Identification of Potent and Selective Inhibitors to Investigate the Role of the Epithelial Sodium Channels in Neurodegeneration

Transcription

1 Identification of Potent and Selective Inhibitors to Investigate the Role of the Epithelial Sodium Channels in Neurodegeneration Victoria Miller, John Atack & Martin Gosling 1

2 Overview Biological and chemical motivation for project - ENaC channel as a target for neurodegeneration - Current pharmacological tools available Generation of reagents and primary screen - Cell line generation - Assay development - Comparison of assay platforms - Primary screen outcomes Follow up Hit confirmation and SAR - Patch clamp studies 2

3 ENaC: an amiloride-sensitive sodium channel DEG/ENaC family of non voltagegated sodium channels A Chicken ASIC1a Channel, PDB 2QTS B Sensitivity to amiloride Heterotrimer composed of α/δ, β and γ subunits Subunits contain two transmembrane domains Taken from Jasti, et al.(2007). 3

4 ENaC: an amiloride-sensitive sodium channel a Data is taken from Waldmann, et al. (1995) b Data is taken from Yamamura, et al. (2006) 4

5 ENaC: an amiloride-sensitive sodium channel Tissue distribution of ENaCδmRNA analysed by Northern blot Highest of all ENaC subunits Exclusively neuronal a Data is taken from Waldmann, et al. (1995) b Data is taken from Yamamura, et al. (2006) 5

6 ENaC: an amiloride-sensitive sodium channel Tissue distribution of ENaCδmRNA analysed by Northern blot Higher single channel conductance (12 ps vs 4 ps) Longer opening time of channel Highest of all ENaC subunits Exclusively neuronal Decreased sensitivity to amiloride (IC 50 = 2 µm vs <1 µm ) Acid-sensing capacity a Data is taken from Waldmann, et al. (1995) b Data is taken from Yamamura, et al. (2006) 6

7 ENaC δ: biological and chemical motivation Potential role in the integration of ischemia-related signals in inflamed and hypoxic tissues Lowered ph Increased ENaC channel open probability (P o ) Na + loading into cells Neuronal cell death Amiloride 7

8 ENaC δ: biological and chemical motivation Poor selectivity: abg >>> dbg Poor pharmacokinetic properties Low CNS penetration Amiloride Common to all of its derivatives eg. phenamil, benzamil 8

9 Aims Identify a novel potent and selective inhibitor of the ENaC δ channel, to investigate channel function and assess potential as neuroprotective agents 9

10 Development of heterologous expression system HEK293 cell line stably expressing ENaC β(p618ay620l)γ(p624stop)(gsk, Stevenage) 10

11 Development of heterologous expression system HEK293 cell line stably expressing ENaC β(p618ay620l)γ(p624stop)(gsk, Stevenage) Transiently transduce cells with BacMam baculovirus containing third ENaC subunit (α/δ) BacMam Baculovirus: - Based on double-stranded DNA insect viruses (baculovirus) - Modified with a mammalian expression cassette (e.g. CMV) upstream of gene of interest 11 Adapted from Thermo Fisher Scientific, (2014).

12 Development of heterologous expression system MOI 0 ENaC δ-gfp Cytotoxic MOI Adapted from Thermo Fisher Scientific, (2014).

13 Membrane potential assay Detects ion channel modulation by increasing or decreasing the fluorescent signal as cellular membrane potential changes Quencher Charged Dye OUT 2s e.g Blocker IN e.g Agonist 13

14 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation μcell/fdss MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout Read duration In-well control? Analysis of data 14

15 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation μcell/fdss MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout Read duration In-well control? Analysis of data 15

16 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) Transduce cells 6-8 hr hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 16

17 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) Transduce cells 6-8 hr hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 17

18 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) Transduce cells 6-8 hr hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 18

19 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) Transduce cells 6-8 hr hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 19

20 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) Transduce cells 6-8 hr hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 20

21 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation Seed cell plate μcell/fdss Seed cell plate 2s MP dye loading Compound plate Dye formulation Dye concentration Dye loading time Conc. of standards Layout Speed of dispense DMSO tolerance 6-8 hr hr (ON) 20 min Transduce cells Load MP dye Assay 6-8 hr hr (ON) 20 min Transduce cells Load MP dye Assay Readout Read duration In-well control? Analysis of data 21

22 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation μcell/fdss MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout Read duration In-well control? Analysis of data 22

23 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout 23 Read duration In-well control? Analysis of data Taken from Hamamatsu, (2016).

24 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout 24 Read duration In-well control? Analysis of data Taken from Hamamatsu, (2016).

25 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation One compound plate μcell/fdss Two compound plates MP dye loading Dye formulation Dye concentration Dye loading time 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout Read duration In-well control? Analysis of data 25

26 Membrane potential assay Cell plating and viral transduction Cell seeding density Virus titre Viral Incubation FlexStation One compound plate μcell/fdss Two compound plates MP dye loading Dye formulation Dye concentration Dye loading time 96-well 96-well 384-well 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Readout Read duration In-well control? Analysis of data 26

27 Membrane potential assay Cell plating and viral transduction MP dye loading Cell seeding density Virus titre Viral Incubation Test compound Amiloride max Dye formulation Dye concentration Dye loading time :00.0 5: :00.0 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Day FlexStation μcell/ FDSS Single-addition Single-addition Double-addition Readout Read duration In-well control? Analysis of data

28 Membrane potential assay Cell plating and viral transduction MP dye loading Cell seeding density Virus titre Viral Incubation Test compound Amiloride max Dye formulation Dye concentration Dye loading time :00.0 5: :00.0 Amiloride Benzamil EIPA 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Drug Amiloride EC 50 value (µm) ± 0.14(n=3) Readout Read duration In-well control? Analysis of data Benzamil EIPA ± 0.13(n=3) n/a (n=3) 28

29 Membrane potential assay FlexStation μcell/ FDSS Dye concentration 1 x (> 1000) 1/3 x (< 500) Assay window < 2-fold > 3-fold Compound plates 1 2 2s Cell plate 96-well 96-well OR 384-well Time per 96-well plate 30 min (single-addition) 5 min (single-addition) 10 min (double addition) Time for screen n=1 2-3 days < 1 day 29

30 Count Membrane potential assay 21 componds Inhibition % 30

31 Count Membrane potential assay 21 componds Inhibition % 31

32 Patch clamp validation 2s The Dynaflow platform is an automated microfluidic system 16 glass microchannels Requires small volumes of compound Adsorption-free Fast solution exchange Reusable Dynaflow Resolve Chip 32 Taken from Cellectricon, (2016).

33 Patch clamp validation 2s The Dynaflow platform is an automated microfluidic system 16 glass microchannels Requires small volumes of compound Adsorption-free Fast solution exchange Reusable Dynaflow Resolve Chip 33 Taken from Cellectricon, (2016).

34 Patch clamp validation 2s The Dynaflow platform is an automated microfluidic system 16 glass microchannels Requires small volumes of compound Adsorption-free Fast solution exchange Reusable Dynaflow Resolve Chip Taken from Cellectricon, (2016).

35 Patch clamp validation Amiloride Benzamil Amiloride 2s Drug EC 50 value (µm) Amiloride Benzamil 1.46 ± 0.03 (n=11) 0.56 ± 0.03 (n=11) 35

36 Patch clamp validation Amiloride Benzamil Amiloride Compound X 2s Drug EC 50 value (µm) Amiloride Benzamil 1.46 ± 0.03 (n=11) 0.56 ± 0.03 (n=11) Compound X ± 0.02 (n=8) 36

37 Patch clamp validation Amiloride 2s ENaC channel EC 50 value (µm) δβγ αβδ 1.46 ± 0.03 (n=11) 0.47 ± 0.04 (n=8) 37

38 Patch clamp validation Amiloride Compound X 2s ENaC channel EC 50 value (µm) ENaC Channel EC 50 value (µm) δβγ αβδ 1.46 ± 0.03 (n=11) 0.47 ± 0.04 (n=8) δβγ αβδ ± 0.14 (n=3) ± 0.13 (n=3) 38

39 Current and future work Ongoing SAR e-phys-based around Compound X Main limitation of membrane potential assay is high false positive hit rate Additional automated e-phys screen of a larger 12K compound diversity set - identification of second compound series 39

40 Identification of Potent and Selective Inhibitors to Investigate the Role of the Epithelial Sodium Channels in Neurodegeneration Victoria Miller, John Atack & Martin Gosling Sussex Drug Discovery Centre (SDDC) 40