Identification of WP1066, an inhibitor of JAK2 and STAT3, as a KV 1.3 potassium channel blocker.

Abstract

KV 1.3 potassium channels play a predominant role in regulating calcium signalling that is essential for the activation and proliferation of effector memory T (TEM ) cells. This ion channel has been recognized as a promising therapeutic target against various autoimmune diseases. In a high-throughput screening programme, WP1066 was identified as a KV 1.3 channel inhibitor. Using molecular biology and electrophysiological methods, the mechanism(s) underlying WP1066 blockade of Kv1.3 channels was investigated. Using TEM cell proliferation assay and mouse delayed-type hypersensitivity (DTH) model, the effects of WP1066 were examined. WP1066 blocked KV 1.3 channels in a dose-dependent manner with an IC50 of 3.2μM and induced a hyperpolarizing shift of the steady-state inactivation curve. This blockade was use-dependent, as WP1066 interacted preferentially with channels in their open state, rather than the closed state or inactivated state. When the residues located in the S6 domain scaffolding the inner vestibule, were sequentially mutated, the potency of WP1066 was significantly impaired, especially by mutations A413C and I420C, indicating a higher affinity of interacting sites for WP1066. Moreover, WP1066 effectively suppressed mouse TEM cell proliferation in vitro and mouse DTH reaction in vivo. The results presented here have identified WP1066 as a KV 1.3 channel blocker with an open-state-dependent property, providing fundamental evidence for the application of WP1066 in further immunomodulatory studies targeting KV 1.3 channels.