Inhibitors Phosphodiesterase (PDE)

Integrated Technologies for the Characterization of 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

Compounds Libraries Assay Development Molecular Screening Selectivity Panels

Drug Safety Assessment

Pathway Analysis

Mechanism of Action

Cellular Functional Assay

Drug Combinations

Ex-vivo tissue analysis In Vivo Imaging Pre-Efficacy Studies Marker PK-ADME Identification/Detection

22 LabChip™ Mobility-Shift Assay Target Classes

Kinetic or endpoint assays for target classes including:

Kinases Epigenetic Targets Phosphodiesterases – HATs Phosphatases – HDACs Proteases – Methyl Transferases Lipid Kinases – Demethylases DNA/RNA Binding Proteins

33 Introduction

Phosphodiesterases are enzymes that play a major role in cell signaling and function, by regulating levels of the second messengers cAMP and cGMP

Dual cAMP Specificity Specific

PDE1 PDE4 PDE2 PDE7 PDE3 PDE8 PDE10 PDE11 PDE5 PDE6 PDE9 cGMP PDE4B location, duration, amplitude Specific (catalytic domain)

44 Clinical PDE Inhibitors

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: 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)

55 PDE’s potential as drug targets

Regulate second messengers

High PDE activity in many tissues

Many isoforms connected to different physiological functions 21 PDE gene products Isoform selective inhibitors

PDE’s relatively unique in substrate binding

66 Caliper Life Sciences LabChip® Platform

Key benefits: No antibodies or radioactivity Substrate Stopped rxn or real -time kinetic measurements Product 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

77 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

iFL-cAMP MW 926.9

cGMP

S S

iFL-cGMP MW 942.9

88 PDE LC Assay: Materials and Methods

Reaction volume of 25 µµµL 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

Reaction buffer: 100 mM Hepes pH 7.5, 5 mM MgCl 2, 0.002% Brij-35

One hour incubation time at room temperature

Caliper’s Reviewer software measures % conversion

PDE1A cGMP Km PDE10A cAMP Km

1.25 0.3 1.00

0.75 0.2

0.50 0.1 0.25 VV (pmol/min)(pmol/min) VV (pmol/min)(pmol/min) 0.00 0.0 0 5 10 15 20 0 1 2 3 4 5

[cGMP] (uM) [cAMP] (uM) A VMAX 1.477 A KM 6.230 VMAX 0.2503 KM 0.3602

Substrate Km determination for PDE1A with cGMP, and PDE10A with cAMP. These are representative of the 20 PDE substrate Km’s tested. 1111 Determination of inhibitor IC 50 s for various PDEs.

110 100 110 90 100 90 80 80 70 70 60 60 50 50 40 PDE1A 40 PDE10A 30 30 % Specific Activity Specific % 20 cGMP Activity Specific % 20 cAMP 10 10 0 0 -10 -10 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 log [drug] (M) log [drug] (M)

PDE2A Reference Compound PDE4D IC (nM) Reference Compound PDE3A IC 50 (nM)PDE4A1A PDE4B1 PDE4D2 50 110 110 110 110 100 100 3 110 100 100 Trequinsin 70 90 110 100 90 90 1,80090 100 80 90 80 8-methoxy-IBMX 80 80 70 Dipyridamole 90 2,700 80 70 70 70 60 80 Trequinsin70 22,000 60 60 60 50 70 60 50 50 50 60 50 40 3,500 40 50 40 40 30 8-methoxy-IBMX Zaprinast40 26,000 % Specific Activity 30 40 30 30 20 Zaprinast% Specific Activity 30 %Specific Activity %Specific Activity 20 30 % Specific %Specific Activity

%Specific Activity 19,000 20 20 20 10 Pentoxifylline 10 20 10 10 >100,00010 0 Pentoxifylline0 10 0 0 0 -10 -10 0 -10 -10 -10 -9 -8 -7 -6 -5 -4 96,000 Rolipram-10 -10 -9 -8 -7 -6 -5 -4 -10 -10 -9 -8 -7 -6 -5 -4 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 log [drug] (M) Rolipram -10 -9 -8 -7 -6 -5 -4 >100,000 log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) Reference Compound IC (nM) >100,000 50 Reference Compound IC (nM) Reference Compound IC (nM) 50 50 Reference Compound IC 50 (nM) Reference Compound IC 50 (nM) Trequinsin 374 Reference Compound IC 50 (nM) Trequinsin 150 Trequinsin 590 Rolipram 1,050 Trequinsin 0.16 Trequinsin 240 Rolipram 630 Rolipram 57 8-methoxy-IBMX 3,400 Dipyridamole 3,960 Trequinsin 136 8-methoxy-IBMX 4,800 Rolipram 490 Dipyridamole 2,800 Dipyridamole 6,300 Zaprinast 42,000 Dipyridamole 1,400 Rolipram 93,000 Dipyridamole 4,900 Pentoxifylline 30,000 Zaprinast 41,000 Pentoxifylline 53,000 8-methoxy-IBMX 5,400 Zaprinast >100,000 Pentoxifylline >100,000 8-methoxy-IBMX 30,000 8-methoxy-IBMX 91,000 50,000 Pentoxifylline >100,000 8-methoxy-IBMX >100,000 Pentoxifylline Zaprinast 55,000 Zaprinast 50,000 Dipyridamole >100,000 Zaprinast >100,000

PDE5A1 PDE7A1 PDE8A1 110 110 PDE9 PDE11A4 100 110 110 110 PDE Panel: 100 90 100 100 100 90 80 90 90 90 80 70 80 80 80 70 60 70 70 70 60 50 60 60 60 50 40 50 50 50 now 20 assays 40 30 40 40 40

30 % Specific Activity 30 % SpecificActivity 20 30 30 % Specific Activity %Specific Activity 20 % SpecificActivity 10 20 20 20 10 0 10 10 10 0 -10 0 0 0 -10 -10 -9 -8 -7 -6 -5 -4 -10 -10 -10 -10 -9 -8 -7 -6 -5 -4 log [drug] (M) -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M)

Reference Compound IC 50 (nM) Reference Compound IC 50 (nM) BRL-50481 660 Reference Compound IC 50 (nM) Reference Compound IC 50 (nM) Reference Compound IC 50 (nM) Zaprinast 200 Trequinsin >100,000 Trequinsin 5,500 Zaprinast 15,000 Dipyridamole 1,300 Dipyridamole 770 8-methoxy-IBMX >100,000 Dipyridamole 8,400 8-methoxy-IBMX 17,000 Trequinsin 10,000 Trequinsin 1,900 Rolipram >100,000 Rolipram >100,000 Dipyridamole >100,000 8-methoxy-IBMX 25,000 8-methoxy-IBMX 5,500 Pentoxifylline >100,000 Rolipram >100,000 Zaprinast 25,000 Pentoxifylline 44,000 Zaprinast >100,000

1212 Compound Specificity

•Compound specificity critical to drug development

•Side effects

0.16 nM (PDE3) 100 uM (PDE7)

1313 Assay DMSO Sensitivity

DMSO Tolerance

100 PDE 3A 80 PDE 5A PDE 10A 60

40 %% Activity Activity

20 •Limit PDE assays to 0 1% DMSO if possible

.0 .7 .4 .1 .4 .7 0 0 1.4 2.1 2.8 3 4 5 6 7.9 % DMSO

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

1414 Z Prime

PDE4B1PDE4B Z Z Prime Prime

Z'=0.80 PDE Z’ PDE1A 0.63 50 PDE2A 0.69

40 PDE3A 0.74 PDE4A1A 0.74 30 PDE4B1 0.80 20 PDE4D2 0.82 10

%% ConversionConversion PDE4D3 0.86 % Conversion Conversion % % 0 PDE5A1 0.74 0 50 100 150PDE7A1 200 0.63 PDE8A1 0.81 Well Number PDE9A2 0.62 PDE10A1 0.70 Z prime determination for PDE4B1. PDE11A4 0.75

Average 0.73

1515 Collaborating with customers to improve the health and safety of people and their environment

In-Depth PDEi Research

IC 50 Determination Tight-Binding?

YES Reversible NO

Linear Progress Curve Covalent Inhibition NO NO

YES YES Time-Dependent Inhibition Analysis Mechanism Based Classical Steady-State YES Anaysis NO

Further Characterization Poor 1717 Adapted from Copeland, R.A. (2005) Candidate PDE3A Mechanism of Action Study

60 Trequinsin Reversibility •time dependent example 45 PDE3A [Trequinsin], as 30 . multiple of IC50 15 0 0 25 50 75 100 125 50 0X Time, Min 10X LinearTrequinsin inhibition Linearity over time 40 30X of PDE3A by Trequinsin [Trequinsin] as 100X 30 multiple of IC50

20 50 0

% Conversion •no time 0.5x 10 40 dependence 1x 0 30 0 10 20 30 40 50 60 2x Time, minutes 20 4x %% Conversion Conversion 10 Determination of reversibility 0 for Trequinsin for PDE3A 0 10 20 30 40 50 60 70 80 90 100 Enzyme is incubated with inhibitor Time, minutes concentration indicated above and 100X Determination of reaction enzyme. Assay is then diluted to 1X enzyme with substrate. For reversible progress linearity with Trequinsin inhibitors, conversion rate after Reaction linearity with inhibitor is a prerequisite for 10X →0.1X inhibitor dilution is restored. determination of the Ki and mode of inhibition.

1818 PDE3A Mechanism of Action Study

PDE3A Curve Fit Trequinsin

Trequinsin (nM) 15 0 40 80 10 160 320 V (pmol/min) (pmol/min) V V 5 480

0 0 1 2 3 4 5 6 7 [cAMP] (uM) 0 40 80 160 320 VMAX 21.04 17.11 480 19.30 15.91 11.78 4.385 KM 0.7232 0.8597 1.307 2.361 4.186 2.187 Michaelis-Menton Plot of Trequinsin for PDE3A Inhibitor and substrate concentrations are varied around the IC50 and Km, respectively. 1919 PDE3A Mechanism of Action Study

Lineweaver-Burk Plot TrequinsinLinew witheav ePDE3Ar-Burk Trequinsin (nM) 1.4 [I] = 0.000 [I] =40.0040 1.2 [I] =80.0080 [I] =160.00160 Trequinsin Ki at PDE3A [I] =320.00320 [I] =480.00480 5 1.0

4 0.8 3 o V

/ 0.6

1 Slope 0.011 2 Km,Km, uM uM Y-int = 0.53 0.4 Ki = 46 pM 1 r2 = 0.99 0.2 0 0 70 140 210 280 350 0 [Trequinsin] (pM)

-0.2 -2 -1 0 1 2 3 Competitive inhibition 1/[S]

- 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].

2020 Analysis of irreversible inhibitors

Ki and kinact determination using 3D Fit Modeling

Input: IC50 + time

Output:

Ki 1.72E -09 (M)

kinact 1.03E -02 (min -1)

Kinact – inactivation rate constant at ∞ inactivation concentration

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 XLfit TM (IDBS) and 3D Fit modeling with the Krippendorff equation.

2121 PDE Cellular Assay Characterization

Schematic diagram of a PDE cellular assay PDE2A cellular inhibition measured in HEK 293 using a transfected CRE-Luciferase reporter cells co-transfected with a PDE2A expression construct to measure cAMP signaling. vector and a CRE-Luciferase construct. Luciferase activity determined after 48 hours

2222 PDE Immunoreactivity Assay

Low-resolution raw 4X image showing PDE2 immunoreactivity in coronal section of mouse brain.

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.).

2323 PDEScreen™ Research Services from CDAS

A complete suite of PDE offerings

PDEScreen™ HTS Customized screening projects to meet clients’ expectations of throughput, process & workflow. 2 week turnaround

PDEScreen™ IC50 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 Caliper Discovery Alliances & compounds interact with enzymatic Services targets. Discovery and Profiling services >1000 in vitro / 100 in vivo assays

2424 Summary

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

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) 2525 For more information, please contact:

Caliper Discovery Alliances & Services: [email protected]

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

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