Integrated Technologies for the

Characterization of

Phosphodiesterase (PDE) Inhibitors

11 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer

Phosphodiesterase (PDE) Inhibitors

Edmond Massuda, Laurel Provencher, Abbie Esterman,

Benjamin Lineberry, Lisa Fleet, Chris Spence, Dhanrajan

Tiruchinapalli, Scott Perschke, Hao Chen, and Seth Cohen

Caliper Discovery Alliances and Services (CDAS)

PKI Global Health Summit - June 8, 2012

PerkinElmer Accelerates Drug Discovery

Molecular Screening

Mechanism of Action

Compounds Libraries

Drug Safety Assessment

Selectivity PanelsAssay Development

Pathway Analysis

22

Mechanism of Action

In Vivo Imaging Pre-Efficacy StudiesPK-ADME

Cellular Functional Assay

Drug Combinations

Marker Identification/Detection

Ex-vivo tissue analysis

LabChip™ Mobility-Shift Assay Target Classes

� Epigenetic Targets

– HATs– HDACs– Methyl Transferases– Demethylases

� Kinases

� Phosphodiesterases

� Phosphatases

� Proteases

� Lipid Kinases

� DNA/RNA Binding Proteins

Kinetic or endpoint assays for target classes including:

33

� DNA/RNA Binding Proteins

Phosphodiesterases are enzymes that play a major role in cell

signaling and function, by regulating levels of the second

messengers cAMP and cGMP

Introduction

Dual

SpecificitycAMP

44

PDE4B(catalytic domain)

PDE4

PDE7

PDE8

PDE1

PDE2

PDE3

PDE10

PDE11

PDE5

PDE6

PDE9

SpecificitycAMP

Specific

cGMP

Specificlocation, duration, amplitude

Success of therapeutic inhibitors has validated PDEs as important drug targets

� Viagra/Cialis/Levitra (PDE5)

� Milrinone – heart failure (PDE3)

� Cilostazol – claudication (PDE3)

� Dipyridamole – anti platelet aggregation, stroke

Studies in clinical trials:

Clinical PDE Inhibitors

55

Studies in clinical trials:

� TPI1100 – inflammation

– PDE4/7 inhibitor (Topigen)

� IC224 series – ADHD, Parkinson’s

– PDE1 inhibitors (ICOS/Lilly)

� NDxxxx series – depression, chronic inflammation, atherosclerosis

– PDE4 inhibitors (Via Pharma, Celgene, and Nycomed)

� PF-2545920 – schizophrenia

– PDE10 inhibitors (Pfizer)

Regulate second messengers

High PDE activity in many tissues

Many isoforms connected to different physiological functions

� 21 PDE gene products

PDE’s potential as drug targets

66

� 21 PDE gene products

� Isoform selective inhibitors

PDE’s relatively unique in substrate binding

Caliper Life Sciences LabChip® Platform

Substrate

Key benefits:

� No antibodies or radioactivity

� Stopped rxn or real-time kinetic measurements

77

Product� Stopped rxn or real-time kinetic measurements

� Superior data quality:

- Direct measurement of substrates & products

- Ratiometric method

- High signal-to-noise ratios

- Fewer false positive and negative

� Screening with cAMP/cGMP around PDE Km

� HTS assay format (384 wells)

� Activation assays also available

Assay Principle – Substrate/Products structures

� Hydrolization of Labeled cAMP and cGMP creates charge difference with products

� Allows for independent detection of substrates and products

� Enzyme percent conversion is defined as a measure of ration P/(P+S)

cAMP

S

S

88

MW 942.9iFL-cGMP

cGMP

SS

P

MW 926.9iFL-cAMP

P

Reaction volume of 25 µµµµL in a 384 well plate

PDE enzymes available from BPS Bioscience, Inc

iFL-cAMP or iFL-cGMP substrate (Caliper)

Substrate concentrations near or below substrate Km

PDE LC Assay: Materials and Methods

99

Substrate concentrations near or below substrate Km

Reaction buffer: 100 mM Hepes pH 7.5, 5 mM MgCl2, 0.002% Brij-35

One hour incubation time at room temperature

Caliper’s Reviewer software measures % conversion

1010 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer

PDE10A cAMP Km

0.1

0.2

0.3

V (

pm

ol/

min

)

PDE1A cGMP Km

0.50

0.75

1.00

1.25

V (

pm

ol/

min

)PDE Substrate Km’s

1111

0 1 2 3 4 50.0

0.1

VMAX

KM

A

0.2503

0.3602

[cAMP] (uM)V

(p

mo

l/m

in)

0 5 10 15 200.00

0.25

VMAX

KM

A

1.477

6.230

[cGMP] (uM)

V (

pm

ol/

min

)

Substrate Km determination for PDE1A with cGMP, and PDE10A with cAMP. These are representative of the 20 PDE substrate Km’s tested.

Determination of inhibitor IC50s for various PDEs.

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

% S

pecific

Activity

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

log [drug] (M)

% S

pecific

Activity

PDE1A

cGMP

PDE10A

cAMP

1212

Trequinsin

Reference Compound IC50 (nM)

1,800

22,000

26,000

>100,000

>100,000

8-methoxy-IBMX

Zaprinast

Pentoxifylline

Rolipram

log [drug] (M)

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)

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

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

40

50

60

70

80

90

100

110

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

PDE2A

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

Trequinsin

Reference Compound IC50 (nM)

590

3,400

6,300

41,000

8-methoxy-IBMX

Dipyridamole

Zaprinast

log [drug] (M)

% S

pec

ific

Ac

tivit

y

PDE3A

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

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

50

60

70

80

90

100

110

Trequinsin

Rolipram

Reference Compound IC50 (nM)

240

490

4,900

>100,000

>100,000

>100,0008-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

50

60

70

80

90

100

110

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

PDE4D2

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

Trequinsin

Rolipram

Reference Compound IC50 (nM)

150

630

2,800

30,000

30,000

55,000

8-methoxy-IBMX

Dipyridamole

Zaprinast

Pentoxifylline

log [drug] (M)

% S

pe

cif

ic A

cti

vit

y

PDE7A1

-9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

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

PDE9

-9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

Reference Compound IC50 (nM)

15,000

17,000

>100,000

>100,000

8-methoxy-IBMX

Zaprinast

Rolipram

Dipyridamole

log [drug] (M)

% S

pe

cif

ic A

cti

vit

y

PDE4D3

PDE11A4

-10 -9 -8 -7 -6 -5 -4

-10

0

10

20

30

40

50

60

70

80

90

100

110

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

40

50

60

70

80

90

100

110

Trequinsin

Reference Compound IC50 (nM)

660

>100,000

>100,000

>100,000

8-methoxy-IBMX

BRL-50481

Rolipram

log [drug] (M)

% S

pe

cif

ic A

cti

vit

y

PDE4B1PDE4A1A

PDE5A1 PDE8A1PDE Panel:now 20 assays

Compound Specificity

•Compound specificity critical to drug development

•Side effects

1313

0.16 nM (PDE3) 100 uM (PDE7)

DMSO Tolerance

40

60

80

100PDE 3A

PDE 5A

PDE 10A

% A

cti

vit

y

Assay DMSO Sensitivity

1414

0.0

0.7

1.4 2.1 2.8 3.4

4.1

5.4

6.7

7.9

0

20

40

% DMSO

% A

cti

vit

y

Concentrations of DMSO were analyzed

for each assay. These are representative of the 20 PDE DMSO tolerances tested.

•Limit PDE assays to

1% DMSO if possible

Z Prime

PDE Z’

PDE1A 0.63

PDE2A 0.69

PDE3A 0.74

PDE4A1A 0.74

PDE4B1 0.80

PDE4B Z Prime

Z'=0.80

30

40

50

% C

on

ve

rsio

n%

Convers

ion

PDE4B1 Z Prime

1515

Z prime determination for PDE4B1.

PDE4B1 0.80

PDE4D2 0.82

PDE4D3 0.86

PDE5A1 0.74

PDE7A1 0.63

PDE8A1 0.81

PDE9A2 0.62

PDE10A1 0.70

PDE11A4 0.75

Average 0.73

0

10

20

0 50 100 150 200

Well Number

% C

on

ve

rsio

n%

Convers

ion

Collaborating with customers to improve the health and safety of people and their environment

1616 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer

In-Depth PDEi Research

Lead Optimization: Compound Evaluation Flowchart

IC50 Determination

Tight-Binding?

NOYES

Linear Progress Curve

Reversible

Covalent Inhibition

Biochemical analyses typically performed during the characterization of lead compounds for

drug discovery

1717

YESYES

YES

NO

NO

NO

Linear Progress Curve Covalent Inhibition

Poor

CandidateFurther Characterization

Mechanism BasedClassical Steady-State

Anaysis

Time-Dependent

Inhibition Analysis

Adapted from Copeland, R.A. (2005)

Trequinsin ReversibilityPDE3A

10

20

30

40

50 0X

10X

30X

100X

[Trequinsin], asmultiple of IC50

% C

on

ve

rsio

n

Trequinsin Linearity

40

50

1x

0.5x

0

[Trequinsin] asmultiple of IC50

% C

on

ve

rsio

n

Linear inhibition over timeof PDE3A by Trequinsin

0 25 50 75 100 1250

15

30

45

60

.

Time, Min

•time dependent example

•no time dependence

PDE3A Mechanism of Action Study

1818

0 10 20 30 40 50 600

Time, minutes

Determination of reversibility

for Trequinsin for PDE3AEnzyme is incubated with inhibitor

concentration indicated above and 100X

enzyme. Assay is then diluted to 1X

enzyme with substrate. For reversible

inhibitors, conversion rate after

10X→0.1X inhibitor dilution is restored.

Determination of reaction progress linearity with TrequinsinReaction linearity with inhibitor is a prerequisite for determination of the Ki and mode of inhibition.

0 10 20 30 40 50 60 70 80 90 1000

10

20

30

4x

2x

1x

Time, minutes

% C

on

ve

rsio

n

PDE3A Mechanism of Action Study

PDE3A Curve FitTrequinsin

10

15

20

40

0

80

Trequinsin (nM)

V (

pm

ol/m

in)

1919

0 1 2 3 4 5 6 70

5

10

VMAX

KM

0

21.04

0.7232

40

17.11

0.8597

80

19.30

1.307

160

15.91

2.361

320

11.78

4.186

480

4.385

2.187

160

320

480

[cAMP] (uM)

V (

pm

ol/m

in)

Michaelis-Menton Plot of Trequinsin for PDE3A

Inhibitor and substrate concentrations are varied around the IC50 and Km, respectively.

PDE3A Mechanism of Action Study

Trequinsin Ki at PDE3A

2

3

4

5

Slope 0.011Y-int = 0.53K

m,

uM

0.6

0.8

1.0

1.2

1.4Lineweaver-Burk

1/V

o

[I] = 0.00

[I] =40.00

[I] =80.00

[I] =160.00

[I] =320.00

[I] =480.00

0

40

80

160

320

480

Lineweaver-Burk Plot

Trequinsin with PDE3A Trequinsin (nM)

2020

0 70 140 210 280 3500

1

2 Y-int = 0.53Ki = 46 pM

r2 = 0.99

[Trequinsin] (pM)

Km

, u

M

- Determination of Trequinsin Ki for PDE3A and mode of inhibition.

- Data is plotted in a Lineweaver-Burk Plot and the Ki is determined by slope of Km vs [I].

Competitive inhibition-2 -1 0 1 2 3

-0.2

0

0.2

0.4

1/[S]

Ki and kinact determination using 3D Fit Modeling

Ki 1.72E-09 (M)

k

Input:

IC50 + time

Output:

Analysis of irreversible inhibitors

2121

kinact 1.03E-02 (min-1)

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 kinact

were estimated using XLfitTM (IDBS) and 3D Fit modeling with the Krippendorff equation.

Kinact – inactivation rate

constant at ∞ inactivation concentration

PDE Cellular Assay Characterization

2222

Schematic diagram of a PDE cellular assay using a transfected CRE-Luciferase reporter construct to measure cAMP signaling.

PDE2A cellular inhibition measured in HEK 293 cells co-transfected with a PDE2A expression vector and a CRE-Luciferase construct.Luciferase activity determined after 48 hours

PDE Immunoreactivity Assay

Low-resolution raw 4X image showing

PDE2 immunoreactivity in coronal section

of mouse brain.

2323

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.).

PDEScreen™ Research Services from CDAS

PDEScreen™ HTS� Customized screening projects to meet

clients’ expectations of throughput, process & workflow.

� 2 week turnaround

PDEScreen™ IC50� Compound inhibitor potency

A complete suite of PDE offerings

2424

� Compound inhibitor potency determined in your selection of PDE assays.

PDEScreen™ PROFILEing� Extensive panel of 20 human PDE

assays

PDEScreen™ Mechanism of Action (MOA)� Understand the manner in which your

compounds interact with enzymatic targets.

Caliper Discovery Alliances &

Services

Discovery and Profiling services

>1000 in vitro / 100 in vivo assays

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.

Mechanism of action of PDE inhibitors can be determined with Labchip®

technology

Summary

2525

PDE2 immunostaining in the mouse brain was acquired using the Vectra/Nuance

Imaging system (Caliper Life Sciences/PerkinElmer, Inc). PDE2 showed discrete

staining in 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 (20 PDE’s)

For more information, please contact:

Caliper Discovery Alliances & Services:

CDASinvitro@caliperls.com

or visit http://www.caliperls.com/products/contract-research/

2626

or visit http://www.caliperls.com/products/contract-research/