目的:对于检测纤维状形式的α-突触核蛋白(αSyn)和4-重复tau的化合物,这在许多神经退行性疾病中至关重要。这里,我们的目标是开发一种有效的基于表面等离子体共振(SPR)的测定法,以促进可以结合这些原纤维的小分子的表征。
方法:进行SPR测量以表征荧光配体/化合物对重组淀粉样β(Aβ)42,K18-tau,全长2N4R-tau和αSyn原纤维。进行计算机模拟建模以检查配体在αSyn原纤维上的结合袋。用荧光配体和特异性抗体对帕金森病患者和小鼠模型的死后脑组织切片进行免疫荧光染色。
结果:我们优化了Aβ42,K18-tau,在SPR传感器芯片上处于受控聚集状态的全长2N4R-tau和αSyn原纤维并用于评估它们与配体的结合。结合动力学分析的SPR结果表明,所有原纤维都存在至少两个结合位点,包括发光共轭低聚噻吩,苯并噻唑衍生物,非荧光亚甲蓝和兰索拉唑。对αSyn(6H6B)的计算机模拟研究揭示了四个结合位点,在表面上优先考虑一个位点。免疫荧光染色验证了pS129-αSyn阳性在帕金森病患者和αSyn预形成原纤维注射小鼠的大脑中的检测,arcAβ小鼠中的6E10阳性Aβ,和pR5小鼠中的AT-8/AT-100阳性。
结论:与Aβ42,K18/全长2N4R-tau和αSyn原纤维结合的小分子的SPR测量表明存在多个结合位点。这种方法可以为神经退行性疾病相关蛋白质病的化合物提供有效的表征。
OBJECTIVE: 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.
METHODS: SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds toward recombinant amyloid-beta (Aβ)42, 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.
RESULTS: We optimized the protocol for the immobilization of Aβ42, 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.
CONCLUSIONS: SPR measurements of small molecules binding to Aβ42, 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.