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

Identification of Potent and Selective Inhibitors to Investigate the Role of the Epithelial Sodium Channels in Neurodegeneration Victoria Miller, John Atack & Martin Gosling 1 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

Aims Identify a novel potent and selective inhibitor of the ENaC δ channel, to investigate channel function and assess potential as neuroprotective agents 9 www.sussex.ac.uk/sddc

Development of heterologous expression system HEK293 cell line stably expressing ENaC β(p618ay620l)γ(p624stop)(gsk, Stevenage) 10 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc Adapted from Thermo Fisher Scientific, (2014).

Development of heterologous expression system MOI 0 ENaC δ-gfp Cytotoxic MOI 100 12 www.sussex.ac.uk/sddc Adapted from Thermo Fisher Scientific, (2014).

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 16-24 hr (ON) Transduce cells 6-8 hr 16-24 hr (ON) Transduce cells Readout Read duration In-well control? Analysis of data 16 www.sussex.ac.uk/sddc

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 16-24 hr (ON) 20 min Transduce cells Load MP dye Assay 6-8 hr 16-24 hr (ON) 20 min Transduce cells Load MP dye Assay Readout Read duration In-well control? Analysis of data 21 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc Read duration In-well control? Analysis of data Taken from Hamamatsu, (2016).

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 www.sussex.ac.uk/sddc Read duration In-well control? Analysis of data Taken from Hamamatsu, (2016).

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

Membrane potential assay Cell plating and viral transduction MP dye loading Cell seeding density Virus titre Viral Incubation Test compound 25000 20000 0 10000 Amiloride max Dye formulation Dye concentration Dye loading time 5000 0 0:00.0 5:00.0 10:00.0 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Day FlexStation μcell/ FDSS Single-addition Single-addition Double-addition 1 0.38 0.46 0.83 Readout 2 0.41 0.52 0.67 Read duration In-well control? Analysis of data 3 0.29 0.41 0.72 27 www.sussex.ac.uk/sddc

Membrane potential assay Cell plating and viral transduction MP dye loading Cell seeding density Virus titre Viral Incubation Test compound 25000 20000 0 10000 Amiloride max Dye formulation Dye concentration Dye loading time 5000 0 0:00.0 5:00.0 10:00.0 Amiloride Benzamil EIPA 2s Compound plate Conc. of standards Layout Speed of dispense DMSO tolerance Drug Amiloride EC 50 value (µm) 25.18 ± 0.14(n=3) Readout Read duration In-well control? Analysis of data Benzamil EIPA 15.30 ± 0.13(n=3) n/a (n=3) 28 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

Count Membrane potential assay 21 componds Inhibition % 30 www.sussex.ac.uk/sddc

Count Membrane potential assay 21 componds Inhibition % 31 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc Taken from Cellectricon, (2016).

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 www.sussex.ac.uk/sddc Taken from Cellectricon, (2016).

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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 34 www.sussex.ac.uk/sddc Taken from Cellectricon, (2016).

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 www.sussex.ac.uk/sddc

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 145.10 ± 0.02 (n=8) 36 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 122.4 ± 0.14 (n=3) 212.3 ± 0.13 (n=3) 38 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc

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 www.sussex.ac.uk/sddc