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

Abstract

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_Ca3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of K_Ca3.1 and K_Ca2.3 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_Ca2.2 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_Ca2.2 and K_Ca3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the K_Ca2.2 and K_Ca.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and K_Ca2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any K_Ca2.2 selective compounds. Based on the full-length K_Ca3.1 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_Ca3.1.

Keywords: KCa activators; KCa2.2; KCa3.1; Rosetta; SKA-111; calcium-activated potassium channels; calmodulin binding domain.