A "Target Class" Screen to Identify Activators of Two-Pore Domain Potassium (K2P) Channels.

Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a "target class" approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous screening of multiple targets. Importantly, this system, by allowing precise titration of channel function, allows optimization to facilitate the identification of activators. A representative set of channels (THIK-1, TWIK-1, TREK-2, TASK-3, and TASK-2) were screened against a library of Food and Drug Administration (FDA)-approved compounds and the LifeArc Index Set. Activators were then analyzed in concentration-response format across all channels to assess selectivity. Using the target class approach to investigate the K2P channels has enabled us to determine which of the K2Ps are amenable to small-molecule activation, de-risk multiple channels from a technical point of view, and identify a diverse range of previously undescribed pharmacology.

Pranlukast is a novel small molecule activator of the two-pore domain potassium channel TREK2.

TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast's activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation.

Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3.

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K+ channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3.

Localization of agonist-sensitive PtdIns(3,4,5)P3 reveals a nuclear pool that is insensitive to PTEN expression.

Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3] is a lipid second messenger, produced by Type I phosphoinositide 3-kinases (PI 3-kinases), which mediates intracellular responses to many growth factors. Although PI 3-kinases are implicated in events at both the plasma membrane and intracellular sites, including the nucleus, direct evidence for the occurrence of PtdIns(3,4,5)P3 at non-plasma membrane locations is limited. We made use of the pleckstrin homology (PH) domain of general receptor for phosphoinositides (Grp1) to detect PtdIns(3,4,5)P3 in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of PtdIns(3,4,5)P3 that increased up to 15-fold upon stimulation with platelet-derived growth factor (PDGF). The signal was sensitive to PI 3-kinase inhibitors and present mainly at plasma membranes, including lamellipodia, and in a surprisingly large pool within the nuclear matrix. Comparatively little labeling was observed in endomembranes. A similar distribution of PtdIns(3,4,5)P3 was observed in U87MG cells, which lack the PtdIns(3,4,5)P3 phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane PtdIns(3,4,5)P3, but not the nuclear pool of this lipid even when PTEN was targeted to nuclei. These data have important implications for the versatility of PI 3-kinase signaling and for the proposed functions of PTEN in the nucleus.