Rudolf Virchow Center, Center for Integrative and Translational Bioimaging, University of Wuerzburg, Josef-Schneider-Str. 2, Germany, 97080, Wuerzburg, Germany.
Dynamic Biosensors GmbH Germany, Lochhamer Strasse 15, 82152, Martinsried/Planegg, Germany.
Commun Biol. 2022 Oct 7;5(1):1070. doi: 10.1038/s42003-022-03997-3.
Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.
多价蛋白相互作用物是探测蛋白功能和探索新型药物策略的一种有吸引力的模式。目前，用于有效开发多价结合物的最先进方法和工作流程的通量和精度受到表面固定化、荧光标记和样品消耗的限制。我们以抑制性突触的主调控因子神经胶质蛋白作为基准，举例说明了荧光近程感应 (FPS) 在多价肽结构的系统动力学和热力学优化中的应用。高通量合成了 100 多种具有不同组合二聚体、四聚体和八聚体结构的肽，结合直接 FPS 测量，与三种互补技术相比，以高精度和低样品消耗的方式解析了结合物的结合速率、解离速率和离解常数。该数据集及其基于机器学习的分析阐明了特定结构特征与结合动力学之间的关系，从而鉴定出具有前所未有的蛋白抑制能力的结合物；因此，突出了 FPS 用于多价抑制剂合理设计的价值。
