Abstract
BackgroundKCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development.MethodsHere we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders.ResultsUsing the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 μM.ConclusionOur results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.
Highlights
KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression
General description of the surface plasmon resonance (SPR)-based screening method To efficiently screen a library of chemical compounds for binding to the intracellular PAS and C-linker/cyclic nucleotide-binding homology (CNBH) domains of KCNH channels we developed a SPR-based screening method
Structural comparison of the isolated domains with the recent cryo-electron microscopy structures of the full-length EAG, EAG-related gene (ERG) and HCN1 channels indicates that the fold of the PAS and Clinker/cyclic nucleotide binding (CNB)(CNBH) domains is essentially the same in the isolated form and as part of the full-length channel structures [34, 40,41,42,43]
Summary
KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. The CNBH domain shares sequence and structural similarity with the cyclic nucleotide binding (CNB) domains of the hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, and other CNB domain containing proteins, such as CAP and PKA [21, 24, 25]. Both PAS and CNB domains frequently function as ligand-binding domains in other proteins [23, 26,27,28,29]. The presence of cavities suitable for small molecule binding in the PAS and CNBH domains of KCNH channels suggests that there is a potential for discovery of small molecule binders and necessitates the development of novel methods for their identification [33]
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