The Trials and Tribulations of Structure Assisted Design of KCa Channel Activators

Calcium-activated K+ channels constitute attractive targets for the treatment of neurological and cardiovascular diseases. To explain why certain 2-aminobenzothiazole/oxazole-type K-Ca activators (SKAs) are K(Ca)3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of K(Ca)3.1 and K(Ca)2.3 in complex with CaM using Rosetta modeling software. We here attempted to employ this atomistic level understanding of K-Ca activator binding to switch selectivity around and design K(Ca)2.2 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 K(Ca)2.2 and K(Ca)3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the K(Ca)2.2 and K-Ca.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and K(Ca)2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any K(Ca)2.2 selective compounds. Based on the full-length K(Ca)3.1 structure it was recently demonstrated that the C-terminal crystal dimer was an artefact and suggested that the real binding pocket for the K-Ca 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 K-Ca 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 K(Ca)3.1.