Identification of potent and selective inhibitors of the epithelial sodium channel d

Members of the DEG/ENaC family have been recently implicated in the neurodegeneration associated with a number of CNS disease states. More specifically the neuronally-expressed ENaC d channel has been linked to the integration of ischemia- related signals in inflamed and hypoxic tissues (Ji, et al., 2000; Ji & Benos, 2004). Although a recent study reported clinical efficacy of the prototypical ENaC inhibitor amiloride in multiple sclerosis (Friese, et al., 2007; Vergo, et al., 2011), this drug was optimised for the non-CNS ENaC variant (aß?). To date, a small number of compounds are described in the literature as ENaC blockers but all share a structural similarity to amiloride. These amiloride analogues all exhibit activity at multiple ion channels and have poor pharmacokinetic properties with respect to CNS penetration. As such our aim is to identify novel potent and selective inhibitors of the ENaC d channel which could be used to probe channel function. A heterologous expression system was developed to overexpress the ENaC dß? channel in a Human Embryonic Kidney (HEK) 293 cell line. This implemented a BacMam baculoviral delivery system to transiently express ENaC d subunit in HEK293 cell which stably expressed ENaC ß and ? subunits, reconstituting channel function. This expression system has been used to establish both a novel membrane potential-based fluorescence assay and an automated electrophysiological-based assay to screen for regulators of ENaC d channel function. Primary hits have been subsequently triaged using conventional whole cell patch clamp electrophysiology. This has been used to support an SAR-based approach to improve potency and selectivity in the development of a tool compound to investigate the ENaC dß? channel.