Abstract
Calcium-activated K+ channels constitute attractive targets for the treatment of neurological and cardiovascular diseases. To explain why certain 2-aminobenzothiazole/oxazole-type KCa activators (SKAs) are KCa3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of KCa3.1 and KCa2.3 in complex with CaM using Rosetta modeling software. We here attempted to employ this atomistic level understanding of KCa activator binding to switch selectivity around and design KCa2.2 selective activators as potential anticonvulsants. In this structure-based drug design approach we used RosettaLigand docking and carefully compared the binding poses of various SKA compounds in the KCa2.2 and KCa3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the KCa2.2 and KCa.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and KCa2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any KCa2.2 selective compounds. Based on the full-length KCa3.1 structure it was recently demonstrated that the C-terminal crystal dimer was an artefact and suggested that the “real” binding pocket for the KCa activators is located at the S4-S5 linker. We here confirmed this structural hypothesis through mutagenesis and now offer a new, corrected binding site model for the SKA-type KCa channel activators. SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine) is binding in the interface between the CaM N-lobe and the S4-S5 linker where it makes van der Waals contacts with S181 and L185 in the S45A helix of KCa3.1.
Highlights
Small- and intermediate-conductance calcium-activated potassium channels (KCa) are voltage independent and are gated by the binding of calcium to calmodulin, which functions as their calciumsensing beta subunit (Xia et al, 1998; Fanger et al, 1999)
E54 was further stabilized by an extensive hydrogen bond network with R362, E295 and N300 in the KCa3.1 channel, which we hypothesized to be responsible for the KCa3.1 selectivity of SKA-121 and SKA-111
We docked eight more 2-amino-naphthobenzothiazole derivatives (SKA-31, SKA-44, SKA-45, SKA-72, SKA-73, SKA-107, SKA-117, and SKA-120) into the KCa2.2 and KCa3.1 homology models and found that these compounds exhibited the same hydrogen bond network as SKA-121 and SKA-111 in KCa3.1 (Supplementary Figure 1). Based on these docking poses we hypothesized that disruption of the hydrogen bond between the –NH2 group of the benzothiazole ring, and the CaM M51 and E54 residues, which are present in both the KCa3.1 and KCa2.2 models, might be a way to achieve KCa2.2 selectivity
Summary
Small- and intermediate-conductance calcium-activated potassium channels (KCa) are voltage independent and are gated by the binding of calcium to calmodulin, which functions as their calciumsensing beta subunit (Xia et al, 1998; Fanger et al, 1999). The idea behind the later hypothesis is that by enhancing K+ efflux and lowering intracellular K+ it might be possible to reset the “ionic checkpoint” and boost anti-tumor T cell functions in tumor infiltrating T cells, which have been shown to have increased intracellular K+ concentrations suppressing their ability to activate (Eil et al, 2016). In support of this exciting therapeutic postulate, it has recently been demonstrated that pharmacological KCa3.1 activation can restore the ability of cancer patient derived CD8+ T cells to chemotax (Chimote et al, 2018)
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