integrated technologies for the characterization of
TRANSCRIPT
Integrated technologies for the characterization of phosphodiesterase (PDE) inhibitors
Caliper Life Sciences, a PerkinElmer Company, 7170 Standard Drive, Hanover, Maryland, 21076 USA
Edmond Massuda, Lisa Fleet, Benjamin Lineberry, Laurel Provencher, Abbie Esterman, Dhanrajan Tiruchinapalli, Faith Gawthrop, Christopher Spence, Rajneesh P. Uzgare, Scott Perschke, Seth Cohen, and Hao Chen. 618.01/XX63
ResultsAbstractPhosphodiesterases (PDEs) are a class of signal transduction enzymes regulating various cellular functions and disease
progressions in a number of central or peripheral nervous system-related disorders. For example, these enzymes are
involved in neurological diseases including psychosis in schizophrenia, multiple sclerosis and other neurodegenerative
conditions. Thus, safe and highly selective PDE inhibitors or modulators are becoming an important class of disease
modifying therapeutic agents. We have developed an integrated platform which includes Caliper LabChip™ microfluidic
mobility-shift assays measuring fluorescent analogs of cAMP and cGMP in conjunction with a cellular assay
characterizing intracellular signal transduction in cells modulated by PDE inhibitors. These technologies are useful in
Results
PDE1A
40
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
PDE2A
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
PDE3A
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
PDE4B1PDE4A1A
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
40
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
PDE3BPDE1B
Percent of Maximum Activity by Time
Ki and kinact determination using 3D Fit Model
characterizing intracellular signal transduction in cells modulated by PDE inhibitors. These technologies are useful in
target selection and validation, lead compound selectivity and mode of action, and for measuring cellular activities
involved in mediation of signal transduction and cell functions, all of which are critical components for assessing
potential efficacy and/or adverse effects of PDE inhibitors.-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
Trequinsin
Reference Compound IC50 (nM)
1,800
22,000
26,000
>100,000
>100,000
8-methoxy-IBMX
Zaprinast
Pentoxifylline
Rolipram
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
Trequinsin
Reference Compound IC50 (nM)
590
3,400
6,300
41,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
Trequinsin
Rolipram
Reference Compound IC50 (nM)
0.16
4,800
93,000
>100,000
>100,000
>100,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
Trequinsin
Rolipram
Reference Compound IC50 (nM)
240
490
4,900
>100,000
>100,000
>100,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
Trequinsin
Rolipram
Reference Compound IC50 (nM)
374
1,050
3,960
42,000
53,000
91,0008-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE7A
40
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
PDE9A2
40
50
60
70
80
90
100
110
% S
pec
ific
Acti
vit
y
PDE4D3PDE10A2
30
40
50
60
70
80
90
100
110
% S
pec
ific
Ac
tiv
ity
PDE11A
30
40
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
40
50
60
70
80
90
100
110
% S
pecif
ic A
cti
vit
y
30
40
50
60
70
80
90
100
110
% S
pe
cif
ic A
cti
vit
y
30
40
50
60
70
80
90
100
110
% S
pec
ific
Acti
vit
y
PDE5A1 PDE8A1
-10 -8 -6 -4 -2
-10
0
10
20
30
40
50
Zaprinast
8-methoxy-IBMX
Vinpocetine
Reference Com pound IC50 (nM)
1900
10,000
14,000
35,000
Trequinsin
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-15-14-13-12-11-10 -9 -8 -7 -6 -5 -4 -3
-10
0
10
20
30
40
Rolipram
Trequinsin
Zaprinast
Re fere nce Com pound IC50 (nM)
0.25
6600
>100,000
>100,000
8-methoxy -IBMX
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
Introduction
Phosphodiesterases are enzymes that play a major role in cell signaling and function by regulating levels of the second messengers cAMP and cGMP. The success of therapeutic inhibitors of PDE 5 and 3, such as Sildenafil, Tadalafil, and Milrinone in the treatment of erectile
Figure 12. Time dependent enzyme inactivation with a PDE4 inhibitor and determination of the Ki and kinact using the Krippendorff equation, above (J Biomol Screen. 14, 2009, pp. 913-923). Ki and k were estimated using XLfitTM (IDBS) and 3D Fit modeling with the Krippendorff equation.
Ki 1.72E-09 (M)
kinact 1.03E-02 (min-1)
In vitro →→→→ In vivo
•Biochemical Screen
•Mechanism of Action
Figure 5. Determination of inhibitor IC50s for 14 PDEs.
-9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Rolipram
Reference Compound IC50 (nM)
5,500
8,400
>100,000
>100,000
>100,000
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-9 -8 -7 -6 -5 -4
-10
0
10
20
30
Reference Compound IC50 (nM)
15,000
17,000
>100,000
>100,000
8-methoxy-IBMX
Zaprinast
Rolipram
Dipyridamole
log [drug] (M)
% S
pec
ific
Acti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Rolipram
Reference Compound IC50 (nM)
70
2,700
3,500
19,000
96,000
>100,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pec
ific
Ac
tiv
ity
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Reference Compound IC50 (nM)
1,300
10,000
25,000
25,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Rolipram
Reference Compound IC50 (nM)
57
136
1,400
5,400
50,000
50,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pecif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Reference Compound IC50 (nM)
200
770
1,900
5,500
44,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
Trequinsin
Reference Compound IC50 (nM)
660
>100,000
>100,000
>100,000
8-methoxy-IBMX
BRL-50481
Rolipram
log [drug] (M)
% S
pec
ific
Acti
vit
y
inhibitors of PDE 5 and 3, such as Sildenafil, Tadalafil, and Milrinone in the treatment of erectile dysfunction and congestive heart failure has validated these enzymes as an important class of drug targets. Therefore, effective assays for identification, characterization, and profiling of compounds modulating specific PDE activities are crucial components of drug discovery efforts in most therapeutic areas. For drug characterization, target selectivity is critical to avoid side effects resulting from the inhibition of other PDEs.
(J Biomol Screen. 14, 2009, pp. 913-923). Ki and kinact were estimated using XLfitTM (IDBS) and 3D Fit modeling with the Krippendorff equation.
X AMP
PDE
cAMP
PKA
Transcription
•Mechanism of Action
•Cell Functional Validation
•In vivo Pharmacodynamics
•Tissue Imaging
Caliper Life Sciences offers the Labchip® EZ Reader II platform which simplifies high throughput screening of phosphodiesterase assays as well as other target classes including kinases, epigenetics, proteases... In addition, Caliper Discovery Alliances & Services, the contract research service arm of Caliper offers complete compound screening and characterization programs producing a full-fledged discovery platform. PDEs hydrolyze the 3’ cyclic phosphate bond of cAMPand cGMP causing the net charge of the molecule to decrease, which allows electrophoretic separation of product from substrate. By using Caliper’s fluorescently labeled iFL-cAMP and iFL-cGMP substrates, the separation can be easily measured on the EZ Reader II, and thousands of compounds can be screened per day. A panel of 14 PDE assays was developed to determine inhibitor potency and selectivity. Recently, we have expanded our service capabilities to provide a unique comprehensive platform for the discovery & characterization of phosphodiesterase inhibitors. We present here the validation of PDE cellular assays which measure cAMP signaling, and PDE immunostaining in cell lines or mouse tissue sections. Combined to mechanism of action studies for both reversible and irreversible inhibitors, these technologies enable a comprehensive platform to study the in vitro to in vivo characteristics of PDE inhibitors.
Figure 13. Schematic diagram of PDE cellular assay using a transfected CRE-Luciferase reporter construct to measure cAMP signaling.
CRE Luciferase
CREB
Figure 15. Low-resolution raw 4X image showing PDE2 immunoreactivity in coronal section of mouse brain.
Figure 14. PDE2A cellular inhibition was measured in HEK 293 cells co-transfected with a PDE2A expression vector and a CRE-Luciferase construct. After 48 hours, luciferase activity was determined.
log [drug] (M)
to mechanism of action studies for both reversible and irreversible inhibitors, these technologies enable a comprehensive platform to study the in vitro to in vivo characteristics of PDE inhibitors.
Figure 16. PDE2 immunoreactivity in coronal section of mouse brain, with discrete staining pattern in many regions. (i) Section of the mouse brain shows prominent expression of PDE2A in the forebrain structures, including the cortex, piriform cortex, hippocampus and Habenulo-interpeduncular. These 20X high-resolution images were spectrally unmixed using Vectra/Nuance image-analysis system (Caliper LifeSciences/PerkinElmer, Inc.).
• The Labchip® EZ Reader II platform enables high quality and high throughput PDE enzyme assays
• Fluorescent analogs of cAMP and cGMP can be utilized as substrates for PDE assays and are available directly
from Caliper Life Sciences/PerkinElmer, Inc.
• A panel of 14 robust, reproducible PDE assays has been characterized
Summary
• A panel of 14 robust, reproducible PDE assays has been characterized
• Mechanism of action of PDE inhibitors can be determined with Labchip® technology
• PDE2 immunostaining in the mouse brain was acquired using the Vectra/Nuance Imaging system (Caliper Life
Sciences/PerkinElmer, Inc). PDE2 showed discrete staining specific regions of the brain.
• Multiplex tissue imaging can correlate PDE and CREB to allow pharmacodynamic characterization of drug effects.
• A complete suite of contract research services for PDE drug discovery and development is available.
Figure 4b. Trequinsin specificity for each of the PDE’s.
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