Direct Activation ofβ-Cell K_ATPChannels with a Novel Xanthine Derivative

Abstract

ATP-regulated potassium (K ATP ) channel complexes of inward rectifier potassium channel (K ir ) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet b-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The K ATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved K ir 6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing K ATP channels. Here we report the serendipitous discovery in a highthroughput screen of a novel activator of K ir 6.2/SUR1: VU0071063The xanthine derivative rapidly and dose-dependently activates K ir 6.2/SUR1 with a half-effective concentration (EC 50 ) of approximately 7 mM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1-over SUR2A-containing K ir 6.1 or K ir 6.2 channels, as well as K ir 2.1, K ir 2.2, K ir 2.3, K ir 3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native K ir 6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic b cells. VU0071063 represents a novel tool/compound for investigating b-cell physiology, K ATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

Discovery and Characterization of a Potent and Selective Inhibitor of Aedes aegypti Inward Rectifier Potassium Channels

“…For Tl + flux assays on Kir6.2/SUR1 expressing cells, test compounds were diluted in assay buffer containing diazoxide (250 µM final) to induce channel activation [20]. Concentration-response curves (CRCs) were generated by screening compounds at 3-fold dilution series in 4-point (1 µM–30 µM) or 11-point (1 nM–30 µM) CRCs.…”

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

“…gambiae Malpighian tubules (Genbank Accession # KJ596497)17 was sub-cloned into the pcDNA5/TO expression vector (Life Technologies) and used for stable cell line generation, as described previously4445. Expression constructs for mammalian Kir channels were developed and validated in previous studies by our group464748.…”

“…Transiently transfected HEK-293T cells expressing An Kir1 cells were voltage clamped in the whole-cell configuration of the patch clamp technique, as described previously174748. The extracellular bath solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 glucose, 10 HEPES free acid, pH 7.4, 290 mOsm/kg H 2 O.…”

ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels

“…The 3.7-fold shift in IC 50 determined with patch clamp electrophysiology, as compared with Tl + flux, is consistent with previous observations of other Kir channel inhibitors 18–20 . Quantitative Tl + flux assays were utilized to evaluate the selectivity of VU714 for Kir7.1 over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, Kir4.1, and Kir6.2/SUR1, as reported previously 16, 21, 22 . The concentration-response curves (CRCs) shown in Fig.…”

“…Transfections and whole-cell patch clamp experiments were performed essentially as described previously 22 . Briefly, HEK-293T cells were transfected with plasmids encoding WT or mutant Kir7.1 and EGFP (transfection marker) using Lipofectamine LTX (Life Technologies).…”