR data, several kinetically
differing substrates are needed that
sense drug-interactions at any of the
metabolism-affecting sites in these
isozymes.
B
A
Viv
III
Potential Sites
of Oxidation
% Inhibition
I
References
1. Ekins, S. et al. (1998). Int. J. Clin. Pharmacol. Ther., 36, 642
2. Harlow, G.R. & Halpert, J.R. (1998). PNAS U S A, 95, 6636
3. Houston, J.B. & Kenworthy, K.E. (2000). Drug Metab. Dispos., 28, 246
4. Koley, A.P. et al. (1997). J. Biol. Chem., 272, 3149
5. Korzekwa, K.R. et al. (1998). Biochemistry, 37, 4137
6. Shou, M. et al. (1999). Biochem. J., 340, 845
7. Thummel, K.E. & Wilkinson, G.R. (1998). Annu. Rev. Pharmacol. Toxicol., 38, 389
8. Ueng, Y.F. et al. (1997). Biochemistry, 36, 370
Summary & Discussion
Aurora's VividTM substrates are benzyloxymethyl and alkyloxymethyl derivatives of highly fluorescent phenolic dyes (Panel I). Initially, the ether substrates show little or no fluorescence. Metabolism by CYP450 isozymes liberates
TM
fluorescent dyes with high extinction coefficients and high aqueous fluorescence quantum yields permitting sensitive detection of CYP450 isozyme activity. The excellent kinetic behavior of the Vivid 3A4 substrates (Panel III) is
possibly due to the increased freedom of rotation and movement of the methylenes carrying the abstractable hydrogens relative to the more rigid benzyl ether substrates. This may permit better access of these hydrogens to the
heme in the CYP450 active site. As depicted in Panel I, abstraction of any one of the four of the aliphatic hydrogens in the benzyloxymethyl moiety leads to a fluorescent product, possibly increasing the probability of metabolism by
CYP3A4 isozyme, which is known to metabolize substrates at multiple sites.
TM
TM
Aurora has developed a diverse set of fluorogenic substrates for CYP3A4 (Panel II & III). Vivid 3A4 substrates show excellent to good selectivity for CYP3A isozymes permitting their use in human liver microsomal
TM
preparation as well as in recombinant CYP3A4 preparations (Panels II, V & VI). Vivid 3A4 substrates are highly sensitive probes for drug interactions with this isozyme and permit assay miniaturization to < 2µl volumes (Panel
VIII) and enable automated high throughput screening. A benefit of this set of substrates is that it permits both, high throughput screening of entire compound collections using well defined recombinant enzyme preparations as well
as permitting the follow-up analysis in human liver microsomal preparations using the same probe substrates (Panels IV, V & VI).
VividTM 3A4 Red interacts with CYP3A4 in a manner that reports drugs that can partially inhibit the enzyme or cause enzyme activation (Panels V B, C & D) in the recombinant CYP450 enzyme system. In a screen of >100 drugs
(Panel IV), VividTM 3A4 Blue and Cyan pick up additional inhibitory CYP450-drug interactions, while VividTM 3A4 Green is comparatively less sensitive. This property of VividTM 3A4 Green is useful in determining whether a drug is
a turnover dependent inhibitor of CYP3A4 (Panel VII). In human liver microsomes, the situation is similarly complex, in that inhibitors have differential effect on the metabolism of VividTM 3A4 probe substrates (Panels V & VI).
It has been shown that inhibition of drug probe metabolism is less pronounced in human liver microsomal preparations than in recombinant CYP450 isozyme preparations (7). Similarly, metabolism of VividTM 3A4 substrates is
generally less affected by test compounds in human liver microsomal preparations (Panels V & VI).
The spreadsheet (above) lists the mean apparent Ki values of inhibition of CYP3A4 by a panel of drugs as assessed by their
TM
inhibition of Vivid 3A4 substrate metabolism. The source of CYP3A4 activity was either from recombinant human isozyme (RHI)
preparations or pooled human liver microsomes (HLM). Shown for comparison are value ranges from the recent literature for
inhibition of CYP3A4 drug substrate metabolism by human liver microsomes.
AuroraTM has also developed a diverse set of fluorogenic substrates for 2C9 (kinetics: Panel IX). VividTM 2C9 Blue interacts with CYP2C9 in a manner that reports drugs that can partially inhibit the enzyme or cause enzyme
activation (Panels X B & C) in recombinant isozyme preparations, while VividTM 2C9 Red and Green experience full inhibition, though at different drug dosages. Potent CYP2C9 inhibitors that were present in a set of > 100 drugs
TM
were found with all three Vivid 2C9 substrates (Panel XII).
Lit. K: Thummel, K.E. & Wilkinson, G.R. (1998). Annu. Rev. Pharmacol. Toxicol., 38, 389.
Poster presented at Gordon Research Conference on Drug Metabolism, Holderness School, Plymouth NH, July 9-14, 2000.