integrated technologies for the characterization of ... · phosphodiesterases (pdes) are a class of...

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


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


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


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


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


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


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


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


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


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


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


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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