Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Methods Mol Biol. 2021;2248:121-137. doi: 10.1007/978-1-0716-1130-2_9.
Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor-receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor-receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.
肿瘤坏死因子受体 1（TNFR1）的抑制作用是治疗自身免疫和炎症性疾病的数十亿美元产业。由于目前的抗 TNF 疗法由于配体的全球抑制而导致危险的副作用，因此受体特异性的 TNFR1 信号抑制是一个强烈追求的策略。为了直接监测活细胞中受体的结构变化，我们通过将绿色和红色荧光蛋白融合到 TNFR1 上来设计了荧光共振能量转移（FRET）生物传感器。FRET 生物传感器在活细胞中的表达可检测受体-受体相互作用和受体结构动力学。使用 TNFR1 FRET 生物传感器，结合高精度和高通量荧光寿命检测技术，我们开发了一种基于时间分辨 FRET 的高通量筛选平台，用于发现直接针对和调节 TNFR1 功能的小分子。使用这种方法在筛选多个药物文库中，我们发现了一种竞争性抑制剂，可破坏受体-受体相互作用，以及改变受体结构状态的别构调节剂。这使科学家能够通过与化合物对受体结构的扰动相关的生物物理方法进行高通量筛选，以发现具有高特异性调节 TNFR1 信号的新型先导化合物。
