title: Validation and optimization of novel high-throughput assays for human epithelial sodium channels.
abstract: The epithelial sodium channel (ENaC) plays a crucial role in salt and water homeostasis and is primarily involved in sodium reabsorption in the kidney and lung. Modulators of ENaC function, particularly within lung epithelia, could offer potential treatments for a number of diseases. As a constitutively active sodium channel, ENaC expression at the cell membrane is highly regulated through rapid turnover. This short half-life of the channel at the membrane and cytotoxicity from overexpression pose a problem for reagent generation and assay development in drug discovery. We have generated an HEK293 stable cell line expressing ENaC β and γ subunits containing the PY motif trafficking mutations found in Liddle's syndrome to overcome rapid channel turnover at the membrane. A BacMam virus was used to transiently express the ENaC α subunit to reconstitute channel function to reduce the toxicity associated with long-term overexpression. We have configured a 384-well FLIPR membrane potential antagonist assay for high-throughput screening and an IonWorks Quattro electrophysiology antagonist assay that is predictive of potency values derived from primary lung epithelial cell short-circuit measurements. The triage strategy for compound screening and profiling against this target using these assays has resulted in the discovery of novel chemotypes.
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK mark.x.chen@gsk.com.
authors.forename: Mao Xiang
authors.initials: MX
authors.lastname: Chen
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: Kelly
authors.initials: K
authors.lastname: Gatfield
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Ward
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: David
authors.initials: D
authors.lastname: Downie
authors.affiliation: Green Chemistry Performance Unit, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: Helen F
authors.initials: HF
authors.lastname: Sneddon
authors.affiliation: Target and Pathway Validation, GlaxoSmithKline R&D, Upper Providence, Philadelphia, PA, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Walsh
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Powell
authors.affiliation: Neurobiology DPU, GlaxoSmithKline R&D, Upper Merion, Philadelphia, PA, USA.
authors.forename: Dramane
authors.initials: D
authors.lastname: Laine
authors.affiliation: Neurobiology DPU, GlaxoSmithKline R&D, Upper Merion, Philadelphia, PA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Carr
authors.affiliation: Biological Sciences, GlaxoSmithKline R&D, Stevenage, Herts, UK.
authors.forename: Derek
authors.initials: D
authors.lastname: Trezise
mesh_terms: D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D062708:Epithelial Sodium Channel Agonists / Q000494:pharmacology*; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology*; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D020545:Respiratory Mucosa / Q000378:metabolism; D054852:Small Molecule Libraries
chemical_list: D062708:Epithelial Sodium Channel Agonists; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D054852:Small Molecule Libraries
keywords: FLIPR; IonWorks; channel; epithelial; sodium