Objective

Target-class drug discovery is a complementary approach to target-based or phenotypic screening strategies. It allows the simultaneous interrogation of a family of targets, increasing the likelihood of identifying novel pharmacological agents by driving synergies in assay development, target structure and ligand development. We have developed a cellular system to identify activators across all of the described subclasses of a family of ion channels, the two-pore domain potassium channels (K2Ps).

K2Ps are characterised by their four transmembrane domain, two-pore topology. They function to carry background (or leak) potassium current in a variety of cell types, including those with important pathophysiological roles, and primarily act to maintain resting membrane potential. However, 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.

Generation of cell lines stably over-expressing ion channels can be challenging and the ability to identify ion channel activators can be compromised by systems in which the target is over-expressed. To avoid these issues we have developed a novel cellular assay system which utilises baculovirus (‘BacMam’) to express ion channels in mammalian cells. BacMam allows the precise titration of expression of the gene of interest. This has enabled us to generate cell systems in which we are able to intricately and robustly select a level of K2P expression in functional assays, optimized for the identification of channel activators. It also offers a significant time savings when developing multiple cellular reagents at one time.

Using an initial representative group of channels, this cellular assay system was used to screen a 10k compound set against multiple K2Ps, using thallium flux as a measure of channel activity. Our ‘target-class’ approach allowed us to rapidly screen across the K2P family, with all ‘hit’ compounds being screened at all targets, in concentration-response format. This enabled us to quickly assess which channels were tractable to activation (which channels were ‘druggable’) and allowed the simultaneous analysis of multiple channels for small molecule selectivity. A number of diverse and novel activators were identified, however, not all channels were found to be druggable. A wide range of pharmacological profiles were also identified, including a number of activators which did show selectivity at K2Ps.

Using a ‘target-class’ approach to screening has allowed the identification of K2P drug targets which are amenable to small molecule activation and has provided selective starting points for chemical optimization. This approach has allowed exploration of less well validated targets, using automated screening, to determine which targets are ‘druggable’ and could potentially deliver novel therapeutics.