Biochemistry and Molecular Biology Graduate Program, The University of Texas Medical Branch, Galveston, TX, USA.
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX, USA.
Methods Mol Biol. 2021;2188:191-228. doi: 10.1007/978-1-0716-0818-0_10.
As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein-protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na (Nav) channel Nav1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.
作为细胞功能的关键参与者，离子通道是针对广泛健康状况的药物发现和治疗开发的重要靶标。因此，开发在生理条件下重组离子通道大分子复合物的测定法并筛选这些复合物中蛋白质-蛋白质相互作用的化学修饰剂是药物发现活动中的当务之急。对于大多数离子通道来说，在异源哺乳动物细胞中表达其孔形成亚基现在已经成为常规程序。然而，在生理环境中重新构建蛋白质通道复合物仍然具有挑战性，限制了我们基于变构机制识别工具和探针的能力，这可能导致对通道功能的更具针对性和更精确的调节。在这里，我们描述了使用荧光素酶互补测定法（LCA）稳定地重建电压门控 Na（Nav）通道 Nav1.6与其辅助蛋白成纤维细胞生长因子 14（FGF14）之间相互作用的测定法开发步骤，然后在 384 孔板中进行测定法的微型化和优化，用于针对蛋白质-通道相互作用进行细胞内高通量筛选（HTS）。这种优化的 LCA 可以随后用于通过剂量依赖性研究快速估计命中的效力和功效，从而在更耗时的验证研究之前对命中进行排名。最后，我们介绍了使用半自动平面膜片钳电生理学进行快速功能命中验证研究的方法。我们的稳健的细胞内 HTS 平台可以适应任何合适的离子通道复合物，以使用激酶抑制剂探索细胞信号转导的调节途径，以及筛选小分子用于探针开发和药物再利用以针对新的靶标/医学领域。总体而言，该测定法的灵活性允许用户快速探索与通道病相关疾病的治疗选择，从而在药物发现早期阶段快速生成假说，并在更耗时的体内研究之前缩小目标。
