University of Minnesota, Minneapolis, MN, USA.
Photonic Pharma LLC, Minneapolis, MN, USA.
ASN Neuro. 2023 Jan-Dec;15:17590914231184086. doi: 10.1177/17590914231184086.
There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC of 200 nM and casanthranol supported a 85.5% rescue with a projected EC of 34.2 μM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.
目前迫切需要能够挽救由α-突触核蛋白（aSyn）错误折叠和寡聚化引起的病理生理表型的小分子。基于我们之前的 aSyn 细胞荧光寿命（FLT）-Förster 共振能量转移（FRET）生物传感器，我们开发了一种包含红移 mCyRFP1/mMaroon1（OFP/MFP）FRET 对的可诱导细胞模型。这种新的 aSyn FRET 生物传感器提高了信噪比，减少了非特异性背景 FRET，并使 FRET 信号相对于我们之前的 GFP/RFP aSyn 生物传感器增加了 4 倍（瞬时转染）和 2 倍（稳定，可诱导的细胞系）。诱导系统具有更大的时间控制和可扩展性，允许精细调整生物传感器的表达，并最大限度地减少由于过度表达 aSyn 引起的细胞毒性。使用这些可诱导的 aSyn-OFP/MFP 生物传感器，我们筛选了 Selleck 拥有的 2684 种商业上可获得的、FDA 批准的化合物库，并确定原花青素和 casanthranol 为新型命中化合物。二次测定验证了这些化合物调节 aSyn FLT-FRET 的能力。功能测定探测细胞毒性和 aSyn 纤维化表明它们能够抑制接种的 aSyn 纤维化。原花青素完全挽救了 aSyn 纤维诱导的细胞毒性，EC50 为 200 nM， casanthranol 支持 85.5%的挽救，预测 EC50 为 34.2 μM。此外，原花青素为验证我们的 aSyn 生物传感器在未来高通量筛选工业规模（百万化合物）化学库中的性能提供了有价值的工具化合物。
