Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC, USA.
Department of Oncology, Georgetown University Medical Center, Washington, DC, USA.
BMC Pharmacol Toxicol. 2019 Jul 17;20(1):42. doi: 10.1186/s40360-019-0324-8.
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. 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.
Here 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.
Using 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.
Our 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.
KCNH 家族的钾通道负责多种生理功能，从神经元兴奋性和心脏收缩的调节到癌症进展的调节。KCNH 通道在其 N 端含有 Per-Arn-Sim（PAS）结构域，在其 C 端含有环核苷酸结合同源性（CNBH）结构域。这些细胞内结构域塑造了 KCNH 通道的功能，是药物开发的重要靶点。
本文描述了一种基于表面等离子体共振（SPR）的筛选方法，旨在鉴定三种 KCNH 通道亚家族（Ether-à-go-go（EAG）、EAG 相关基因（ERG）和 EAG 样 K+（ELK））的 PAS 和 CNBH 结构域的小分子结合物。该方法包括 PAS 和 CNBH 结构域的纯化、纯化结构域在 SPR 传感器芯片上的固定以及使用 SPR 响应的变化作为结合的报告，筛选化学文库中的小分子与固定化结构域的结合。该方法的优点包括用于实施筛选所需的少量纯化 PAS 和 CNBH 结构域、直接评估小分子与 PAS 和 CNBH 结构域的结合以及易于评估小分子结合物对 KCNH 亚家族的特异性。
使用基于 SPR 的方法，我们针对三种 KCNH 通道亚家族的 PAS 和 CNBH 结构域筛选了 2560 种化合物的 Spectrum Collection 文库，并鉴定了一组与 PAS 或 CNBH 结构域结合的小分子。为了进一步评估筛选的有效性，我们测试了一种鉴定出的 mEAG PAS 结构域特异性小分子结合物对 EAG 通道电流的功能影响。十一烯酸以浓度依赖的方式抑制 EAG 通道的电流，IC50 约为 1μM。
我们的结果表明，基于 SPR 的方法非常适合鉴定 KCNH 通道的小分子结合物，也可以促进其他离子通道的药物发现。
