Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.
DZHK (German Centre for Cardiovascular Research), Berlin, Germany.
Methods Mol Biol. 2022;2483:117-139. doi: 10.1007/978-1-0716-2245-2_8.
A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits.
A-激酶锚定蛋白（AKAPs）是一类多功能支架蛋白。它们与蛋白激酶、激酶底物和其他信号分子直接发生蛋白-蛋白相互作用。每个 AKAP 与一组特定的蛋白相互作用伙伴相互作用，而这些伙伴在不同的细胞区室和细胞之间可以有所不同。因此，AKAP 可以在特定的细胞环境中空间和时间协调信号转导过程。AKAP 依赖性蛋白-蛋白相互作用参与了大量的生理过程，包括心血管、神经和免疫系统的过程。AKAP 的失调及其相互作用与广泛的疾病有关，例如心脏病，如心力衰竭。然而，对于特定的 AKAP 依赖性蛋白-蛋白相互作用的功能理解存在着深刻的不足。部分原因是缺乏专门针对特定蛋白-蛋白相互作用的靶向药物。肽类和非肽类抑制剂是阐明 AKAP 依赖性蛋白-蛋白相互作用功能的宝贵分子工具。此外，这种相互作用破坏剂可能为以 AKAP 依赖性蛋白-蛋白相互作用为潜在药物靶点的疾病治疗开辟新的概念。在这里，我们描述了用于鉴定 AKAP 依赖性蛋白-蛋白相互作用小分子破坏剂的筛选方法。例如，AKAP18 与蛋白激酶 A（PKA）和 AKAP-Lbc 与 RhoA 的相互作用。我们讨论了用于小分子文库筛选的均相时间分辨荧光（HTRF）和 AlphaScreen 测定法，以及人类诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）作为鉴定命中物的细胞系统。
