Functional Genomics Center, University of Zurich & ETH Zurich, Zürich, Switzerland.
Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden.
Eur J Nucl Med Mol Imaging. 2024 Nov;51(13):3960-3977. doi: 10.1007/s00259-024-06806-7. Epub 2024 Jul 2.
There is an unmet need for compounds to detect fibrillar forms of alpha-synuclein (αSyn) and 4-repeat tau, which are critical in many neurodegenerative diseases. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based assay to facilitate the characterization of small molecules that can bind these fibrils.
SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds toward recombinant amyloid-beta (Aβ), K18-tau, full-length 2N4R-tau and αSyn fibrils. In silico modeling was performed to examine the binding pockets of ligands on αSyn fibrils. Immunofluorescence staining of postmortem brain tissue slices from Parkinson's disease patients and mouse models was performed with fluorescence ligands and specific antibodies.
We optimized the protocol for the immobilization of Aβ, K18-tau, full-length 2N4R-tau and αSyn fibrils in a controlled aggregation state on SPR-sensor chips and for assessing their binding to ligands. The SPR results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes, benzothiazole derivatives, nonfluorescent methylene blue and lansoprazole. In silico modeling studies for αSyn (6H6B) revealed four binding sites with a preference for one site on the surface. Immunofluorescence staining validated the detection of pS129-αSyn positivity in the brains of Parkinson's disease patients and αSyn preformed-fibril injected mice, 6E10-positive Aβ in arcAβ mice, and AT-8/AT-100-positivity in pR5 mice.
SPR measurements of small molecules binding to Aβ, K18/full-length 2N4R-tau and αSyn fibrils suggested the existence of multiple binding sites. This approach may provide efficient characterization of compounds for neurodegenerative disease-relevant proteinopathies.
目前需要开发能够检测α-突触核蛋白（αSyn）和 4 重复tau 纤维形式的化合物，因为这些纤维形式在许多神经退行性疾病中至关重要。本研究旨在开发一种有效的表面等离子体共振（SPR）分析方法，以促进能够结合这些纤维的小分子的特性分析。
通过 SPR 测量来描述荧光配体/化合物与重组淀粉样蛋白-β（Aβ）、K18-tau、全长 2N4R-tau 和 αSyn 纤维的结合特性。采用计算模拟的方法，研究配体在 αSyn 纤维上的结合口袋。使用荧光配体和特异性抗体对帕金森病患者和小鼠模型的脑组织切片进行免疫荧光染色。
我们优化了 SPR 传感器芯片上的 Aβ、K18-tau、全长 2N4R-tau 和 αSyn 纤维的固定方案，使纤维处于受控聚集状态，并用于评估它们与配体的结合情况。分析结合动力学的 SPR 结果表明，所有纤维都至少有两个结合位点，包括发光共轭寡聚噻吩、苯并噻唑衍生物、非荧光亚甲蓝和兰索拉唑。针对 αSyn（6H6B）的计算模拟研究显示，在表面上有四个结合位点，其中一个具有优先性。免疫荧光染色验证了在帕金森病患者和注射αSyn 预制纤维的小鼠的大脑中检测到 pS129-αSyn 阳性，在 arcAβ 小鼠中检测到 6E10 阳性的 Aβ，在 pR5 小鼠中检测到 AT-8/AT-100 阳性。
SPR 测量小分子与 Aβ、K18/全长 2N4R-tau 和 αSyn 纤维的结合表明存在多个结合位点。这种方法可能为神经退行性疾病相关蛋白病提供化合物的有效特性分析。
