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Integrated Technologies for the Characterization Of Integrated technologies for the characterization of phosphodiesterase (PDE) inhibitors 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 Caliper Life Sciences, a PerkinElmer Company, 7170 Standard Drive, Hanover, Maryland, 21076 USA Abstract Phosphodiesterases (PDEs) are a class of signal transduction enzymes regulating various cellular functions and disease Results Results 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 PDE1A PDE1B PDE2A PDE3A PDE3B PDE4A1A PDE4B1 Ki and kinact determination using 3D Fit Model 110 110 110 110 110 100 110 110 100 100 100 Percent of Maximum Activity by Time 100 conditions. Thus, safe and highly selective PDE inhibitors or modulators are becoming an important class of disease 100 90 100 90 90 90 90 90 80 90 80 80 80 80 modifying therapeutic agents. We have developed an integrated platform which includes Caliper LabChip™ microfluidic 80 70 80 70 70 70 70 70 60 70 60 60 60 60 mobility-shift assays measuring fluorescent analogs of cAMP and cGMP in conjunction with a cellular assay 60 60 50 50 50 50 50 50 50 40 40 40 40 characterizing intracellular signal transduction in cells modulated by PDE inhibitors. These technologies are useful in 40 40 40 30 30 30 30 %% SpecificSpecific Activity Activity 30 30 30 %%SpecificSpecific Activity Activity %%SpecificSpecific Activity Activity %% SpeSpe cific cific ActivityActivity 20 20 %% SpecificSpecific Activity Activity %% SpecificSpecific Activity Activity 20 20 target selection and validation, lead compound selectivity and mode of action, and for measuring cellular activities %% SpeSpe cific cific ActivityActivity 20 20 20 10 10 10 10 10 10 10 0 0 0 0 involved in mediation of signal transduction and cell functions, all of which are critical components for assessing 0 0 0 -10 -10 -10 -10 -10 -10 -10 -10 -9 -8 -7 -6 -5 -4 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -10 -8 -6 -4 -2 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 potential efficacy and/or adverse effects of PDE inhibitors. log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) Reference Compound IC 50 (nM) Reference Compound IC (nM) Reference Compound IC (nM) Reference Compound IC (nM) Reference Compound IC 50 (nM) Reference Compound IC (nM) 50 50 50 50 Reference Compound IC 50 (nM) 8-methoxy-IBMX 1,800 Trequinsin 590 Trequinsin 0.16 Trequinsin 240 Trequinsin 22,000 8-methoxy-IBMX 1900 Trequinsin 0.25 Trequinsin 374 8-methoxy-IBMX 3,400 8-methoxy-IBMX 4,800 Rolipram 490 Zaprinast 26,000 8-methoxy-IBMX 6600 Rolipram 1,050 Trequinsin 10,000 Dipyridamole 6,300 Rolipram 93,000 Dipyridamole 4,900 >100,000 Dipyridamole 3,960 Pentoxifylline Zaprinast 14,000 Zaprinast 41,000 Zaprinast >100,000 Rolipram >100,000 Pentoxifylline >100,000 >100,000 Zaprinast 42,000 Rolipram Vinpocetine 35,000 Pentoxifylline >100,000 Zaprinast >100,000 8-methoxy-IBMX >100,000 Pentoxifylline 53,000 Dipyridamole >100,000 Zaprinast >100,000 8-methoxy-IBMX 91,000 Ki 1.72E -09 (M) PDE4D3 PDE5A1 PDE7A PDE8A1 kinact 1.03E -02 (min -1) 110 PDE11A 110 PDE9A2 PDE10A2 110 110 110 100 110 110 100 100 100 100 100 Introduction 90 100 90 90 90 90 90 80 90 80 80 80 80 80 70 In vitro → In vivo 80 70 →→ 70 70 70 70 70 60 60 60 Phosphodiesterases are enzymes that play a major role in cell signaling and function by 60 60 60 60 50 50 50 •Biochemical Screen 50 50 50 regulating levels of the second messengers cAMP and cGMP. The success of therapeutic 50 40 40 40 40 40 40 Figure 12. Time dependent enzyme inactivation with a PDE4 inhibitor and determination of the Ki and k using the Krippendorff equation, above 40 30 inact 30 30 inhibitors of PDE 5 and 3, such as Sildenafil , Tadalafil , and Milrinone in the treatment of erectile %% SpecificSpecific Activity Activity 30 • 30 30 %% Specific Specific Activity Activity Mechanism of Action %% SpecificSpecific Activity Activity TM Mechanism of Action 30 %% SpecificSpecific Activity Activity 20 %% SpecificSpecific Activity Activity 20 %% SpecificSpecific Activity Activity %%SpecificSpecific Activity Activity 20 (J Biomol Screen. 14, 2009, pp. 913 -923). Ki and k were estimated using XLfit (IDBS) and 3D Fit modeling with the Krippendorff equation. 20 20 20 inact dysfunction and congestive heart failure has validated these enzymes as an important class of 20 10 10 10 • 10 10 10 10 Cell Functional Validation 0 0 drug targets. Therefore, effective assays for identification, characterization, and profiling of 0 0 0 0 0 -10 -10 -10 -10 -9 -8 -7 -6 -5 -4 -10 • -10 -10 -9 -8 -7 -6 -5 -4 -10 -10 compounds modulating specific PDE activities are crucial components of drug discovery efforts In vivo Pharmacodynamics -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 log [drug] (M) -10 -9 -8 -7 -6 -5 -4 log [drug] (M) log [drug] (M) in most therapeutic areas. For drug characterization, target selectivity is critical to avoid side • log [drug] (M) log [drug] (M) log [drug] (M) log [drug] (M) Tissue Imaging Reference Compound IC (nM) 50 Reference Compound IC 50 (nM) effects resulting from the inhibition of other PDEs. Reference Compound IC 50 (nM) Reference Compound IC 50 (nM) Trequinsin 70 Reference Compound IC (nM) Reference Compound IC 50 (nM) Reference Compound IC (nM) 50 Zaprinast 200 50 Rolipram 57 BRL-50481 660 Dipyridamole 2,700 PDE 5,500 Dipyridamole 1,300 Dipyridamole 770 Trequinsin Zaprinast 15,000 8-methoxy-IBMX 3,500 Trequinsin 136 Trequinsin >100,000 Caliper Life Sciences offers the Labchip® EZ Reader II platform which simplifies high throughput screening of Dipyridamole 8,400 17,000 Zaprinast 19,000 Trequinsin 10,000 Dipyridamole 1,400 Trequinsin 1,900 8-methoxy-IBMX 8-methoxy-IBMX >100,000 Rolipram >100,000 8-methoxy-IBMX 25,000 8-methoxy-IBMX 5,400 8-methoxy-IBMX 5,500 Dipyridamole >100,000 Pentoxifylline 96,000 Rolipram >100,000 Pentoxifylline >100,000 Rolipram >100,000 Zaprinast 25,000 phosphodiesterase assays as well as other target classes including kinases, epigenetics, proteases... In addition, Caliper Pentoxifylline 50,000 Pentoxifylline 44,000 Rolipram >100,000 >100,000 Discovery Alliances & Services, the contract research service arm of Caliper offers complete compound screening and Zaprinast 50,000 Zaprinast cAMP characterization programs producing a full-fledged discovery platform. PDEs hydrolyze the 3’ cyclic phosphate bond of cAMP X AMP and cGMP causing the net charge of the molecule to decrease, which allows electrophoretic separation of product from Figure 5. Determination of inhibitor IC 50 s for 14 PDEs. 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 PKA 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 Transcription 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. CREB CRE Luciferase log [drug] (M) Figure 13. Schematic diagram of PDE cellular Figure 14. PDE2A cellular inhibition was measured in Figure 15. Low-resolution raw 4X image assay using a transfected CRE-Luciferase HEK 293 cells co-transfected with a PDE2A expression showing PDE2 immunoreactivity in coronal reporter construct to measure cAMP signaling. vector and a CRE-Luciferase construct. After 48 section of mouse brain. hours, luciferase activity was determined. 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.). 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 . • 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. a PerkinElmer company.
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