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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 11 © 2009 PerkinElmer PerkinElmer Accelerates Drug Discovery 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 P iFL-cAMP MW 926.9 cGMP S S P 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 99 1010 © 2009 PerkinElmer PDE Substrate Km’s 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 VMAX 0.2503 KM 6.230 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. 110 100 110 100 90 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 Trequinsin 70 100 100 90 110 100 90 90 1,80090 8-methoxy-IBMX 100 80 90 80 80 80 70 Dipyridamole 90 2,700 80 70 70 70 60 80 Trequinsin70 22,000 60 60 60 50 70 60 50 3,500 8-methoxy-IBMX 60 50 50 50 40 40 50 Zaprinast 40 26,00040 30 40 % Specific Activity 30 40 30 30 Zaprinast% Specific Activity 20 19,000 30 %Specific Activity %Specific Activity 20 30 % Specific Activity 20 20 20 10 %Specific Activity Pentoxifylline >100,000 10 20 10 10 10 0 Pentoxifylline0 10 96,000 0 0 0 -10 -10 0 -10 -10 -10 -9 -8 -7 -6 -5 -4 Rolipram-10 >100,000 -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 >100,000-4 log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) Reference Compound IC (nM) 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 Pentoxifylline 50,000 Pentoxifylline >100,000 8-methoxy-IBMX >100,000 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 Prime Z Prime Z'=0.80 PDE Z’ PDE1A 0.63 50 PDE2A 0.69 PDE3A 0.74 40 PDE4A1A 0.74 30 PDE4B1 0.80 20 PDE4D2 0.82 10 %% ConversionConversion PDE4D3 0.86 % Conversion Conversion % % 0 PDE5A1 0.74 0 50 100 150 200 PDE7A1 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 1616 © 2009 PerkinElmer Lead Optimization: Compound Evaluation Flowchart Biochemical analyses typically performed during the characterization of lead compounds for drug discovery 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 20 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 80 160 320 480 VMAX 21.04 17.11 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.
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