Development of novel therapeutics for treating edema and SeSAME/EAST syndrome targeting heteromeric Kir4.1/5.1 potassium channels

Abstract

Heterotetrameric inward rectifier potassium (Kir) channels composed of Kir4.1 (KCNJ10) and Kir5.1 (KCNJ16) play key roles in regulation of sodium, potassium, and water balance by the kidney. Loss‐of‐function mutations in KCNJ10lead to SeSAME/EAST syndrome, which is characterized by neurological dysfunction (e.g. epilepsy, ataxia, and sensorineural deafness) and renal salt wasting. In the distal convoluted kidney, Kir4.1/5.1 channels appear to regulate the phosphorylation state and hence activity of the electroneutral sodium‐chloride cotransporter, NCC, through a membrane potential‐intracellular chloride‐WNK kinase‐SPAK kinase‐dependent pathway. The upregulation of distal nephron sodium chloride reabsorption capacity in the setting of loop diuretic resistance suggests that peripherally restricted Kir4.1/5.1‐specific inhibitors might help reduce extracellular fluid volume and edema in congestive heart failure and chronic kidney disease patients. In addition, small‐molecule activators might rescue the function of mutant Kir4.1/5.1 channels in SeSAME/EAST syndrome patients carrying specific mutations in KCNJ10. However, the small‐molecule pharmacology of Kir4.1/5.1 is limited to neurological drugs (e.g. amitriptyline) with off‐target effects on Kir channels, including Kir4.1/5.1, making it impossible to test these hypotheses. We therefore set out to perform a high‐throughput screen of approximately 110,000 compounds from the Vanderbilt Institute of Chemical Biology library for small‐molecule modulators of Kir4.1/5.1. We first established and extensively characterized a monoclonal HEK‐293 cell line that stably expresses human Kir4.1 and Kir5.1 from a bicistronic vector, and then developed and validated a fluorescence‐based thallium‐flux assay of Kir4.1/5.1 channel function in 384‐well format. To date, we have screened more than 60,000 compounds using this assay and have identified 420 putative inhibitors and 354 putative activators of Kir4.1/5.1. One inhibitor, termed VU690, inhibits Kir4.1/5.1 with an IC50 (in “gold standard” manual patch clamp experiments) of 900 nM, and is highly selective for Kir4.1/5.1 over Kir4.1, Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir6.2/SUR1, and Kir7.1. Medicinal chemistry aimed at improving the potency and DMPK properties of VU690 has been initiated. Importantly, we have also verified with patch clamp electrophysiology the activity of the first‐in‐class activator of Kir4.1/5.1, termed VU206. Our next immediate goals include completing the 110,000‐compound screen, verifying the activity of inhibitors and activators of Kir4.1/5.1, evaluating their potency and selectivity using fluorescence‐ and electrophysiology‐based assays, and leveraging these newly discovered compound for exploring the integrative physiology and therapeutic potential of Kir4.1/5.1 channels in the treatment of edema and SeSAME/EAST syndrome, as well as other diseases associated with dysfunction of Kir4.1‐containing channels.Support or Funding InformationNIH R01 DK120821, T32 T32NS007491‐19