Braun Anthony R, Liao Elly E, Horvath Mian, Kalra Prakriti, Acosta Karen, Young Malaney C, Kochen Noah Nathan, Lo Chih Hung, Brown Roland, Evans Michael D, Pomerantz William C K, Rhoades Elizabeth, Luk Kelvin, Cornea Razvan L, Thomas David D, Sachs Jonathan N
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
NPJ Parkinsons Dis. 2021 Jun 28;7(1):52. doi: 10.1038/s41531-021-00195-6.
We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.
我们开发了一种高通量药物发现平台，利用荧光α-突触核蛋白（αSN）生物传感器测量荧光共振能量转移（FRET），以检测活细胞中的自发预纤维寡聚体。我们的两种αSN FRET生物传感器为αSN的寡聚化和构象提供了互补的见解，以提高治疗帕金森病药物发现活动的成功率。我们通过荧光寿命而非传统的荧光强度来测量FRET，提供了具有更高分辨率和精度的结构读数。这有助于识别那些在寡聚体状态集合中引起细微但显著构象变化的化合物，而这些变化使用基于强度的FRET很容易被忽略。我们筛选了一个包含1280种化合物的小分子文库，鉴定出21种使寿命变化超过5个标准差的化合物。其中两种化合物在保护SH-SY5Y细胞免受αSN诱导的死亡方面具有纳摩尔效力，比已知抑制剂提高了近十倍。我们在小鼠原代神经元αSN病理学检测（小鼠αSN预形成纤维的磷酸化）中测试了几种化合物的疗效，结果显示其中两种化合物能够挽救病理学变化。通过生物物理和生化分析对这些命中化合物进行了进一步表征，以探索其潜在作用机制。体外αSN寡聚化、单分子FRET和蛋白质观察氟核磁共振实验表明，这些化合物调节αSN寡聚体而非单体。随后的聚集分析进一步表明，这些化合物还能阻止或阻断αSN纤维的组装。
