Category: Assay Development and Screening
Two-pore domain potassium channels (K2Ps) are characterised by their four transmembrane domain, two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles they have proved a difficult target class to modulate with small molecules and there is a lack of useful specific pharmacological tools which target K2Ps. This in turn has limited the interrogation of the precise physiological function of K2Ps and efforts to generate K2P targeting therapeutics.
Our aim was to develop a system to identify activators across all the described subclasses of K2P channels. Generation of cell lines stably over-expressing ion channels can be challenging for a number of reasons including inherent toxicity. Moreover, the ability to identify channel activators can be compromised by systems in which the target is expressed at high levels. To avoid these issues we utilized baculovirus (‘BacMam’) to deliver Ion channels into mammalian cells. ‘BacMam’ confers a number of advantages, including safety and reduced time, compared to generating stable cell lines, but importantly it allows the precise titration of expression of the gene of interest. This enabled us to generate cell systems in which we were able to intricately and robustly select a level of K2P expression in functional assays, optimized for the identification of channel activators.
Using an initial representative group of channels (THIK1, TWIK1, TREK2, TASK3 and TASK2) this system was used to screen a 10k representative set of the full LifeArc compound collection and a library of 1k FDA approved compounds. A number of diverse activators were identified. For a subset of active compounds potency was compared between the thallium flux system and automated patch-clamp screening systems. This was done to establish functional translation of compounds identified using thallium flux and other assay formats. Activators were also screened in concentration-response format across all channels to investigate selectivity. Comparisons of activators is complex due to differences in potency, efficacy and activity to known standards. Strategies to delineate selectivity will be discussed.
An exemplar novel activator identified was Terbinafine, which was shown to be a TASK3 activator using the thallium flux system and had a pEC50 of 6.2. Activity was confirmed using whole cell patch clamp electrophysiology. It is the first identified selective TASK3 activator, displaying no activation at other K2Ps screened. Interestingly, Terbinafine showed an increased level of activation versus the pathophysiological G236A variant compared to the wild type TASK channel.
Paul Wright– Senior Scientist, LifeArc, Stevenage, England, United Kingdom
Stevenage, England, United Kingdom
Paul Wright is part of the Assay Development and Screening group at LifeArc (formerly MRC Technology). Paul specialises in the development of cell-based assays to study ion channels and GPCRs and has a particular interest in difficult targets and novel screening technologies. Paul completed a PhD in Neuroscience at the Institute of Psychiatry, London and post-doctoral training at Harvard Medical school and the University of Massachusetts Medical school.